The recruitment of TRiC chaperonin in rotavirus viroplasms correlates with virus replication.

Rotavirus (RV) replication takes place in the viroplasms, cytosolic inclusions that allow the synthesis of virus genome segments and their encapsidation in the core shell, followed by the addition of the second layer of the virion. The viroplasms are composed of several viral proteins, including NSP5, which serves as the main building block. Microtubules, lipid droplets, and miRNA-7 are among the host components recruited in viroplasms. We investigated the interaction between RV proteins and host components of the viroplasms by performing a pull-down assay of lysates from RV-infected cells expressing NSP5-BiolD2. Subsequent tandem mass spectrometry identified all eight subunits of the tailless complex polypeptide I ring complex (TRiC), a cellular chaperonin responsible for folding at least 10% of the cytosolic proteins. Our confirmed findings reveal that TRiC is brought into viroplasms and wraps around newly formed double-layered particles. Chemical inhibition of TRiC and silencing of its subunits drastically reduced virus progeny production. Through direct RNA sequencing, we show that TRiC is critical for RV replication by controlling dsRNA genome segment synthesis, particularly negative-sense single-stranded RNA. Importantly, cryo-electron microscopy analysis shows that TRiC inhibition results in defective virus particles lacking genome segments and polymerase complex (VP1/VP3). Moreover, TRiC associates with VP2 and NSP5 but not with VP1. Also, VP2 is shown to be essential for recruiting TRiC in viroplasms and preserving their globular morphology. This study highlights the essential role of TRiC in viroplasm formation and in facilitating virion assembly during the RV life cycle. IMPORTANCE: The replication of rotavirus takes place in cytosolic inclusions termed viroplasms. In these inclusions, the distinct 11 double-stranded RNA genome segments are co-packaged to complete a genome in newly generated virus particles. In this study, we show for the first time that the tailless complex polypeptide I ring complex (TRiC), a cellular chaperonin responsible for the folding of at least 10% of the cytosolic proteins, is a component of viroplasms and is required for the synthesis of the viral negative-sense single-stranded RNA. Specifically, TRiC associates with NSP5 and VP2, the cofactor involved in RNA replication. Our study adds a new component to the current model of rotavirus replication, where TRiC is recruited to viroplasms to assist replication.

Molten Globule Driven and Self-downmodulated Phase Separation of a Viral Factory Scaffold.

Viral factories of liquid-like nature serve as sites for transcription and replication in most viruses. The respiratory syncytial virus factories include replication proteins, brought together by the phosphoprotein (P) RNA polymerase cofactor, present across non-segmented negative stranded RNA viruses. Homotypic liquid-liquid phase separation of RSV-P is governed by an α-helical molten globule domain, and strongly self-downmodulated by adjacent sequences. Condensation of P with the nucleoprotein N is stoichiometrically tuned, defining aggregate-droplet and droplet-dissolution boundaries. Time course analysis show small N-P nuclei gradually coalescing into large granules in transfected cells. This behavior is recapitulated in infection, with small puncta evolving to large viral factories, strongly suggesting that P-N nucleation-condensation sequentially drives viral factories. Thus, the tendency of P to undergo phase separation is moderate and latent in the full-length protein but unleashed in the presence of N or when neighboring disordered sequences are deleted. This, together with its capacity to rescue nucleoprotein-RNA aggregates suggests a role as a "solvent-protein".

Specific Residues in the C-Terminal Domain of the Human Metapneumovirus Phosphoprotein Are Indispensable for Formation of Viral Replication Centers and Regulation of the Function of the Viral Polymerase Complex.

Human metapneumovirus (HMPV) is a negative-strand RNA virus that frequently causes respiratory tract infections in infants, the elderly, and the immunocompromised. A hallmark of HMPV infection is the formation of membraneless, liquid-like replication and transcription centers in the cytosol termed inclusion bodies (IBs). The HMPV phosphoprotein (P) and nucleoprotein (N) are the minimal viral proteins necessary to form IB-like structures, and both proteins are required for the viral polymerase to synthesize RNA during infection. HMPV P is a homotetramer with regions of intrinsic disorder and has several known and predicted phosphorylation sites of unknown function. In this study, we found that the P C-terminal intrinsically disordered domain (CTD) must be present to facilitate IB formation with HMPV N, while either the N-terminal intrinsically disordered domain or the central oligomerization domain was dispensable. Alanine substitution at a single tyrosine residue within the CTD abrogated IB formation and reduced coimmunoprecipitation with HMPV N. Mutations to C-terminal phosphorylation sites revealed a potential role for phosphorylation in regulating RNA synthesis and P binding partners within IBs. Phosphorylation mutations which reduced RNA synthesis in a reporter assay produced comparable results in a recombinant viral rescue system, measured as an inability to produce infectious viral particles with genomes containing these single P mutations. This work highlights the critical role HMPV P plays in facilitating a key step of the viral life cycle and reveals the potential role for phosphorylation in regulating the function of this significant viral protein. IMPORTANCE Human metapneumovirus (HMPV) infects global populations, with severe respiratory tract infections occurring in infants, the elderly, and the immunocompromised. There are currently no FDA-approved therapeutics available to prevent or treat HMPV infection. Therefore, understanding how HMPV replicates is vital for the identification of novel targets for therapeutic development. During HMPV infection, viral RNA synthesis proteins localize to membraneless structures called inclusion bodies (IBs), which are sites of genome replication and transcription. The HMPV phosphoprotein (P) is necessary for IBs to form and for the virus to synthesize RNA, but it is not known how this protein contributes to IB formation or if it is capable of regulating viral replication. We show that the C-terminal domain of P is the location of a molecular interaction driving IB formation and contains potential phosphorylation sites where amino acid charge regulates the function of the viral polymerase complex.

Visualization of Marek's Disease Virus Genomes in Living Cells during Lytic Replication and Latency.

Visualization of the herpesvirus genomes during lytic replication and latency is mainly achieved by fluorescence in situ hybridization (FISH). Unfortunately, this technique cannot be used for the real-time detection of viral genome in living cells. To facilitate the visualization of the Marek's disease virus (MDV) genome during all stages of the virus lifecycle, we took advantage of the well-established tetracycline operator/repressor (TetO/TetR) system. This system consists of a fluorescently labeled TetR (TetR-GFP) that specifically binds to an array of tetO sequences. This tetO repeat array was first inserted into the MDV genome (vTetO). Subsequently, we fused TetR-GFP via a P2a self-cleaving peptide to the C-terminus of the viral interleukin 8 (vIL8), which is expressed during lytic replication and latency. Upon reconstitution of this vTetO-TetR virus, fluorescently labeled replication compartments were detected in the nucleus during lytic replication. After validating the specificity of the observed signal, we used the system to visualize the genesis and mobility of the viral replication compartments. In addition, we assessed the infection of nuclei in syncytia as well as lytic replication and latency in T cells. Taken together, we established a system allowing us to track the MDV genome in living cells that can be applied to many other DNA viruses.

Turnip mosaic virus co-opts the vacuolar sorting receptor VSR4 to promote viral genome replication in plants by targeting viral replication vesicles to the endosome.

Accumulated experimental evidence has shown that viruses recruit the host intracellular machinery to establish infection. It has recently been shown that the potyvirus Turnip mosaic virus (TuMV) transits through the late endosome (LE) for viral genome replication, but it is still largely unknown how the viral replication vesicles labelled by the TuMV membrane protein 6K2 target LE. To further understand the underlying mechanism, we studied the involvement of the vacuolar sorting receptor (VSR) family proteins from Arabidopsis in this process. We now report the identification of VSR4 as a new host factor required for TuMV infection. VSR4 interacted specifically with TuMV 6K2 and was required for targeting of 6K2 to enlarged LE. Following overexpression of VSR4 or its recycling-defective mutant that accumulates in the early endosome (EE), 6K2 did not employ the conventional VSR-mediated EE to LE pathway, but targeted enlarged LE directly from cis-Golgi and viral replication was enhanced. In addition, VSR4 can be N-glycosylated and this is required for its stability and for monitoring 6K2 trafficking to enlarged LE. A non-glycosylated VSR4 mutant enhanced the dissociation of 6K2 from cis-Golgi, leading to the formation of punctate bodies that targeted enlarged LE and to more robust viral replication than with glycosylated VSR4. Finally, TuMV hijacks N-glycosylated VSR4 and protects VSR4 from degradation via the autophagy pathway to assist infection. Taken together, our results have identified a host factor VSR4 required for viral replication vesicles to target endosomes for optimal viral infection and shed new light on the role of N-glycosylation of a host factor in regulating viral infection.

MicroRNA 122 Affects both the Initiation and the Maintenance of Hepatitis C Virus Infections.

A liver-specific microRNA, miR-122, anneals to the hepatitis C virus (HCV) genomic 5' terminus and is essential for virus replication in cell culture. However, bicistronic HCV replicons and full-length RNAs with specific mutations in the 5' untranslated region (UTR) can replicate, albeit to low levels, without miR-122. In this study, we have identified that HCV RNAs lacking the structural gene region or having encephalomyocarditis virus internal ribosomal entry site (EMCV IRES)-regulated translation had reduced requirements for miR-122. In addition, we found that a smaller proportion of cells supported miR-122-independent replication compared a population of cells supporting miR-122-dependent replication, while viral protein levels per positive cell were similar. Further, the proportion of cells supporting miR-122-independent replication increased with the amount of viral RNA delivered, suggesting that establishment of miR-122-independent replication in a cell is affected by the amount of viral RNA delivered. HCV RNAs replicating independently of miR-122 were not affected by supplementation with miR-122, suggesting that miR-122 is not essential for maintenance of an miR-122-independent HCV infection. However, miR-122 supplementation had a small positive impact on miR-122-dependent replication, suggesting a minor role in enhancing ongoing virus RNA accumulation. We suggest that miR-122 functions primarily to initiate an HCV infection but has a minor influence on its maintenance, and we present a model in which miR-122 is required for replication complex formation at the beginning of an infection and also supports new replication complex formation during ongoing infection and after infected cell division. IMPORTANCE The mechanism by which miR-122 promotes the HCV life cycle is not well understood, and a role in directly promoting genome amplification is still debated. In this study, we have shown that miR-122 increases the rate of viral RNA accumulation and promotes the establishment of an HCV infection in a greater number of cells than in the absence of miR-122. However, we also confirm a minor role in promoting ongoing virus replication and propose a role in the initiation of new replication complexes throughout a virus infection. This study has implications for the use of anti-miR-122 as a potential HCV therapy.

Rab1b-GBF1-ARF1 Secretory Pathway Axis Is Required for Birnavirus Replication.

Birnaviruses are members of the Birnaviridae family, responsible for major economic losses to poultry and aquaculture. The family is composed of nonenveloped viruses with a segmented double-stranded RNA (dsRNA) genome. Infectious bursal disease virus (IBDV), the prototypic family member, is the etiological agent of Gumboro disease, a highly contagious immunosuppressive disease in the poultry industry worldwide. We previously demonstrated that IBDV hijacks the endocytic pathway for establishing the viral replication complexes on endosomes associated with the Golgi complex (GC). Here, we report that IBDV reorganizes the GC to localize the endosome-associated replication complexes without affecting its secretory functionality. By analyzing crucial proteins involved in the secretory pathway, we showed the essential requirement of Rab1b for viral replication. Rab1b comprises a key regulator of GC transport and we demonstrate that transfecting the negative mutant Rab1b N121I or knocking down Rab1b expression by RNA interference significantly reduces the yield of infectious viral progeny. Furthermore, we showed that the Rab1b downstream effector Golgi-specific BFA resistance factor 1 (GBF1), which activates the small GTPase ADP ribosylation factor 1 (ARF1), is required for IBDV replication, since inhibiting its activity by treatment with brefeldin A (BFA) or golgicide A (GCA) significantly reduces the yield of infectious viral progeny. Finally, we show that ARF1 dominant negative mutant T31N overexpression hampered IBDV infection. Taken together, these results demonstrate that IBDV requires the function of the Rab1b-GBF1-ARF1 axis to promote its replication, making a substantial contribution to the field of birnavirus-host cell interactions. IMPORTANCE Birnaviruses are unconventional members of the dsRNA viruses, with the lack of a transcriptionally active core being the main differential feature. This structural trait, among others that resemble those of the plus single-stranded (+ssRNA) viruses features, suggests that birnaviruses might follow a different replication program from that conducted by prototypical dsRNA members and the hypothesis that birnaviruses could be evolutionary links between +ssRNA and dsRNA viruses has been argued. Here, we present original data showing that IBDV-induced GC reorganization and the cross talk between IBDV and the Rab1b-GBF1-ARF1 mediate the intracellular trafficking pathway. The replication of several +ssRNA viruses depends on the cellular protein GBF1, but its role in the replication process is not clear. Thus, our findings make a substantial contribution to the field of birnavirus-host cell interactions and provide further evidence supporting the proposed evolutionary connection role of birnaviruses, an aspect which we consider especially relevant for researchers working in the virology field.

The Large Nonstructural Protein (NS1) of Human Bocavirus 1 Directly Interacts with Ku70, Which Plays an Important Role in Virus Replication in Human Airway Epithelia.

Human bocavirus 1 (HBoV1), an autonomous human parvovirus, causes acute respiratory tract infections in young children. HBoV1 infects well-differentiated (polarized) human airway epithelium cultured at an air-liquid interface (HAE-ALI). HBoV1 expresses a large nonstructural protein, NS1, that is essential for viral DNA replication. HBoV1 infection of polarized human airway epithelial cells induces a DNA damage response (DDR) that is critical to viral DNA replication involving DNA repair with error-free Y-family DNA polymerases. HBoV1 NS1 or the isoform NS1-70 per se induces a DDR. In this study, using the second-generation proximity-dependent biotin identification (BioID2) approach, we identified that Ku70 is associated with the NS1-BioID2 pulldown complex through a direct interaction with NS1. Biolayer interferometry (BLI) assay determined a high binding affinity of NS1 with Ku70, which has an equilibrium dissociation constant (KD) value of 0.16 µM and processes the strongest interaction at the C-terminal domain. The association of Ku70 with NS1 was also revealed during HBoV1 infection of HAE-ALI. Knockdown of Ku70 and overexpression of the C-terminal domain of Ku70 significantly decreased HBoV1 replication in HAE-ALI. Thus, our study provides, for the first time, a direct interaction of parvovirus large nonstructural protein NS1 with Ku70. IMPORTANCE Parvovirus infection induces a DNA damage response (DDR) that plays a pivotal role in viral DNA replication. The DDR includes activation of ATM (ataxia telangiectasia mutated), ATR (ATM- and RAD3-related), and DNA-PKcs (DNA-dependent protein kinase catalytic subunit). The large nonstructural protein (NS1) often plays a role in the induction of DDR; however, how the DDR is induced during parvovirus infection or simply by the NS1 is not well studied. Activation of DNA-PKcs has been shown as one of the key DDR pathways in DNA replication of HBoV1. We identified that HBoV1 NS1 directly interacts with Ku70, but not Ku80, of the Ku70/Ku80 heterodimer at high affinity. This interaction is also important for HBoV1 replication in HAE-ALI. We propose that the interaction of NS1 with Ku70 recruits the Ku70/Ku80 complex to the viral DNA replication center, which activates DNA-PKcs and facilitates viral DNA replication.

Analysis of a crucial interaction between the coronavirus nucleocapsid protein and the major membrane-bound subunit of the viral replicase-transcriptase complex.

The coronavirus nucleocapsid (N) protein comprises two RNA-binding domains connected by a central spacer, which contains a serine- and arginine-rich (SR) region. The SR region engages the largest subunit of the viral replicase-transcriptase, nonstructural protein 3 (nsp3), in an interaction that is essential for efficient initiation of infection by genomic RNA. We carried out an extensive genetic analysis of the SR region of the N protein of mouse hepatitis virus in order to more precisely define its role in RNA synthesis. We further examined the N-nsp3 interaction through construction of nsp3 mutants and by creation of an interspecies N protein chimera. Our results indicate a role for the central spacer as an interaction hub of the N molecule that is partially regulated by phosphorylation. These findings are discussed in relation to the recent discovery that nsp3 forms a molecular pore in the double-membrane vesicles that sequester the coronavirus replicase-transcriptase.

Coronavirus RNA Synthesis Takes Place within Membrane-Bound Sites.

Infectious bronchitis virus (IBV), a gammacoronavirus, is an economically important virus to the poultry industry, as well as a significant welfare issue for chickens. As for all positive strand RNA viruses, IBV infection causes rearrangements of the host cell intracellular membranes to form replication organelles. Replication organelle formation is a highly conserved and vital step in the viral life cycle. Here, we investigate the localization of viral RNA synthesis and the link with replication organelles in host cells. We have shown that sites of viral RNA synthesis and virus-related dsRNA are associated with one another and, significantly, that they are located within a membrane-bound compartment within the cell. We have also shown that some viral RNA produced early in infection remains within these membranes throughout infection, while a proportion is trafficked to the cytoplasm. Importantly, we demonstrate conservation across all four coronavirus genera, including SARS-CoV-2. Understanding more about the replication of these viruses is imperative in order to effectively find ways to control them.

Contribution of autophagy machinery factors to HCV and SARS-CoV-2 replication organelle formation.

Positive-strand RNA viruses replicate in close association with rearranged intracellular membranes. For hepatitis C virus (HCV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), these rearrangements comprise endoplasmic reticulum (ER)-derived double membrane vesicles (DMVs) serving as RNA replication sites. Cellular factors involved in DMV biogenesis are poorly defined. Here, we show that despite structural similarity of viral DMVs with autophagosomes, conventional macroautophagy is dispensable for HCV and SARS-CoV-2 replication. However, both viruses exploit factors involved in autophagosome formation, most notably class III phosphatidylinositol 3-kinase (PI3K). As revealed with a biosensor, PI3K is activated in cells infected with either virus to produce phosphatidylinositol 3-phosphate (PI3P) while kinase complex inhibition or depletion profoundly reduces replication and viral DMV formation. The PI3P-binding protein DFCP1, recruited to omegasomes in early steps of autophagosome formation, participates in replication and DMV formation of both viruses. These results indicate that phylogenetically unrelated HCV and SARS-CoV-2 exploit similar components of the autophagy machinery to create their replication organelles.

Viroplasms: Assembly and Functions of Rotavirus Replication Factories.

Viroplasms are cytoplasmic, membraneless structures assembled in rotavirus (RV)-infected cells, which are intricately involved in viral replication. Two virus-encoded, non-structural proteins, NSP2 and NSP5, are the main drivers of viroplasm formation. The structures (as far as is known) and functions of these proteins are described. Recent studies using plasmid-only-based reverse genetics have significantly contributed to elucidation of the crucial roles of these proteins in RV replication. Thus, it has been recognized that viroplasms resemble liquid-like protein-RNA condensates that may be formed via liquid-liquid phase separation (LLPS) of NSP2 and NSP5 at the early stages of infection. Interactions between the RNA chaperone NSP2 and the multivalent, intrinsically disordered protein NSP5 result in their condensation (protein droplet formation), which plays a central role in viroplasm assembly. These droplets may provide a unique molecular environment for the establishment of inter-molecular contacts between the RV (+)ssRNA transcripts, followed by their assortment and equimolar packaging. Future efforts to improve our understanding of RV replication and genome assortment in viroplasms should focus on their complex molecular composition, which changes dynamically throughout the RV replication cycle, to support distinct stages of virion assembly.

Rotavirus Viroplasm Biogenesis Involves Microtubule-Based Dynein Transport Mediated by an Interaction between NSP2 and Dynein Intermediate Chain.

Rotaviruses are the causative agents of severe and dehydrating gastroenteritis in children, piglets, and many other young animals. They replicate their genomes and assemble double-layered particles in cytoplasmic electron-dense inclusion bodies called "viroplasms." The formation of viroplasms is reportedly associated with the stability of microtubules. Although material transport is an important function of microtubules, whether and how microtubule-based transport influences the formation of viroplasms are still unclear. Here, we demonstrate that small viroplasms move and fuse in living cells. We show that microtubule-based dynein transport affects rotavirus infection, viroplasm formation, and the assembly of transient enveloped particles (TEPs) and triple-layered particles (TLPs). The dynein intermediate chain (DIC) is shown to localize in the viroplasm and to interact directly with nonstructural protein 2 (NSP2), indicating that the DIC is responsible for connecting the viroplasm to dynein. The WD40 repeat domain of the DIC regulates the interaction between the DIC and NSP2, and the knockdown of the DIC inhibited rotaviral infection, viroplasm formation, and the assembly of TEPs and TLPs. Our findings show that rotavirus viroplasms hijack dynein transport for fusion events, required for maximal assembly of infectious viral progeny. This study provides novel insights into the intracellular transport of viroplasms, which is involved in their biogenesis. IMPORTANCE Because the viroplasm is the viral factory for rotavirus replication, viroplasm formation undoubtedly determines the effective production of progeny rotavirus. Therefore, an understanding of the virus-host interactions involved in the biogenesis of the viroplasm is critical for the future development of prophylactic and therapeutic strategies. Previous studies have reported that the formation of viroplasms is associated with the stability of microtubules, whereas little is known about its specific mechanism. Here, we demonstrate that rotavirus viroplasm formation takes advantage of microtubule-based dynein transport mediated by an interaction between NSP2 and the DIC. These findings provide new insight into the intracellular transport of viroplasms.

Semliki Forest Virus Chimeras with Functional Replicase Modules from Related Alphaviruses Survive by Adaptive Mutations in Functionally Important Hot Spots.

Alphaviruses (family Togaviridae) include both human pathogens such as chikungunya virus (CHIKV) and Sindbis virus (SINV) and model viruses such as Semliki Forest virus (SFV). The alphavirus positive-strand RNA genome is translated into nonstructural (ns) polyprotein(s) that are precursors for four nonstructural proteins (nsPs). The three-dimensional structures of nsP2 and the N-terminal 2/3 of nsP3 reveal that these proteins consist of several domains. Cleavage of the ns-polyprotein is performed by the strictly regulated protease activity of the nsP2 region. Processing results in the formation of a replicase complex that can be considered a network of functional modules. These modules work cooperatively and should perform the same task for each alphavirus. To investigate functional interactions between replicase components, we generated chimeras using the SFV genome as a backbone. The functional modules corresponding to different parts of nsP2 and nsP3 were swapped with their counterparts from CHIKV and SINV. Although some chimeras were nonfunctional, viruses harboring the CHIKV N-terminal domain of nsP2 or any domain of nsP3 were viable. Viruses harboring the protease part of nsP2, the full-length nsP2 of CHIKV, or the nsP3 macrodomain of SINV required adaptive mutations for functionality. Seven mutations that considerably improved the infectivity of the corresponding chimeric genomes affected functionally important hot spots recurrently highlighted in previous alphavirus studies. These data indicate that alphaviruses utilize a rather limited set of strategies to survive and adapt. Furthermore, functional analysis revealed that the disturbance of processing was the main defect resulting from chimeric alterations within the ns-polyprotein. IMPORTANCE Alphaviruses cause debilitating symptoms and have caused massive outbreaks. There are currently no approved antivirals or vaccines for treating these infections. Understanding the functions of alphavirus replicase proteins (nsPs) provides valuable information for both antiviral drug and vaccine development. The nsPs of all alphaviruses consist of similar functional modules; however, to what extent these are independent in functionality and thus interchangeable among homologous viruses is largely unknown. Homologous domain swapping was used to study the functioning of modules from nsP2 and nsP3 of other alphaviruses in the context of Semliki Forest virus. Most of the introduced substitutions resulted in defects in the processing of replicase precursors that were typically compensated by adaptive mutations that mapped to determinants of polyprotein processing. Understanding the principles of virus survival strategies and identifying hot spot mutations that permit virus adaptation highlight a route to the rapid development of attenuated viruses as potential live vaccine candidates.

Coronavirus infection induces progressive restructuring of the endoplasmic reticulum involving the formation and degradation of double membrane vesicles.

Coronaviruses rearrange endoplasmic reticulum (ER) membranes to form a reticulovesicular network (RVN) comprised predominantly of double membrane vesicles (DMVs) involved in viral replication. While portions of the RVN have been analyzed by electron tomography (ET), the full extent of the RVN is not known, nor how RVN formation affects ER morphology. Additionally the precise mechanism of DMV formation has not been observed. In this work, we examined large volumes of coronavirus-infected cells at multiple timepoints during infection using serial-section ET. We provide a comprehensive 3D analysis of the ER and RVN which gives insight into the formation mechanism of DMVs as well as the first evidence for their lysosomal degradation. We also show that the RVN breaks down late in infection, concurrent with the ER becoming the main budding compartment for new virions. This work provides a broad view of the multifaceted involvement of ER membranes in coronavirus infection.

Mutations in SARS-CoV-2 nsp7 and nsp8 proteins and their predicted impact on replication/transcription complex structure.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) has been identified to be a mutation hot spot, with the P323L mutation being commonly observed in viral genomes isolated from North America. RdRp forms a complex with nonstructural proteins nsp7 and nsp8 to form the minimal replication/transcription machinery required for genome replication. As mutations in RdRp may affect formation of the RdRp-nsp7-nsp8 supercomplex, we analyzed viral genomes to identify mutations in nsp7 and nsp8 protein sequences. Based on in silico analysis of predicted structures of the supercomplex comprising of native and mutated proteins, we demonstrate that specific mutations in nsp7 and nsp8 proteins may have a role in stabilization of the replication/transcription complex.

Japanese encephalitis virus capsid protein interacts with non-lipidated MAP1LC3 on replication membranes and lipid droplets.

Microtubule-associated protein 1 light chain 3 (MAP1LC3) is a protein with a well-defined function in autophagy, but still incompletely understood roles in several other autophagy-independent processess. Studies have shown MAP1LC3 is a host-dependency factor for the replication of several viruses. Japanese encephalitis virus (JEV), a neurotropic flavivirus, replicates on ER-derived membranes that are marked by autophagosome-negative non-lipidated MAP1LC3 (LC3-I). Depletion of LC3 exerts a profound inhibition on virus replication and egress. Here, we further characterize the role of LC3 in JEV replication, and through immunofluorescence and immunoprecipitation show that LC3-I interacts with the virus capsid protein in infected cells. This association was observed on capsid localized to both the replication complex and lipid droplets (LDs). JEV infection decreased the number of LDs per cell indicating a link between lipid metabolism and virus replication. This capsid-LC3 interaction was independent of the autophagy adaptor protein p62/Sequestosome 1 (SQSTM1). Further, no association of capsid was seen with the Gamma-aminobutyric acid receptor-associated protein family, suggesting that this interaction was specific for LC3. High-resolution protein-protein docking studies identified a putative LC3-interacting region in capsid, 56FTAL59, and other key residues that could mediate a direct interaction between the two proteins.

Differential Splicing of Human Adenovirus 5 E1A RNA Expressed in cis versus in trans.

Human adenovirus (HAdV) is used extensively as a vector for gene delivery for a variety of purposes, including gene therapy and vaccine development. Most adenoviral vectors used for these approaches have a deletion of early region 1 (E1), which is complemented by the cell line. Most commonly, these are 293 cells for HAdV serotype 2 or 5. The 293 cells have the left end of HAdV5 integrated into chromosome 19 and express the E1 genes and protein IX. We observed that viruses with the E1 region deleted often grow less well on 293 cells than E1 wild-type viruses. Therefore, we investigated whether this poor growth is caused by splicing differences between the E1A RNA provided by the cell line (in trans) and the E1A RNA provided by the infecting viral genome (in cis). We observed that E1A RNA that was expressed from the genomes of 293 cells was spliced differently during infection with an E1A-deleted dl312 virus than E1A RNA from the same cells infected with dl309 or wt300. Importantly, 293 cells were not able to fully complement the late E1A transcripts, specifically 11S, 10S, and 9S RNA, which express the E1A217R, E1A171R, and E1A55R isoforms, respectively. We observed that these splicing differences likely arise due to different subnuclear localizations of E1A RNA. E1A RNA expressed from the viral genome was localized to viral replication centers, while E1A RNA expressed from the cell's genome was not. This loss of the late E1A mRNAs and their associated proteins impacts viral growth, gene expression, and protein levels. Complementation of the late E1A mRNAs in 293 cells restored some of the growth defect observed with dl312 and resulted in higher virus growth.IMPORTANCE Human adenovirus has become an important tool for medicine and research, and 293 cells and various similar cell lines are used extensively for virus production in situations where high viral yields are important. Such complementing cell lines are used for the production of viral vectors and vaccines, which often have deletions and replacements in various viral genes. Deletions in essential genes, such as E1, are often complemented by the cell line that is used for virus propagation in trans Here, we show that even complete genetic complementation of a viral gene does not result in full protein complementation, a defect that compromises virus growth. This is particularly important when high viral yields are crucial, as in virus production for vaccine development or gene therapy.

Phosphatidylinositol 3-Phosphate Mediates the Establishment of Infectious Bursal Disease Virus Replication Complexes in Association with Early Endosomes.

Infectious bursal disease virus (IBDV) is the archetypal member of the family Birnaviridae and the etiological agent of Gumboro disease, a highly contagious immunosuppressive infection of concern to the global poultry sector for its adverse health effects in chicks. Unlike most double-stranded RNA (dsRNA) viruses, which enclose their genomes within specialized cores throughout their viral replication cycle, birnaviruses organize their bisegmented dsRNA genome in ribonucleoprotein (RNP) structures. Recently, we demonstrated that IBDV exploits endosomal membranes for replication. The establishment of IBDV replication machinery on the cytosolic leaflet of endosomal compartments is mediated by the viral protein VP3 and its intrinsic ability to target endosomes. In this study, we identified the early endosomal phosphatidylinositol 3-phosphate [PtdIns(3)P] as a key host factor of VP3 association with endosomal membranes and consequent establishment of IBDV replication complexes in early endosomes. Indeed, our data reveal a crucial role for PtdIns(3)P in IBDV replication. Overall, our findings provide new insights into the replicative strategy of birnaviruses and strongly suggest that it resembles those of positive-strand RNA (+ssRNA) viruses, which replicate in association with host membranes. Furthermore, our findings support the role of birnaviruses as evolutionary intermediaries between +ssRNA and dsRNA viruses and, importantly, demonstrate a novel role for PtdIns(3)P in the replication of a dsRNA virus.IMPORTANCEInfectious bursal disease virus (IBDV) infects chicks and is the causative agent of Gumboro disease. During IBDV outbreaks in recent decades, the emergence of very virulent variants and the lack of effective prevention/treatment strategies to fight this disease have had devastating consequences for the poultry industry. IBDV belongs to the peculiar family Birnaviridae Unlike most dsRNA viruses, birnaviruses organize their genomes in ribonucleoprotein complexes and replicate in a core-independent manner. We recently demonstrated that IBDV exploits host cell endosomes as platforms for viral replication, a process that depends on the VP3 viral protein. In this study, we delved deeper into the molecular characterization of IBDV-endosome association and investigated the role of host cell phosphatidylinositide lipids in VP3 protein localization and IBDV infection. Together, our findings demonstrate that PtdIns(3)P serves as a scaffold for the association of VP3 to endosomes and reveal its essential role for IBDV replication.