The Bradford assay is a quick and fairly sensitive method for measuring the concentrations of proteins. It is based on the shift in absorbance maximum of Coomassie Brilliant Blue G-250 dye from 465 to 595 nm following binding to denatured proteins in solution.

A protein shredder that occurs in cell membranes of brain cells apparently also indirectly regulates the fat metabolism.

Determining the concentration of protein samples generally is accomplished either by measuring the UV absorbance at 280 nm or by reacting the protein quantitatively with dyes and/or metal ions (Bradford, Lowry, or BCA assays). For purified proteins, UV absorbance remains the most popular method because it is fast, convenient, and reproducible; it does not consume the protein; and it requires no additional reagents, standards, or incubations. No method of protein concentration determination is perfect because each is subject to a different set of constraints such as interference of buffer components and contaminating proteins in direct UV determination (A₂₈₀) or reactivity of individual proteins and buffer components with the detecting reagents in colorimetric assays. In cases in which protein concentration is critical (e.g., determination of catalytic rate constants for an enzyme), it may be advisable to compare the results of several assays.

Atherosclerotic cardiovascular diseases have roots in childhood. Modification of dietary fat intake influences serum lipid and lipoprotein concentrations. Reduction of saturated fat intake is recommended to promote cardiovascular health.

Dietary counseling had a beneficial effect on saturated fat intake from ages 7 months to 19 years. The counseling reduced serum low-density lipoprotein cholesterol concentrations in both genders. It also decreased computationally estimated concentrations of intermediate-density lipoprotein cholesterol, very low-density lipoprotein–triglycerides and apolipoprotein B in boys. (Read the full article)

Pediatric osteoarticular infections can be treated with successful microbiologic and clinical outcomes with a transition from parenteral to oral therapy. The best way to determine the timing of this transition is neither well studied nor standardized.

A total of 193 (99.5%) of 194 pediatric patients with acute bacterial osteoarticular infections were successfully transitioned to oral therapy, determined by using a combination of clinical findings and C-reactive protein levels, representing the largest single-center data set analyzed. (Read the full article)

Biomarkers such as C-reactive protein (CRP) and procalcitonin are elevated in children with severe bacterial infections. Children with severe malnutrition are at increased risk of bacterial infections and early markers for the diagnosis of infection in these children are needed.

Despite elevated values in severely malnourished children with invasive bacterial infection or infectious diarrhea, CRP and procalcitonin have limited diagnostic value. CRP could predict death in these children with a good negative predictive value. (Read the full article)

Myxovirus resistance protein A (MxA) is a protein induced during viral infections. A few small-scale studies have suggested that MxA could be used as a marker of viral infection in clinical routine practice.

This study involves the largest patient population thus far and confirms the usefulness of MxA for diagnosing viral infections in children consulting the emergency department in a clinical routine setting. (Read the full article)

KBP-7072 is a semi-synthetic aminomethylcycline with broad-spectrum activity against Gram-positive and Gram-negative pathogens including multidrug resistant bacterial strains. The pharmacokinetics (PK) of KBP-7072 after oral and intravenous (IV) administration of single and multiple doses were investigated in animal models including during fed and fasted states and also evaluated the protein binding and excretion characteristics. In Sprague-Dawley (SD) rats, Beagle dogs, and CD-1 mice, KBP-7072 demonstrated a linear PK profile after administration of single oral and IV and multiple oral doses. Oral bioavailability ranged from 12% to 32%. Mean T_max ranged from 0.5 to 4 hours, and mean half-life ranged from approximately 6 to 11 hours. Administration of oral doses in the fed state resulted in a marked reduction in C_max and AUC compared with dosing in fasted animals. The mean bound fractions of KBP-7072 were 77.5%, 69.8%, 64.5%, 69.3%, and 69.2% in mouse, rat, dog, monkey, and human plasma, respectively. Following a single 22.5 mg/kg oral dose of KBP-7072 in SD rats, cumulative excretion in feces was 64% and in urine was 2.5% of the administered dose. The PK results in animal models are consistent with single and multiple ascending dose studies in healthy volunteers and confirm the suitability of KBP-7072 for once daily oral and IV administration in clinical studies.

The rapid evolution of resistance in the malaria parasite to every single drug developed against it calls for the urgent identification of new molecular targets. Using a stain specific for the detection of intracellular amyloid deposits in live cells we have detected the presence of abundant protein aggregates in Plasmodium falciparum blood stages and female gametes cultured in vitro, in the blood stages of mice infected by Plasmodium yoelii, and in the mosquito stages of the murine malaria species Plasmodium berghei. Aggregated proteins could not be detected in early rings, the parasite form that starts the intraerythrocytic cycle. A proteomics approach was followed to pinpoint actual aggregating polypeptides in functional P. falciparum blood stages, which resulted in the identification of 369 proteins, with roles particularly enriched in nuclear import-related processes. Five aggregation-prone short peptides selected from this protein pool exhibited different aggregation propensity according to Thioflavin-T fluorescence measurements, and were observed to form amorphous aggregates and amyloid fibrils in transmission electron microscope images. The results presented suggest that generalized protein aggregation might have a functional role in malaria parasites. Future antimalarial strategies based on the upsetting of the pathogen's proteostasis and therefore affecting multiple gene products could represent the entry to new therapeutic approaches.

ABSTRACT

Human immunodeficiency virus type 1 (HIV-1) establishes lifelong infections in humans, a process that relies on its ability to thwart innate and adaptive immune defenses of the host. Recently, we reported that HIV-1 infection results in a dramatic reduction of the cellular peroxisome pool. Peroxisomes are metabolic organelles that also function as signaling platforms in the innate immune response. Here, we show that the HIV-1 accessory protein Vpu is necessary and sufficient for the depletion of cellular peroxisomes during infection. Vpu induces the expression of four microRNAs that target mRNAs encoding proteins required for peroxisome formation and metabolic function. The ability of Vpu to downregulate peroxisomes was found to be dependent upon the Wnt/β-catenin signaling pathway. Given the importance of peroxisomes in innate immune signaling and central nervous system function, the roles of Vpu in dampening antiviral signaling appear to be more diverse than previously realized. Finally, our findings highlight a potential role for Wnt/β-catenin signaling in peroxisome homeostasis through modulating the production of biogenesis factors.

IMPORTANCE People living with HIV can experience accelerated aging and the development of neurological disorders. Recently, we reported that HIV-1 infection results in a dramatic loss of peroxisomes in macrophages and brain tissue. This is significant because (i) peroxisomes are important for the innate immune response and (ii) loss of peroxisome function is associated with cellular aging and neurodegeneration. Accordingly, understanding how HIV-1 infection causes peroxisome depletion may provide clues regarding how the virus establishes persistent infections and, potentially, the development of neurological disorders. Here, we show that the accessory protein Vpu is necessary and sufficient for the induction of microRNAs that target peroxisome biogenesis factors. The ability of Vpu to downregulate peroxisome formation depends on the Wnt/β-catenin pathway. Thus, in addition to revealing a novel mechanism by which HIV-1 uses intracellular signaling pathways to target antiviral signaling platforms (peroxisomes), we have uncovered a previously unknown link between the Wnt/β-catenin pathway and peroxisome homeostasis.

ABSTRACT

Bacterial flagella are rotating nanomachines required for motility. Flagellar gene expression and protein secretion are coordinated for efficient flagellar biogenesis. Polar flagellates, unlike peritrichous bacteria, commonly order flagellar rod and hook gene transcription as a separate step after production of the MS ring, C ring, and flagellar type III secretion system (fT3SS) core proteins that form a competent fT3SS. Conserved regulatory mechanisms in diverse polar flagellates to create this polar flagellar transcriptional program have not been thoroughly assimilated. Using in silico and genetic analyses and our previous findings in Campylobacter jejuni as a foundation, we observed a large subset of Gram-negative bacteria with the FlhF/FlhG regulatory system for polar flagellation to possess flagellum-associated two-component signal transduction systems (TCSs). We present data supporting a general theme in polar flagellates whereby MS ring, rotor, and fT3SS proteins contribute to a regulatory checkpoint during polar flagellar biogenesis. We demonstrate that Vibrio cholerae and Pseudomonas aeruginosa require the formation of this regulatory checkpoint for the TCSs to directly activate subsequent rod and hook gene transcription, which are hallmarks of the polar flagellar transcriptional program. By reprogramming transcription in V. cholerae to more closely follow the peritrichous flagellar transcriptional program, we discovered a link between the polar flagellar transcription program and the activity of FlhF/FlhG flagellar biogenesis regulators in which the transcriptional program allows polar flagellates to continue to produce flagella for motility when FlhF or FlhG activity may be altered. Our findings integrate flagellar transcriptional and biogenesis regulatory processes involved in polar flagellation in many species.

IMPORTANCE Relative to peritrichous bacteria, polar flagellates possess regulatory systems that order flagellar gene transcription differently and produce flagella in specific numbers only at poles. How transcriptional and flagellar biogenesis regulatory systems are interlinked to promote the correct synthesis of polar flagella in diverse species has largely been unexplored. We found evidence for many Gram-negative polar flagellates encoding two-component signal transduction systems with activity linked to the formation of flagellar type III secretion systems to enable production of flagellar rod and hook proteins at a discrete, subsequent stage during flagellar assembly. This polar flagellar transcriptional program assists, in some manner, the FlhF/FlhG flagellar biogenesis regulatory system, which forms specific flagellation patterns in polar flagellates in maintaining flagellation and motility when activity of FlhF or FlhG might be altered. Our work provides insight into the multiple regulatory processes required for polar flagellation.

ABSTRACT

Pneumocystis, a major opportunistic pathogen in patients with a broad range of immunodeficiencies, contains abundant surface proteins encoded by a multicopy gene family, termed the major surface glycoprotein (Msg) gene superfamily. This superfamily has been identified in all Pneumocystis species characterized to date, highlighting its important role in Pneumocystis biology. In this report, through a comprehensive and in-depth characterization of 459 msg genes from 7 Pneumocystis species, we demonstrate, for the first time, the phylogeny and evolution of conserved domains in Msg proteins and provide a detailed description of the classification, unique characteristics, and phylogenetic relatedness of five Msg families. We further describe, for the first time, the relative expression levels of individual msg families in two rodent Pneumocystis species, the substantial variability of the msg repertoires in P. carinii from laboratory and wild rats, and the distinct features of the expression site for the classic msg genes in Pneumocystis from 8 mammalian host species. Our analysis suggests multiple functions for this superfamily rather than just conferring antigenic variation to allow immune evasion as previously believed. This study provides a rich source of information that lays the foundation for the continued experimental exploration of the functions of the Msg superfamily in Pneumocystis biology.

IMPORTANCE Pneumocystis continues to be a major cause of disease in humans with immunodeficiency, especially those with HIV/AIDS and organ transplants, and is being seen with increasing frequency worldwide in patients treated with immunodepleting monoclonal antibodies. Annual health care associated with Pneumocystis pneumonia costs ~$475 million dollars in the United States alone. In addition to causing overt disease in immunodeficient individuals, Pneumocystis can cause subclinical infection or colonization in healthy individuals, which may play an important role in species preservation and disease transmission. Our work sheds new light on the diversity and complexity of the msg superfamily and strongly suggests that the versatility of this superfamily reflects multiple functions, including antigenic variation to allow immune evasion and optimal adaptation to host environmental conditions to promote efficient infection and transmission. These findings are essential to consider in developing new diagnostic and therapeutic strategies.

ABSTRACT

The nonsense-mediated decay (NMD) pathway presents a challenge for RNA viruses with termination codons that precede extended 3' untranslated regions (UTRs). The umbravirus Pea enation mosaic virus 2 (PEMV2) is a nonsegmented, positive-sense RNA virus with an unusually long 3' UTR that is susceptible to NMD. To establish a systemic infection, the PEMV2 long-distance movement protein p26 was previously shown to both stabilize viral RNAs and bind them for transport through the plant’s vascular system. The current study demonstrated that p26 protects both viral and nonviral messenger RNAs from NMD. Although p26 localizes to both the cytoplasm and nucleolus, p26 exerts its anti-NMD effects exclusively in the cytoplasm independently of long-distance movement. Using a transcriptome-wide approach in the model plant Nicotiana benthamiana, p26 protected a subset of cellular NMD target transcripts, particularly those containing long, structured, GC-rich 3' UTRs. Furthermore, transcriptome sequencing (RNA-seq) revealed that the NMD pathway is highly dysfunctional during PEMV2 infection, with 1,820 (48%) of NMD targets increasing in abundance. Widespread changes in the host transcriptome are common during plant RNA virus infections, and these results suggest that, in at least some instances, virus-mediated NMD inhibition may be a major contributing factor.

IMPORTANCE Nonsense-mediated decay (NMD) represents an RNA regulatory pathway that degrades both natural and faulty messenger RNAs with long 3' untranslated regions. NMD targets diverse families of RNA viruses, requiring that viruses counteract the NMD pathway for successful amplification in host cells. A protein required for long-distance movement of Pea enation mosaic virus 2 (PEMV2) is shown to also protect both viral and host mRNAs from NMD. RNA-seq analyses of the Nicotiana benthamiana transcriptome revealed that PEMV2 infection significantly impairs the host NMD pathway. RNA viruses routinely induce large-scale changes in host gene expression, and, like PEMV2, may use NMD inhibition to alter the host transcriptome in an effort to increase virus amplification.

ABSTRACT

During infection of human parvovirus B19 (B19V), one viral precursor mRNA (pre-mRNA) is transcribed by a single promoter and is alternatively spliced and alternatively polyadenylated. Here, we identified a novel cis-acting sequence (5'-GUA AAG CUA CGG GAC GGU-3'), intronic splicing enhancer 3 (ISE3), which lies 72 nucleotides upstream of the second splice acceptor (A2-2) site of the second intron that defines the exon of the mRNA encoding the 11-kDa viral nonstructural protein. RNA binding motif protein 45 (RBM45) specifically binds to ISE3 with high affinity (equilibrium dissociation constant [K_D] = 33 nM) mediated by its RNA recognition domain and 2-homo-oligomer assembly domain (RRM2-HOA). Knockdown of RBM45 expression or ectopic overexpression of RRM2-HOA in human erythroid progenitor cells (EPCs) expanded ex vivo significantly decreased the level of viral mRNA spliced at the A2-2 acceptor but not that of the mRNA spliced at A2-1 that encodes VP2. Moreover, silent mutations of ISE3 in an infectious DNA of B19V significantly reduced 11-kDa expression. Notably, RBM45 also specifically interacts in vitro with ISE2, which shares the octanucleotide (GGGACGGU) with ISE3. Taken together, our results suggest that RBM45, through binding to both ISE2 and ISE3, is an essential host factor for maturation of 11-kDa-encoding mRNA.

IMPORTANCE Human parvovirus B19 (B19V) is a human pathogen that causes severe hematological disorders in immunocompromised individuals. B19V infection has a remarkable tropism with respect to human erythroid progenitor cells (EPCs) in human bone marrow and fetal liver. During B19V infection, only one viral precursor mRNA (pre-mRNA) is transcribed by a single promoter of the viral genome and is alternatively spliced and alternatively polyadenylated, a process which plays a key role in expression of viral proteins. Our studies revealed that a cellular RNA binding protein, RBM45, binds to two intron splicing enhancers and is essential for the maturation of the small nonstructural protein 11-kDa-encoding mRNA. The 11-kDa protein plays an important role not only in B19V infection-induced apoptosis but also in viral DNA replication. Thus, the identification of the RBM45 protein and its cognate binding site in B19V pre-mRNA provides a novel target for antiviral development to combat B19V infection-caused severe hematological disorders.

ABSTRACT

The ubiquitous parasite Toxoplasma gondii exhibits an impressive ability to maintain chronic infection of its host for prolonged periods. Despite this, little is known regarding whether and how T. gondii bradyzoites, a quasi-dormant life stage residing within intracellular cysts, manipulate the host cell to maintain persistent infection. A previous proteomic study of the cyst wall, an amorphous layer of proteins that forms underneath the cyst membrane, identified MYR1 as a putative cyst wall protein in vitro. Because MYR1 is known to be involved in the translocation of parasite-derived effector proteins into the host cell, we sought to determine whether parasites transitioning toward the bradyzoite life stage retain the capacity to translocate proteins via this pathway. By epitope tagging the endogenous loci of four known effectors that translocate from the parasitophorous vacuole into the host cell nucleus, we show, by immunofluorescence assays, that most effectors accumulate in the host nucleus at early but not late time points after infection, during the tachyzoite-to-bradyzoite transition and when parasites further along the bradyzoite differentiation continuum invade a new host cell. We demonstrate that the suppression of interferon gamma signaling, which was previously shown to be mediated by the effector TgIST, also occurs in the context of prolonged infection with bradyzoites and that TgIST export is a process that occurs beyond the early stages of host cell infection. These findings have important implications regarding how this highly successful parasite maintains persistent infection of its host.

IMPORTANCE Toxoplasma bradyzoites persist within tissue cysts and are refractory to current treatments, serving as a reservoir for acute complications in settings of compromised immunity. Much remains to be understood regarding how this life stage successfully establishes and maintains persistent infection. In this study, we investigated whether the export of parasite effector proteins into the host cell occurs during the development of in vitro tissue cysts. We quantified the presence of four previously described effectors in host cell nuclei at different time points after bradyzoite differentiation and found that they accumulated largely during the early stages of infection. Despite a decline in nuclear accumulation, we found that one of these effectors still mediated its function after prolonged infection with bradyzoites, and we provide evidence that this effector is exported beyond early infection stages. These findings suggest that effector export from within developing tissue cysts provides one potential mechanism by which this parasite achieves chronic infection.

ABSTRACT

The posttranslational Ca²⁺-dependent "clip-and-link" activity of large repeat-in-toxin (RTX) proteins starts by Ca²⁺-dependent structural rearrangement of a highly conserved self-processing module (SPM). Subsequently, an internal aspartate-proline (Asp-Pro) peptide bond at the N-terminal end of SPM breaks, and the liberated C-terminal aspartyl residue can react with a free -amino group of an adjacent lysine residue to form a new isopeptide bond. Here, we report a solution structure of the calcium-loaded SPM (Ca-SPM) derived from the FrpC protein of Neisseria meningitidis. The Ca-SPM structure defines a unique protein architecture and provides structural insight into the autocatalytic cleavage of the Asp-Pro peptide bond through a "twisted-amide" activation. Furthermore, in-frame deletion of the SPM domain from the ApxIVA protein of Actinobacillus pleuropneumoniae attenuated the virulence of this porcine pathogen in a pig respiratory challenge model. We hypothesize that the Ca²⁺-dependent clip-and-link activity represents an unconventional strategy for Gram-negative pathogens to adhere to the host target cell surface.

IMPORTANCE The Ca²⁺-dependent clip-and-link activity of large repeat-in-toxin (RTX) proteins is an exceptional posttranslational process in which an internal domain called a self-processing module (SPM) mediates Ca²⁺-dependent processing of a highly specific aspartate-proline (Asp-Pro) peptide bond and covalent linkage of the released aspartyl to an adjacent lysine residue through an isopeptide bond. Here, we report the solution structures of the Ca²⁺-loaded SPM (Ca-SPM) defining the mechanism of the autocatalytic cleavage of the Asp414-Pro415 peptide bond of the Neisseria meningitidis FrpC exoprotein. Moreover, deletion of the SPM domain in the ApxIVA protein, the FrpC homolog of Actinobacillus pleuropneumoniae, resulted in attenuation of virulence of the bacterium in a pig infection model, indicating that the Ca²⁺-dependent clip-and-link activity plays a role in the virulence of Gram-negative pathogens.

ABSTRACT

Human adenoviruses (HAdVs) have developed mechanisms to manipulate cellular antiviral measures to ensure proper DNA replication, with detailed processes far from being understood. Host cells repress incoming viral genomes through a network of transcriptional regulators that normally control cellular homeostasis. The nuclear domains involved are promyelocytic leukemia protein nuclear bodies (PML-NBs), interferon-inducible, dot-like nuclear structures and hot spots of SUMO posttranslational modification (PTM). In HAdV-infected cells, such SUMO factories are found in close proximity to newly established viral replication centers (RCs) marked by the adenoviral DNA binding protein (DBP) E2A. Here, we show that E2A is a novel target of host SUMOylation, leading to PTMs supporting E2A function in promoting productive infection. Our data show that SUMOylated E2A interacts with PML. Decreasing SUMO-E2A protein levels by generating HAdV variants mutated in the three main SUMO conjugation motifs (SCMs) led to lower numbers of viral RCs and PML-NBs, and these two structures were no longer next to each other. Our data further indicate that SUMOylated E2A binds the host transcription factor Sp100A, promoting HAdV gene expression, and represents the molecular bridge between PML tracks and adjacent viral RCs. Consequently, E2A SCM mutations repressed late viral gene expression and progeny production. These data highlight a novel mechanism used by the virus to benefit from host antiviral responses by exploiting the cellular SUMO conjugation machinery.

IMPORTANCE PML nuclear bodies (PML-NBs) are implicated in general antiviral defense based on recruiting host restriction factors; however, it is not understood so far why viruses would establish viral replication centers (RCs) juxtaposed to such "antiviral" compartments. To understand this enigma, we investigate the cross talk between PML-NB components and viral RCs to find the missing link connecting both compartments to promote efficient viral replication and gene expression. Taken together, the current concept is more intricate than originally believed, since viruses apparently take advantage of several specific PML-NB-associated proteins to promote productive infection. Simultaneously, they efficiently inhibit antiviral measures to maintain the viral infectious program. Our data provide evidence that SUMOylation of the viral RC marker protein E2A represents the basis of this virus-host interface and regulates various downstream events to support HAdV productive infection. These results are the basis of our current attempts to generate and screen for specific E2A SUMOylation inhibitors to constitute novel therapeutic approaches to limit and prevent HAdV-mediated diseases and mortality of immunosuppressed patients.

ABSTRACT

The malaria parasite Plasmodium falciparum traffics the virulence protein P. falciparum erythrocyte membrane protein 1 (PfEMP1) to the surface of infected red blood cells (RBCs) via membranous organelles, known as the Maurer’s clefts. We developed a method for efficient enrichment of Maurer’s clefts and profiled the protein composition of this trafficking organelle. We identified 13 previously uncharacterized or poorly characterized Maurer’s cleft proteins. We generated transfectants expressing green fluorescent protein (GFP) fusions of 7 proteins and confirmed their Maurer’s cleft location. Using co-immunoprecipitation and mass spectrometry, we generated an interaction map of proteins at the Maurer’s clefts. We identified two key clusters that may function in the loading and unloading of PfEMP1 into and out of the Maurer’s clefts. We focus on a putative PfEMP1 loading complex that includes the protein GEXP07/CX3CL1-binding protein 2 (CBP2). Disruption of GEXP07 causes Maurer’s cleft fragmentation, aberrant knobs, ablation of PfEMP1 surface expression, and loss of the PfEMP1-mediated adhesion. GEXP07 parasites have a growth advantage compared to wild-type parasites, and the infected RBCs are more deformable and more osmotically fragile.

IMPORTANCE The trafficking of the virulence antigen PfEMP1 and its presentation at the knob structures at the surface of parasite-infected RBCs are central to severe adhesion-related pathologies such as cerebral and placental malaria. This work adds to our understanding of how PfEMP1 is trafficked to the RBC membrane by defining the protein-protein interaction networks that function at the Maurer’s clefts controlling PfEMP1 loading and unloading. We characterize a protein needed for virulence protein trafficking and provide new insights into the mechanisms for host cell remodeling, parasite survival within the host, and virulence.

ABSTRACT

Satellite viruses, most commonly found in plants, rely on helper viruses to complete their replication cycle. The only known example of a human satellite virus is the hepatitis D virus (HDV), and it is generally thought to require hepatitis B virus (HBV) to form infectious particles. Until 2018, HDV was the sole representative of the genus Deltavirus and was thought to have evolved in humans, the only known HDV host. The subsequent identification of HDV-like agents in birds, snakes, fish, amphibians, and invertebrates indicated that the evolutionary history of deltaviruses is likely much longer than previously hypothesized. Interestingly, none of the HDV-like agents were found in coinfection with an HBV-like agent, suggesting that these viruses use different helper virus(es). Here we show, using snake deltavirus (SDeV), that HBV and hepadnaviruses represent only one example of helper viruses for deltaviruses. We cloned the SDeV genome into a mammalian expression plasmid, and by transfection could initiate SDeV replication in cultured snake and mammalian cell lines. By superinfecting persistently SDeV-infected cells with reptarenaviruses and hartmaniviruses, or by transfecting their surface proteins, we could induce production of infectious SDeV particles. Our findings indicate that deltaviruses can likely use a multitude of helper viruses or even viral glycoproteins to form infectious particles. This suggests that persistent infections, such as those caused by arenaviruses and orthohantaviruses used in this study, and recurrent infections would be beneficial for the spread of deltaviruses. It seems plausible that further human or animal disease associations with deltavirus infections will be identified in the future.

IMPORTANCE Deltaviruses need a coinfecting enveloped virus to produce infectious particles necessary for transmission to a new host. Hepatitis D virus (HDV), the only known deltavirus until 2018, has been found only in humans, and its coinfection with hepatitis B virus (HBV) is linked with fulminant hepatitis. The recent discovery of deltaviruses without a coinfecting HBV-like agent in several different taxa suggested that deltaviruses could employ coinfection by other enveloped viruses to complete their life cycle. In this report, we show that snake deltavirus (SDeV) efficiently utilizes coinfecting reptarena- and hartmaniviruses to form infectious particles. Furthermore, we demonstrate that cells expressing the envelope proteins of arenaviruses and orthohantaviruses produce infectious SDeV particles. As the envelope proteins are responsible for binding and infecting new host cells, our findings indicate that deltaviruses are likely not restricted in their tissue tropism, implying that they could be linked to animal or human diseases other than hepatitis.

ABSTRACT

Hantaviruses are the etiological agent of hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS). The latter is associated with case fatality rates ranging from 30% to 50%. HCPS cases are rare, with approximately 300 recorded annually in the Americas. Recently, an HCPS outbreak of unprecedented size has been occurring in and around Epuyén, in the southwestern Argentinian state of Chubut. Since November of 2018, at least 29 cases have been laboratory confirmed, and human-to-human transmission is suspected. Despite posing a significant threat to public health, no treatment or vaccine is available for hantaviral disease. Here, we describe an effort to identify, characterize, and develop neutralizing and protective antibodies against the glycoprotein complex (Gn and Gc) of Andes virus (ANDV), the causative agent of the Epuyén outbreak. Using murine hybridoma technology, we generated 19 distinct monoclonal antibodies (MAbs) against ANDV GnGc. When tested for neutralization against a recombinant vesicular stomatitis virus expressing the Andes glycoprotein (GP) (VSV-ANDV), 12 MAbs showed potent neutralization and 8 showed activity in an antibody-dependent cellular cytotoxicity reporter assay. Escape mutant analysis revealed that neutralizing MAbs targeted both the Gn and the Gc. Four MAbs that bound different epitopes were selected for preclinical studies and were found to be 100% protective against lethality in a Syrian hamster model of ANDV infection. These data suggest the existence of a wide array of neutralizing antibody epitopes on hantavirus GnGc with unique properties and mechanisms of action.

IMPORTANCE Infections with New World hantaviruses are associated with high case fatality rates, and no specific vaccine or treatment options exist. Furthermore, the biology of the hantaviral GnGc complex, its antigenicity, and its fusion machinery are poorly understood. Protective monoclonal antibodies against GnGc have the potential to be developed into therapeutics against hantaviral disease and are also great tools to elucidate the biology of the glycoprotein complex.

ABSTRACT

The appressoria that are generated by the rice blast fungus Magnaporthe oryzae in response to surface cues are important for successful colonization. Previous work showed that regulators of G-protein signaling (RGS) and RGS-like proteins play critical roles in appressorium formation. However, the mechanisms by which these proteins orchestrate surface recognition for appressorium induction remain unclear. Here, we performed comparative transcriptomic studies of Morgs mutant and wild-type strains and found that M. oryzae Aa91 (MoAa91), a homolog of the auxiliary activity family 9 protein (Aa9), was required for surface recognition of M. oryzae. We found that MoAA91 was regulated by the MoMsn2 transcription factor and that its disruption resulted in defects in both appressorium formation on the artificial inductive surface and full virulence of the pathogen. We further showed that MoAa91 was secreted into the apoplast space and was capable of competing with the immune receptor chitin elicitor-binding protein precursor (CEBiP) for chitin binding, thereby suppressing chitin-induced plant immune responses. In summary, we have found that MoAa91 is a novel signaling molecule regulated by RGS and RGS-like proteins and that MoAa91 not only governs appressorium development and virulence but also functions as an effector to suppress host immunity.

IMPORTANCE The rice blast fungus Magnaporthe oryzae generates infection structure appressoria in response to surface cues largely due to functions of signaling molecules, including G-proteins, regulators of G-protein signaling (RGS), mitogen-activated protein (MAP) kinase pathways, cAMP signaling, and TOR signaling pathways. M. oryzae encodes eight RGS and RGS-like proteins (MoRgs1 to MoRgs8), and MoRgs1, MoRgs3, MoRgs4, and MoRgs7 were found to be particularly important in appressorium development. To explore the mechanisms by which these proteins regulate appressorium development, we have performed a comparative in planta transcriptomic study and identified an auxiliary activity family 9 protein (Aa9) homolog that we named MoAa91. We showed that MoAa91 was secreted from appressoria and that the recombinant MoAa91 could compete with a chitin elicitor-binding protein precursor (CEBiP) for chitin binding, thereby suppressing chitin-induced plant immunity. By identifying MoAa91 as a novel signaling molecule functioning in appressorium development and an effector in suppressing host immunity, our studies revealed a novel mechanism by which RGS and RGS-like proteins regulate pathogen-host interactions.

ABSTRACT

Temperate bacteriophages are common and establish lysogens of their bacterial hosts in which the prophage is stably inherited. It is typical for such prophages to be integrated into the bacterial chromosome, but extrachromosomally replicating prophages have been described also, with the best characterized being the Escherichia coli phage P1 system. Among the large collection of sequenced mycobacteriophages, more than half are temperate or predicted to be temperate, most of which code for a tyrosine or serine integrase that promotes site-specific prophage integration. However, within the large group of 621 cluster A temperate phages, ~20% lack an integration cassette, which is replaced with a parABS partitioning system. A subset of these phages carry genes coding for a RepA-like protein (RepA phages), which we show here is necessary and sufficient for autonomous extrachromosomal replication. The non-RepA phages appear to replicate using an RNA-based system, as a parABS-proximal region expressing a noncoding RNA is required for replication. Both RepA and non-RepA phage-based plasmids replicate at one or two copies per cell, transform both Mycobacterium smegmatis and Mycobacterium tuberculosis, and are compatible with pAL5000-derived oriM and integration-proficient plasmid vectors. Characterization of these phage-based plasmids offers insights into the variability of lysogenic maintenance systems and provides a large suite of plasmids for actinobacterial genetics that vary in stability, copy number, compatibility, and host range.

IMPORTANCE Bacteriophages are the most abundant biological entities in the biosphere and are a source of uncharacterized biological mechanisms and genetic tools. Here, we identify segments of phage genomes that are used for stable extrachromosomal replication in the prophage state. Autonomous replication of some of these phages requires a RepA-like protein, although most lack repA and use RNA-based systems for replication initiation. We describe a suite of plasmids based on these prophage replication functions that vary in copy number, stability, host range, and compatibility. These plasmids expand the toolbox available for genetic manipulation of Mycobacterium and other Actinobacteria, including Gordonia terrae.

ABSTRACT

The capsule is the dominant Streptococcus pneumoniae virulence factor, yet how variation in capsule thickness is regulated is poorly understood. Here, we describe an unexpected relationship between mutation of adcAII, which encodes a zinc uptake lipoprotein, and capsule thickness. Partial deletion of adcAII in three of five capsular serotypes frequently resulted in a mucoid phenotype that biochemical analysis and electron microscopy of the D39 adcAII mutants confirmed was caused by markedly increased capsule thickness. Compared to D39, the hyperencapsulated adcAII mutant strain was more resistant to complement-mediated neutrophil killing and was hypervirulent in mouse models of invasive infection. Transcriptome analysis of D39 and the adcAII mutant identified major differences in transcription of the Sp_0505-0508 locus, which encodes an SpnD39III (ST5556II) type I restriction-modification system and allelic variation of which correlates with capsule thickness. A PCR assay demonstrated close linkage of the SpnD39IIIC and F alleles with the hyperencapsulated adcAII strains. However, transformation of adcAII with fixed SpnD39III alleles associated with normal capsule thickness did not revert the hyperencapsulated phenotype. Half of hyperencapsulated adcAII strains contained the same single nucleotide polymorphism in the capsule locus gene cps2E, which is required for the initiation of capsule synthesis. These results provide further evidence for the importance of the SpnD39III (ST5556II) type I restriction-modification system for modulating capsule thickness and identified an unexpected linkage between capsule thickness and mutation of adcAII. Further investigation will be needed to characterize how mutation of adcAII affects SpnD39III (ST5556II) allele dominance and results in the hyperencapsulated phenotype.

IMPORTANCE The Streptococcus pneumoniae capsule affects multiple interactions with the host including contributing to colonization and immune evasion. During infection, the capsule thickness varies, but the mechanisms regulating this are poorly understood. We have identified an unsuspected relationship between mutation of adcAII, a gene that encodes a zinc uptake lipoprotein, and capsule thickness. Mutation of adcAII resulted in a striking hyperencapsulated phenotype, increased resistance to complement-mediated neutrophil killing, and increased S. pneumoniae virulence in mouse models of infection. Transcriptome and PCR analysis linked the hyperencapsulated phenotype of the adcAII strain to specific alleles of the SpnD39III (ST5556II) type I restriction-modification system, a system which has previously been shown to affect capsule thickness. Our data provide further evidence for the importance of the SpnD39III (ST5556II) type I restriction-modification system for modulating capsule thickness and identify an unexpected link between capsule thickness and adcAII, further investigation of which could further characterize mechanisms of capsule regulation.

ABSTRACT

Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe and fatal acute respiratory disease in humans and remains endemic in the Middle East since first being identified in 2012. There are currently no approved vaccines or therapies available for MERS-CoV. In this study, we evaluated parainfluenza virus 5 (PIV5)-based vaccine expressing the MERS-CoV envelope spike protein (PIV5/MERS-S) in a human DPP4 knockin C57BL/6 congenic mouse model (hDPP4 KI). Following a single-dose intranasal immunization, PIV5-MERS-S induced neutralizing antibody and robust T cell responses in hDPP4 KI mice. A single intranasal administration of 10⁴ PFU PIV5-MERS-S provided complete protection against a lethal challenge with mouse-adapted MERS-CoV (MERS_MA6.1.2) and improved virus clearance in the lung. In comparison, single-dose intramuscular immunization with 10⁶ PFU UV-inactivated MERS_MA6.1.2 mixed with Imject alum provided protection to only 25% of immunized mice. Intriguingly, an influx of eosinophils was observed only in the lungs of mice immunized with inactivated MERS-CoV, suggestive of a hypersensitivity-type response. Overall, our study indicated that PIV5-MERS-S is a promising effective vaccine candidate against MERS-CoV infection.

IMPORTANCE MERS-CoV causes lethal infection in humans, and there is no vaccine. Our work demonstrates that PIV5 is a promising vector for developing a MERS vaccine. Furthermore, success of PIV5-based MERS vaccine can be employed to develop a vaccine for emerging CoVs such as SARS-CoV-2, which causes COVID-19.

ABSTRACT

Packaging of genomic RNA (gRNA) by retroviruses is essential for infectivity, yet the subcellular site of the initial interaction between the Gag polyprotein and gRNA remains poorly defined. Because retroviral particles are released from the plasma membrane, it was previously thought that Gag proteins initially bound to gRNA in the cytoplasm or at the plasma membrane. However, the Gag protein of the avian retrovirus Rous sarcoma virus (RSV) undergoes active nuclear trafficking, which is required for efficient gRNA encapsidation (L. Z. Scheifele, R. A. Garbitt, J. D. Rhoads, and L. J. Parent, Proc Natl Acad Sci U S A 99:3944–3949, 2002, https://doi.org/10.1073/pnas.062652199; R. Garbitt-Hirst, S. P. Kenney, and L. J. Parent, J Virol 83:6790–6797, 2009, https://doi.org/10.1128/JVI.00101-09). These results raise the intriguing possibility that the primary contact between Gag and gRNA might occur in the nucleus. To examine this possibility, we created a RSV proviral construct that includes 24 tandem repeats of MS2 RNA stem-loops, making it possible to track RSV viral RNA (vRNA) in live cells in which a fluorophore-conjugated MS2 coat protein is coexpressed. Using confocal microscopy, we observed that both wild-type Gag and a nuclear export mutant (Gag.L219A) colocalized with vRNA in the nucleus. In live-cell time-lapse images, the wild-type Gag protein trafficked together with vRNA as a single ribonucleoprotein (RNP) complex in the nucleoplasm near the nuclear periphery, appearing to traverse the nuclear envelope into the cytoplasm. Furthermore, biophysical imaging methods suggest that Gag and the unspliced vRNA physically interact in the nucleus. Taken together, these data suggest that RSV Gag binds unspliced vRNA to export it from the nucleus, possibly for packaging into virions as the viral genome.

IMPORTANCE Retroviruses cause severe diseases in animals and humans, including cancer and acquired immunodeficiency syndromes. To propagate infection, retroviruses assemble new virus particles that contain viral proteins and unspliced vRNA to use as gRNA. Despite the critical requirement for gRNA packaging, the molecular mechanisms governing the identification and selection of gRNA by the Gag protein remain poorly understood. In this report, we demonstrate that the Rous sarcoma virus (RSV) Gag protein colocalizes with unspliced vRNA in the nucleus in the interchromatin space. Using live-cell confocal imaging, RSV Gag and unspliced vRNA were observed to move together from inside the nucleus across the nuclear envelope, suggesting that the Gag-gRNA complex initially forms in the nucleus and undergoes nuclear export into the cytoplasm as a viral ribonucleoprotein (vRNP) complex.

ABSTRACT

The multifunctional nature of viral proteins is essentially driven by posttranslational modifications (PTMs) and is key for the successful outcome of infection. For influenza A viruses (IAVs), a composite pattern of PTMs regulates the activity of viral proteins. However, almost none are known that target the PB2 replication protein, except for inducing its degradation. We show here that PB2 undergoes a nonproteolytic ubiquitination during infection. We identified E3 ubiquitin ligases catalyzing this ubiquitination as two multicomponent RING-E3 ligases based on cullin 4 (CRL4s), which are both contributing to the levels of ubiquitinated forms of PB2 in infected cells. The CRL4 E3 ligase activity is required for the normal progression of the viral cycle and for maximal virion production, indicating that the CRL4s mediate a ubiquitin signaling that promotes infection. The CRL4s are recruiting PB2 through an unconventional bimodal interaction with both the DDB1 adaptor and DCAF substrate receptors. While able to bind to PB2 when engaged in the viral polymerase complex, the CRL4 factors do not alter transcription and replication of the viral segments during infection. CRL4 ligases catalyze different patterns of lysine ubiquitination on PB2. Recombinant viruses mutated in the targeted lysines showed attenuated viral production, suggesting that CRL4-mediated ubiquitination of PB2 contributes to IAV infection. We identified K29-linked ubiquitin chains as main components of the nonproteolytic PB2 ubiquitination mediated by the CRL4s, providing the first example of the role of this atypical ubiquitin linkage in the regulation of a viral infection.

IMPORTANCE Successful infection by influenza A virus, a pathogen of major public health importance, involves fine regulation of the multiple functions of the viral proteins, which often relies on post-translational modifications (PTMs). The PB2 protein of influenza A viruses is essential for viral replication and a key determinant of host range. While PTMs of PB2 inducing its degradation have been identified, here we show that PB2 undergoes a regulating PTM signaling detected during infection, based on an atypical K29-linked ubiquitination and mediated by two multicomponent E3 ubiquitin ligases. Recombinant viruses impaired for CRL4-mediated ubiquitination are attenuated, indicating that ubiquitination of PB2 is necessary for an optimal influenza A virus infection. The CRL4 E3 ligases are required for normal viral cycle progression and for maximal virion production. Consequently, they represent potential candidate host factors for antiviral targets.

ABSTRACT

The human gut microbiota (HGM) has far-reaching impacts on human health and nutrition, which are fueled primarily by the metabolism of otherwise indigestible complex carbohydrates commonly known as dietary fiber. However, the molecular basis of the ability of individual taxa of the HGM to address specific dietary glycan structures remains largely unclear. In particular, the utilization of β(1,3)-glucans, which are widespread in the human diet as yeast, seaweed, and plant cell walls, had not previously been resolved. Through a systems-based approach, here we show that the symbiont Bacteroides uniformis deploys a single, exemplar polysaccharide utilization locus (PUL) to access yeast β(1,3)-glucan, brown seaweed β(1,3)-glucan (laminarin), and cereal mixed-linkage β(1,3)/β(1,4)-glucan. Combined biochemical, enzymatic, and structural analysis of PUL-encoded glycoside hydrolases (GHs) and surface glycan-binding proteins (SGBPs) illuminates a concerted molecular system by which B. uniformis recognizes and saccharifies these distinct β-glucans. Strikingly, the functional characterization of homologous β(1,3)-glucan utilization loci (1,3GUL) in other Bacteroides further demonstrated that the ability of individual taxa to utilize β(1,3)-glucan variants and/or β(1,3)/β(1,4)-glucans arises combinatorially from the individual specificities of SGBPs and GHs at the cell surface, which feed corresponding signals to periplasmic hybrid two-component sensors (HTCSs) via TonB-dependent transporters (TBDTs). These data reveal the importance of cooperativity in the adaptive evolution of GH and SGBP cohorts to address individual polysaccharide structures. We anticipate that this fine-grained knowledge of PUL function will inform metabolic network analysis and proactive manipulation of the HGM. Indeed, a survey of 2,441 public human metagenomes revealed the international, yet individual-specific, distribution of each 1,3GUL.

IMPORTANCE Bacteroidetes are a dominant phylum of the human gut microbiota (HGM) that target otherwise indigestible dietary fiber with an arsenal of polysaccharide utilization loci (PULs), each of which is dedicated to the utilization of a specific complex carbohydrate. Here, we provide novel insight into this paradigm through functional characterization of homologous PULs from three autochthonous Bacteroides species, which target the family of dietary β(1,3)-glucans. Through detailed biochemical and protein structural analysis, we observed an unexpected diversity in the substrate specificity of PUL glycosidases and glycan-binding proteins with regard to β(1,3)-glucan linkage and branching patterns. In combination, these individual enzyme and protein specificities support taxon-specific growth on individual β(1,3)-glucans. This detailed metabolic insight, together with a comprehensive survey of individual 1,3GULs across human populations, further expands the fundamental roadmap of the HGM, with potential application to the future development of microbial intervention therapies.

ABSTRACT

RepA is a bacterial protein that builds intracellular amyloid oligomers acting as inhibitory complexes of plasmid DNA replication. When carrying a mutation enhancing its amyloidogenesis (A31V), the N-terminal domain (WH1) generates cytosolic amyloid particles that are inheritable within a bacterial lineage. Such amyloids trigger in bacteria a lethal cascade reminiscent of mitochondrial impairment in human cells affected by neurodegeneration. To fulfill all the criteria to qualify as a prion-like protein, horizontal (intercellular) transmissibility remains to be demonstrated for RepA-WH1. Since this is experimentally intractable in bacteria, here we transiently expressed in a murine neuroblastoma cell line the soluble, barely cytotoxic RepA-WH1 wild type [RepA-WH1(WT)] and assayed its response to exposure to in vitro-assembled RepA-WH1(A31V) amyloid fibers. In parallel, murine cells releasing RepA-WH1(A31V) aggregates were cocultured with human neuroblastoma cells expressing RepA-WH1(WT). Both the assembled fibers and donor-derived RepA-WH1(A31V) aggregates induced, in the cytosol of recipient cells, the formation of cytotoxic amyloid particles. Mass spectrometry analyses of the proteomes of both types of injured cells pointed to alterations in mitochondria, protein quality triage, signaling, and intracellular traffic. Thus, a synthetic prion-like protein can be propagated to, and become cytotoxic to, cells of organisms placed at such distant branches of the tree of life as bacteria and mammalia, suggesting that mechanisms of protein aggregate spreading and toxicity follow default pathways.

IMPORTANCE Proteotoxic amyloid seeds can be transmitted between mammalian cells, arguing that the intercellular exchange of prion-like protein aggregates can be a common phenomenon. RepA-WH1 is derived from a bacterial intracellular functional amyloid protein, engineered to become cytotoxic in Escherichia coli. Here, we have studied if such bacterial aggregates can also be transmitted to, and become cytotoxic to, mammalian cells. We demonstrate that RepA-WH1 is capable of entering naive cells, thereby inducing the cytotoxic aggregation of a soluble RepA-WH1 variant expressed in the cytosol, following the same trend that had been described in bacteria. These findings highlight the universality of one of the central principles underlying prion biology: No matter the biological origin of a given prion-like protein, it can be transmitted to a phylogenetically unrelated recipient cell, provided that the latter expresses a soluble protein onto which the incoming protein can readily template its amyloid conformation.

ABSTRACT

While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the postfusion events that precede RNA replication, such as nucleocapsid disassembly. We describe here a sensitive, conditionally replication-defective yellow fever virus (YFV) entry reporter, YFVSK/Nluc, to quantitively monitor the translation of incoming, virus particle-delivered genomes. We validated that YFVSK/Nluc gene expression can be neutralized by YFV-specific antisera and requires known flavivirus entry pathways and cellular factors, including clathrin- and dynamin-mediated endocytosis, endosomal acidification, YFV E glycoprotein-mediated fusion, and cellular LY6E and RPLP1 expression. The initial round of YFV translation was shown to require cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for nucleocapsid uncoating to occur. Importantly, translation of incoming YFV genomes also required valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a postfusion, pretranslation step in YFV entry. Finally, VCP/p97 activity was required by other flaviviruses in mammalian cells and by YFV in mosquito cells. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus nucleocapsids during a postfusion step in virus entry.

IMPORTANCE Flaviviruses are an important group of RNA viruses that cause significant human disease. The mechanisms by which flavivirus nucleocapsids are disassembled during virus entry remain unclear. Here, we used a yellow fever virus entry reporter, which expresses a sensitive reporter enzyme but does not replicate, to show that nucleocapsid disassembly requires the cellular protein-disaggregating enzyme valosin-containing protein, also known as p97.

ABSTRACT

Reversible repression of HIV-1 5' long terminal repeat (5'-LTR)-mediated transcription represents the main mechanism for HIV-1 to maintain latency. Identification of host factors that modulate LTR activity and viral latency may help develop new antiretroviral therapies. The heterogeneous nuclear ribonucleoproteins (hnRNPs) are known to regulate gene expression and possess multiple physiological functions. hnRNP family members have recently been identified as the sensors for viral nucleic acids to induce antiviral responses, highlighting the crucial roles of hnRNPs in regulating viral infection. A member of the hnRNP family, X-linked RNA-binding motif protein (RBMX), has been identified in this study as a novel HIV-1 restriction factor that modulates HIV-1 5'-LTR-driven transcription of viral genome in CD4⁺ T cells. Mechanistically, RBMX binds to HIV-1 proviral DNA at the LTR downstream region and maintains the repressive trimethylation of histone H3 lysine 9 (H3K9me3), leading to a blockage of the recruitment of the positive transcription factor phosphorylated RNA polymerase II (RNA pol II) and consequential impediment of transcription elongation. This RBMX-mediated modulation of HIV-1 transcription maintains viral latency by inhibiting viral reactivation from an integrated proviral DNA. Our findings provide a new understanding of how host factors modulate HIV-1 infection and latency and suggest a potential new target for the development of HIV-1 therapies.

IMPORTANCE HIV-1 latency featuring silence of transcription from HIV-1 proviral DNA represents a major obstacle for HIV-1 eradication. Reversible repression of HIV-1 5'-LTR-mediated transcription represents the main mechanism for HIV-1 to maintain latency. The 5'-LTR-driven HIV gene transcription can be modulated by multiple host factors and mechanisms. The hnRNPs are known to regulate gene expression. A member of the hnRNP family, RBMX, has been identified in this study as a novel HIV-1 restriction factor that modulates HIV-1 5'-LTR-driven transcription of viral genome in CD4⁺ T cells and maintains viral latency. These findings provide a new understanding of how host factors modulate HIV-1 infection and latency and suggest a potential new target for the development of HIV-1 therapies.

ABSTRACT

Protein homeostasis is critical for proliferation and viability of all organisms. For Candida albicans, protein homeostasis also modulates the transition between yeast and filamentous forms, which is critical for virulence. A key regulator of morphogenesis is the molecular chaperone Hsp90, which mediates proteostasis under physiological and stress conditions. Hsp90 regulates morphogenesis by repressing cyclic AMP-protein kinase A (cAMP-PKA) signaling, such that inhibition of Hsp90 causes filamentation in the absence of an inducing cue. We explored the effect of perturbation of another facet of protein homeostasis and discovered that morphogenesis is also regulated by the proteasome, a large 33-subunit protein complex consisting of a 20S catalytic core and two 19S regulatory particles, which controls degradation of intracellular proteins. We identified a conserved role of the proteasome in morphogenesis as pharmacological inhibition of the proteasome induced filamentation of C. albicans and the related species Candida dubliniensis, Candida tropicalis, Candida krusei, and Candida parapsilosis. For C. albicans, genetic depletion of any of 29 subunits of the 19S or 20S particle induced filamentation. Filaments induced by inhibition of either the proteasome or Hsp90 have shared structural characteristics, such as aberrant nuclear content, and shared genetic dependencies, such as intact cAMP-PKA signaling. Consistent with a functional connection between these facets of protein homeostasis that modulate morphogenesis, we observed that proteasome inhibition results in an accumulation of ubiquitinated proteins that overwhelm Hsp90 function, relieving Hsp90-mediated repression of morphogenesis. Together, our findings provide a mechanism whereby interconnected facets of proteostasis regulate C. albicans morphogenesis.

IMPORTANCE Fungi cause life-threatening infections and pose a serious threat to human health as there are very few effective antifungal drugs. Candida albicans is a major human fungal pathogen and cause of morbidity and mortality in immunocompromised individuals. A key trait that enables C. albicans virulence is its ability to transition between yeast and filamentous forms. Understanding the mechanisms regulating this virulence trait can facilitate the development of much-needed, novel therapeutic strategies. A key regulator of morphogenesis is the molecular chaperone Hsp90, which is crucial for proteostasis. Here, we expanded our understanding of how proteostasis regulates fungal morphogenesis and identified the proteasome as a repressor of filamentation in C. albicans and related species. Our work suggests that proteasome inhibition overwhelms Hsp90 function, thereby inducing morphogenesis. This work provides a foundation for understanding the role of the proteasome in fungal virulence and offers potential for targeting the proteasome to disarm fungal pathogens.

ABSTRACT

Humans encode proteins, called restriction factors, that inhibit replication of viruses such as HIV-1. The members of one family of antiviral proteins, apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3; shortened here to A3), act by deaminating cytidines to uridines during the reverse transcription reaction of HIV-1. The A3 locus encodes seven genes, named A3A to A3H. These genes have either one or two cytidine deaminase domains, and several of these A3s potently restrict HIV-1. A3C, which has only a single cytidine deaminase domain, however, inhibits HIV-1 only very weakly. We tested novel double domain protein combinations by genetically linking two A3C genes to make a synthetic tandem domain protein. This protein created a "super restriction factor" that had more potent antiviral activity than the native A3C protein, which correlated with increased packaging into virions. Furthermore, disabling one of the active sites of the synthetic tandem domain protein resulted in an even greater increase in the antiviral activity—recapitulating a similar evolution seen in A3F and A3G (double domain A3s that use only a single catalytically active deaminase domain). These A3C tandem domain proteins do not have an increase in mutational activity but instead inhibit formation of reverse transcription products, which correlates with their ability to form large higher-order complexes in cells. Finally, the A3C-A3C super restriction factor largely escaped antagonism by the HIV-1 viral protein Vif.

IMPORTANCE As a part of the innate immune system, humans encode proteins that inhibit viruses such as HIV-1. These broadly acting antiviral proteins do not protect humans from viral infections because viruses encode proteins that antagonize the host antiviral proteins to evade the innate immune system. One such example of a host antiviral protein is APOBEC3C (A3C), which weakly inhibits HIV-1. Here, we show that we can improve the antiviral activity of A3C by duplicating the DNA sequence to create a synthetic tandem domain and, furthermore, that the proteins thus generated are relatively resistant to the viral antagonist Vif. Together, these data give insights about how nature has evolved a defense against viral pathogens such as HIV.

ABSTRACT

Cell division requires proper spatial coordination with the chromosome, which undergoes dynamic changes during chromosome replication and segregation. FtsZ is a bacterial cytoskeletal protein that assembles into the Z-ring, providing a platform to build the cell division apparatus. In the model bacterium Caulobacter crescentus, the cellular localization of the Z-ring is controlled during the cell cycle in a chromosome replication-coupled manner. Although dynamic localization of the Z-ring at midcell is driven primarily by the replication origin-associated FtsZ inhibitor MipZ, the mechanism ensuring accurate positioning of the Z-ring remains unclear. In this study, we showed that the Z-ring colocalizes with the replication terminus region, located opposite the origin, throughout most of the C. crescentus cell cycle. Spatial organization of the two is mediated by ZapT, a previously uncharacterized protein that interacts with the terminus region and associates with ZapA and ZauP, both of which are part of the incipient division apparatus. While the Z-ring and the terminus region coincided with the presence of ZapT, colocalization of the two was perturbed in cells lacking zapT, which is accompanied by delayed midcellular positioning of the Z-ring. Moreover, cells overexpressing ZapT showed compromised positioning of the Z-ring and MipZ. These findings underscore the important role of ZapT in controlling cell division processes. We propose that ZapT acts as a molecular bridge that physically links the terminus region to the Z-ring, thereby ensuring accurate site selection for the Z-ring. Because ZapT is conserved in proteobacteria, these findings may define a general mechanism coordinating cell division with chromosome organization.

IMPORTANCE Growing bacteria require careful tuning of cell division processes with dynamic organization of replicating chromosomes. In enteric bacteria, ZapA associates with the cytoskeletal Z-ring and establishes a physical linkage to the chromosomal replication terminus through its interaction with ZapB-MatP-DNA complexes. However, because ZapB and MatP are found only in enteric bacteria, it remains unclear how the Z-ring and the terminus are coordinated in the vast majority of bacteria. Here, we provide evidence that a novel conserved protein, termed ZapT, mediates colocalization of the Z-ring with the terminus in Caulobacter crescentus, a model organism that is phylogenetically distant from enteric bacteria. Given that ZapT facilitates cell division processes in C. crescentus, this study highlights the universal importance of the physical linkage between the Z-ring and the terminus in maintaining cell integrity.

N-terminal (Nt) acetylation (NTA) is an ample and irreversible cotranslational protein modification catalyzed by ribosome-associated Nt-acetyltransferases. NTA on specific proteins can act as a degradation signal (called an Ac/N-degron) for proteolysis in yeast and mammals. However, in plants, the biological relevance of NTA remains largely unexplored. In this study, we reveal that Arabidopsis (Arabidopsis thaliana) SIGMA FACTOR-BINDING PROTEIN1 (SIB1), a transcription coregulator and a positive regulator of salicylic acid-primed cell death, undergoes an absolute NTA on the initiator Met; Nt-acetyltransferase B (NatB) partly contributes to this modification. While NTA results in destabilization of certain target proteins, our genetic and biochemical analyses revealed that plant NatB-involved NTA instead renders SIB1 more stable. Given that the ubiquitin/proteasome system stimulates SIB1 degradation, it seems that the NTA-conferred stability ensures the timely expression of SIB1-dependent genes, mostly related to immune responses. Taking our findings together, here we report a noncanonical NTA-driven protein stabilization in land plants.

Phosphoinositides function as lipid signals in plant development and stress tolerance by binding with partner proteins. We previously reported that Arabidopsis (Arabidopsis thaliana) phosphoinositide-specific phospholipase C2 functions in the endoplasmic reticulum (ER) stress response. However, the underlying molecular mechanisms of how phosphoinositides act in the ER stress response remain elusive. Here, we report that a phosphoinositide-binding protein, SMALLER TRICHOMES WITH VARIABLE BRANCHES (SVB), is involved in the ER stress tolerance. SVB contains a DUF538 domain with unknown function; orthologs are exclusively found in Viridiplantae. We established that SVB is ubiquitously expressed in plant tissues and is localized to the ER, Golgi apparatus, prevacuolar compartment, and plasma membrane. The knockout mutants of svb showed enhanced tolerance to ER stress, which was genetically complemented by transducing genomic SVB. SVB showed time-dependent induction after tunicamycin-induced ER stress, which depended on IRE1 and bZIP60 but not bZIP17 and bZIP28 in the unfolded protein response (UPR). A protein–lipid overlay assay showed specific binding of SVB to phosphatidylinositol 3,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate. SVB is therefore suggested to be the plant-specific phosphoinositide-binding protein whose expression is controlled by the UPR through the IRE1-bZIP60 pathway in Arabidopsis.

Members of the light-harvesting complex protein family participate in multiple processes connected with light sensing, light absorption, and pigment binding within the thylakoid membrane. Amino acid residues of the light-harvesting chlorophyll a/b-binding proteins involved in pigment binding have been precisely identified through x-ray crystallography experiments. In vitro pigment-binding studies have been performed with LIGHT-HARVESTING-LIKE3 proteins, and the pigment-binding ability of cyanobacterial high-light-inducible proteins has been studied in detail. However, analysis of pigment binding by plant high-light-inducible protein homologs, called ONE-HELIX PROTEINS (OHPs), is lacking. Here, we report on successful in vitro reconstitution of Arabidopsis (Arabidopsis thaliana) OHPs with chlorophylls and carotenoids and show that pigment binding depends on the formation of OHP1/OHP2 heterodimers. Pigment-binding capacity was completely lost in each of the OHPs when residues of the light-harvesting complex chlorophyll-binding motif required for chlorophyll binding were mutated. Moreover, the mutated OHP variants failed to rescue the respective knockout (T-DNA insertion) mutants, indicating that pigment-binding ability is essential for OHP function in vivo. The scaffold protein HIGH CHLOROPHYLL FLUORESCENCE244 (HCF244) is tethered to the thylakoid membrane by the OHP heterodimer. We show that HCF244 stability depends on OHP heterodimer formation and introduce the concept of a functional unit consisting of OHP1, OHP2, and HCF244, in which each protein requires the others. Because of their pigment-binding capacity, we suggest that OHPs function in the delivery of pigments to the D1 subunit of PSII.

Despite the ecological relevance of diatoms, many aspects of their photosynthetic machinery remain poorly understood. Diatoms differ from the green lineage of oxygenic organisms by their photosynthetic pigments and light-harvesting complex (Lhc) proteins, the latter of which are also called fucoxanthin-chlorophyll proteins (FCP). These are composed of three groups of proteins: Lhcf as the main group, Lhcr that are PSI associated, and Lhcx that are involved in photoprotection. The FCP complexes are assembled in trimers and higher oligomers. Several studies have investigated the biochemical properties of purified FCP complexes, but limited knowledge is available about their interaction with the photosystem cores. In this study, isolation of stable supercomplexes from the centric diatom Thalassiosira pseudonana was achieved. To preserve in vivo structure, the separation of thylakoid complexes was performed by native PAGE and sucrose density centrifugation. Different subpopulations of PSI and PSII supercomplexes were isolated and their subunits identified. Analysis of Lhc antenna composition identified Lhc(s) specific for either PSI (Lhcr 1, 3, 4, 7, 10–14, and Lhcf10) or PSII (Lhcf 1–7, 11, and Lhcr2). Lhcx6_1 was reproducibly found in PSII supercomplexes, whereas its association with PSI was unclear. No evidence was found for the interaction between photosystems and higher oligomeric FCPs, comprising Lhcf8 as the main component. Although the subunit composition of the PSII supercomplexes in comparison with that of the trimeric FCP complexes indicated a close mutual association, the higher oligomeric pool is only weakly associated with the photosystems, albeit its abundance in the thylakoid membrane.

BEAF (Boundary Element-Associated Factor) was originally identified as a Drosophila melanogaster chromatin domain insulator-binding protein, suggesting a role in gene regulation through chromatin organization and dynamics. Genome-wide mapping found that BEAF usually binds near transcription start sites, often of housekeeping genes, suggesting a role in promoter function. This would be a nontraditional role for an insulator-binding protein. To gain insight into molecular mechanisms of BEAF function, we identified interacting proteins using yeast two-hybrid assays. Here, we focus on the transcription factor Serendipity (Sry-). Interactions were confirmed in pull-down experiments using bacterially expressed proteins, by bimolecular fluorescence complementation, and in a genetic assay in transgenic flies. Sry- interacted with promoter-proximal BEAF both when bound to DNA adjacent to BEAF or > 2-kb upstream to activate a reporter gene in transient transfection experiments. The interaction between BEAF and Sry- was detected using both a minimal developmental promoter (y) and a housekeeping promoter (RpS12), while BEAF alone strongly activated the housekeeping promoter. These two functions for BEAF implicate it in playing a direct role in gene regulation at hundreds of BEAF-associated promoters.

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Implanted medical device-associated infections pose significant health risks, as they are often the result of bacterial biofilm formation. Staphylococcus aureus is a leading cause of biofilm-associated infections which persist due to mechanisms of device surface adhesion, biofilm accumulation, and reprogramming of host innate immune responses. We found that the S. aureus fibronectin binding protein A (FnBPA) is required for normal biofilm development in mammalian serum and that the SaeRS two-component system is required for functional FnBPA activity in serum. Furthermore, serum-developed biofilms deficient in FnBPA were more susceptible to macrophage invasion, and in a model of biofilm-associated implant infection, we found that FnBPA is crucial for the establishment of infection. Together, these findings show that S. aureus FnBPA plays an important role in physical biofilm development and represents a potential therapeutic target for the prevention and treatment of device-associated infections.