Human genome-wide RNAi screen reveals a role for nuclear pore proteins in poxvirus morphogenesis

Poxviruses are considered less dependent on host functions than other DNA viruses because of their cytoplasmic site of replication and large genomes, which encode enzymes for DNA and mRNA synthesis. Nevertheless, RNAi screens with two independent human genome-scale libraries have identified more than 500 candidate genes that significantly inhibited and a similar number that enhanced replication and spread of infectious vaccinia virus (VACV). Translational, ubiquitin-proteosome, and endoplasmic reticulum-to-Golgi transport functions, known to be important for VACV, were enriched in the siRNA-inhibiting group, and RNA polymerase II and associated functions were enriched in the siRNA-enhancing group. Additional findings, notably the inhibition of VACV spread by siRNAs to several nuclear pore genes, were unanticipated. Knockdown of nucleoporin 62 strongly inhibited viral morphogenesis, with only a modest effect on viral gene expression, recapitulating and providing insight into previous studies with enucleated cells.     

cGAS-mediated stabilization of IFI16 promotes innate signaling during herpes simplex virus infection

Interferon γ-inducible protein 16 (IFI16) and cGMP-AMP synthase (cGAS) have both been proposed to detect herpesviral DNA directly in herpes simplex virus (HSV)-infected cells and initiate interferon regulatory factor-3 signaling, but it has been unclear how two DNA sensors could both be required for this response. We therefore investigated their relative roles in human foreskin fibroblasts (HFFs) infected with HSV or transfected with plasmid DNA. siRNA depletion studies showed that both are required for the production of IFN in infected HFFs. We found that cGAS shows low production of cGMP-AMP in infected cells, but instead cGAS is partially nuclear in normal human fibroblasts and keratinocytes, interacts with IFI16 in fibroblasts, and promotes the stability of IFI16. IFI16 is associated with viral DNA and targets to viral genome complexes, consistent with it interacting directly with viral DNA. Our results demonstrate that IFI16 and cGAS cooperate in a novel way to sense nuclear herpesviral DNA and initiate innate signaling.The innate immune response is a crucial component of host immunity and is the first line of defense against microbial pathogens, including bacteria and viruses. The initial events during infection of a host cell induce intracellular signaling pathways, resulting in the production of proinflammatory cytokines and IFNs. These effector molecules activate an antiviral state in neighboring cells and recruit immune cells to promote clearance of infection. Viral nucleic acids are potent activators of these signaling pathways and are recognized by a subset of host cell pattern recognition receptors (PRRs). These PRRs include the membrane-bound Toll-like receptors, the cytosolic RIG-I–like receptors, and a broad class of putative DNA sensors, which include both cytosolic and nuclear proteins (1).Unlike viral RNAs, which are distinct from cellular RNAs and therefore recognized by intracellular PRRs, DNA genomes of viruses that replicate in the nucleus are thought to be chemically and structurally similar to host DNA (2–4). It was therefore generally accepted that viral DNA sensing was limited to the cytoplasm where aberrant DNA accumulation would be perceived as “foreign.” However, this dogma has recently been challenged by the identification of DNA-sensing pathways that are active in the nucleus. The Pyrin and HIN-containing interferon γ-inducible protein 16 (IFI16) protein, initially described as a cytosolic DNA sensor (5), has been demonstrated to be nuclear in many cells and to promote the activation of inflammasomes (6, 7) and production of IFNs (8, 9) in response to herpesvirus infection. These initial studies involved short-term siRNA depletion of IFI16; in addition, a recent study showed that long-term knockdown of IFI16 expression led to abrogated IFN responses to not only DNA viruses, such as herpes simplex virus (HSV), but also RNA viruses, such as Sendai virus (10).cGMP-AMP synthase (cGAS) was also identified as a DNA sensor that recognizes cytosolic DNA and subsequently produces cGMP-AMP (cGAMP), a second messenger that induces stimulator of IFN genes (STING)-dependent activation of interferon regulatory factor-3 (IRF-3) signaling (11, 12). HSV-1 infection of murine L929 fibroblasts or human THP-1 cells led to synthesis of cGAMP and dimerization of IRF-3 (11, 12). Furthermore, fibroblasts from cgas^−/− mice showed decreased induction of IFNβ in response HSV-1 infection, indicating a role for cGAS in the innate immune response to herpesviral infection (13).A number of potential DNA sensors have been reported (14, 15), but IFI16 and cGAS appear most crucial for cellular detection of herpesvirus infections. The essentiality of two putative DNA sensors for innate responses to HSV infection raised a question, however, in that it was unclear how both IFI16 and cGAS could be required if serving as redundant DNA sensors. If the two sensors were completely redundant in one or more pathways, there would be no effect of depletion of either protein. Similarly, if one protein was sufficient as a sensor, both would not be required. Cooperativity between the two proteins could lead to a dual requirement. This analysis prompted us to evaluate whether cGAS and IFI16 cooperate to induce IFN expression in a single cell type.In this study we demonstrate that both IFI16 and cGAS are necessary for the induction of IFNβ in primary human foreskin fibroblasts in response to transfected plasmid DNA and herpesvirus infection and that there are substantial differences in the responses to the two stimuli. We obtained evidence that IFI16 and cGAS cooperate in sensing HSV DNA by a mechanism in which IFI16 serves as the primary DNA sensor, and cGAS plays an indirect role in the response to incoming nuclear herpesviral DNA by interacting with IFI16 and promoting its stability rather than through the production of cGAMP.     

Foot-and-Mouth Disease Virus: Molecular Interplays with IFN Response and the Importance of the Model

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals with a significant socioeconomic impact. One of the issues related to this disease is the ability of its etiological agent, foot-and-mouth disease virus (FMDV), to persist in the organism of its hosts via underlying mechanisms that remain to be elucidated. The establishment of a virus–host equilibrium via protein–protein interactions could contribute to explaining these phenomena. FMDV has indeed developed numerous strategies to evade the immune response, especially the type I interferon response. Viral proteins target this innate antiviral response at different levels, ranging from blocking the detection of viral RNAs to inhibiting the expression of ISGs. The large diversity of impacts of these interactions must be considered in the light of the in vitro models that have been used to demonstrate them, some being sometimes far from biological systems. In this review, we have therefore listed the interactions between FMDV and the interferon response as exhaustively as possible, focusing on both their biological effect and the study models used.     

Structural and functional analysis of the interaction between the nucleoporin Nup98 and the mRNA export factor Rae1

The export of mRNAs is a multistep process, involving the packaging of mRNAs into messenger ribonucleoprotein particles (mRNPs), their transport through nuclear pore complexes, and mRNP remodeling events prior to translation. Ribonucleic acid export 1 (Rae1) and Nup98 are evolutionarily conserved mRNA export factors that are targeted by the vesicular stomatitis virus matrix protein to inhibit host cell nuclear export. Here, we present the crystal structure of human Rae1 in complex with the Gle2-binding sequence (GLEBS) of Nup98 at 1.65 Å resolution. Rae1 forms a seven-bladed β-propeller with several extensive surface loops. The Nup98 GLEBS motif forms an ≈50-Å-long hairpin that binds with its C-terminal arm to an essentially invariant hydrophobic surface that extends over the entire top face of the Rae1 β-propeller. The C-terminal arm of the GLEBS hairpin is necessary and sufficient for Rae1 binding, and we identify a tandem glutamate element in this arm as critical for complex formation. The Rae1•Nup98^GLEBS surface features an additional conserved patch with a positive electrostatic potential, and we demonstrate that the complex possesses single-stranded RNA-binding capability. Together, these data suggest that the Rae1•Nup98 complex directly binds to the mRNP at several stages of the mRNA export pathway.     

RNA virus replication depends on enrichment of phosphatidylethanolamine at replication sites in subcellular membranes

Intracellular membranes are critical for replication of positive-strand RNA viruses. To dissect the roles of various lipids, we have developed an artificial phosphatidylethanolamine (PE) vesicle-based Tomato bushy stunt virus (TBSV) replication assay. We demonstrate that the in vitro assembled viral replicase complexes (VRCs) in artificial PE vesicles can support a complete cycle of replication and asymmetrical RNA synthesis, which is a hallmark of (+)-strand RNA viruses. Vesicles containing ∼85% PE and ∼15% additional phospholipids are the most efficient, suggesting that TBSV replicates within membrane microdomains enriched for PE. Accordingly, lipidomics analyses show increased PE levels in yeast surrogate host and plant leaves replicating TBSV. In addition, efficient redistribution of PE leads to enrichment of PE at viral replication sites. Expression of the tombusvirus p33 replication protein in the absence of other viral compounds is sufficient to promote intracellular redistribution of PE. Increased PE level due to deletion of PE methyltransferase in yeast enhances replication of TBSV and other viruses, suggesting that abundant PE in subcellular membranes has a proviral function. In summary, various (+)RNA viruses might subvert PE to build membrane-bound VRCs for robust replication in PE-enriched membrane microdomains.Many steps in the infection cycles of positive-strand RNA viruses, including entry into the cell, replication, virion assembly, and egress, are associated with subcellular membranes (1–4). Therefore, viruses have to interact with different lipids, such as phospholipids and sterols, which affect the biophysical features of membranes, including the fluidity and curvature (5, 6). The subverted cellular membranes could protect the viral RNA from recognition by the host nucleic acid sensors or from destruction by the cellular innate immune system. In addition, membranes facilitate the sequestration of viral and coopted host proteins to increase their local concentrations and promote macromolecular assembly, including formation of the viral replicase complex (VRC) or virion assembly. To optimize viral processes, RNA viruses frequently manipulate lipid composition of various intracellular membranes (6–13). Overall, the interaction between cellular lipids and viral components is emerging as one of the possible targets for antiviral methods against a great number of viruses. Understanding the roles of various lipids in RNA virus infections is important to ultimately control harmful RNA viruses.Among the various lipids, the highly abundant phospholipids are especially targeted by RNA viruses (2). In general, phospholipids likely affect the replication of most RNA viruses, which takes place within membranous structures (1, 3, 4). Accordingly, lipidomics analyses of cells infected with Dengue virus and hepatitis C virus (HCV) (8, 9) revealed enhanced virus-induced lipid biosynthesis, resulting in changes in the global lipid profile of host cells. Also, the less abundant regulatory phosphatidylinositol-4-phosphate (PI4P) was shown to be enriched at sites of enterovirus and HCV replication due to recruitment of cellular lipid kinases (7, 14), suggesting that a microenvironment enriched for PI4P facilitates (+)RNA virus replication. However, our knowledge on the roles of various phospholipids in RNA virus replication is currently incomplete. By using tombusviruses, small model RNA viruses of plants that can replicate in a yeast surrogate host (15), which has the advantage of tolerating large changes in different phospholipid composition, a major role for global phospholipid and sterol biosynthesis, have been revealed (16–18). In this paper, a viral replicase reconstitution assay based on artificial phospholipid vesicles identified the essential role of phosphatidylethanolamine (PE) in replication of Tomato bushy stunt virus (TBSV). It has also been shown that TBSV could recruit and enrich PE to the sites of viral replication in yeast and plant cells. Moreover, genetic changes that either increase or decrease PE levels in yeast greatly stimulated or inhibited TBSV replication, confirming the key role of PE in the formation of TBSV replicase.     

Defensive hypervariable regions confer superinfection exclusion in microviruses

Single-stranded DNA phages of the family Microviridae have fundamentally different evolutionary origins and dynamics than the more frequently studied double-stranded DNA phages. Despite their small size (around 5 kb), which imposes extreme constraints on genomic innovation, they have adapted to become prominent members of viromes in numerous ecosystems and hold a dominant position among viruses in the human gut. We show that multiple, divergent lineages in the family Microviridae have independently become capable of lysogenizing hosts and have convergently developed hypervariable regions in their DNA pilot protein, which is responsible for injecting the phage genome into the host. By creating microviruses with combinations of genomic segments from different phages and infecting Escherichia coli as a model system, we demonstrate that this hypervariable region confers the ability of temperate Microviridae to prevent DNA injection and infection by other microviruses. The DNA pilot protein is present in most microviruses, but has been recruited repeatedly into this additional role as microviruses altered their lifestyle by evolving the ability to integrate in bacterial genomes, which linked their survival to that of their hosts. Our results emphasize that competition between viruses is a considerable and often overlooked source of selective pressure, and by producing similar evolutionary outcomes in distinct lineages, it underlies the prevalence of hypervariable regions in the genomes of microviruses and perhaps beyond.

Numerous studies have found members of the viral family Microviridae as a, if not the, dominating force in the human gut virome (e.g., refs. 1–3). Many human viromes are composed almost entirely of these small, single-stranded DNA (ssDNA) phages, which have fundamentally different evolutionary origins than the more commonly studied double-stranded DNA (dsDNA) phages (4). To date, few aspects of their ecology and evolution as a whole have been studied because most microviruses are known only from metagenomic data rather than from physical isolates (5).Several microviral taxa have been detected as prophages of the Bacteroidetes, Firmicutes, and Proteobacteria (6–9). As prophages, they link their fate to the survival and replication of their bacterial host (10)—“piggybacking-the-winner” rather than “killing-the-winner” by preying on abundant bacteria (11, 12). Therefore, the transition from a lytic to a temperate lifestyle [which, in microviruses, occurred through the acquisition of a short integration motif recognized by bacterial integrase/recombinase systems (9)] imposes new selective pressures on viral populations (13). For example, the lytic microviruses of the subfamily Bullavirinae, exemplified by the venerable phiX174, and the recently discovered temperate members of the subfamily Gokushovirinae both infect Escherichia coli but display very different survival strategies: the former excels at rapid replication and lysis of its bacterial host (14), whereas the latter are slow replicators that can reside in host cultures for long periods of time, with no apparent effect on host fitness (9).Temperate Gokushovirinae, like other prophages, render their bacterial hosts immune to subsequent infection by related viruses (9) via a process referred to as superinfection exclusion or immunity (SiEx) (15–17). SiEx not only serves to defend hosts against further phage infection but also offers an offensive strategy to bacteria. By providing a way to differentiate infected self- from uninfected nonself cells (18), the excision of prophages produces viral particles that infect and kill nonself strains (19, 20). Given the role of SiEx in competition between bacteria harboring different phage types, as well as between the phages themselves, numerous mechanisms of SiEx exist. SiEx has been studied for decades (21, 22) but primarily in double-stranded DNA viruses such as Lambda, P1, T4, and Mu. In these phages, SiEx is typically conferred by accessory proteins that are not essential to viral function and often lost or gained through recombination (e.g., refs. 23–25). However, the small size of microviral genomes and the broadly overlapping gene sets of lytic and temperate microviruses render the evolution of SiEx through the horizontal acquisition of genes unlikely.In this study, we link the evolution of a hypervariable region (HVR) in the DNA pilot protein of microviral capsids to the viral ability to both mediate and overcome SiEx. We demonstrate that multiple divergent variants of this region, resulting from competition between otherwise identical phages, are present in populations of lysogens. By synthesizing hybrid phages composed of segments derived from different gokushoviruses and conducting superinfection experiments, we pinpoint the genomic region responsible for SiEx—a region that is almost uniform in lytic viruses but highly variable in temperate viruses. Our results show that divergent microviruses, as a result of (pro)phage arms races, have converged on the identical defensive strategy based on the evolution of HVRs in the same ancestral, structural gene. These findings advance our understanding of the biology and evolution of the ubiquitous microviruses, whose small genomes lack space to evolve or acquire accessory genes to confer new traits. Rather, already existing genes are repurposed for novel functions while preserving compact genome sizes.     

Positive-strand RNA viruses stimulate host phosphatidylcholine synthesis at viral replication sites

All positive-strand RNA viruses reorganize host intracellular membranes to assemble their viral replication complexes (VRCs); however, how these viruses modulate host lipid metabolism to accommodate such membrane proliferation and rearrangements is not well defined. We show that a significantly increased phosphatidylcholine (PC) content is associated with brome mosaic virus (BMV) replication in both natural host barley and alternate host yeast based on a lipidomic analysis. Enhanced PC levels are primarily associated with the perinuclear ER membrane, where BMV replication takes place. More specifically, BMV replication protein 1a interacts with and recruits Cho2p (choline requiring 2), a host enzyme involved in PC synthesis, to the site of viral replication. These results suggest that PC synthesized at the site of VRC assembly, not the transport of existing PC, is responsible for the enhanced accumulation. Blocking PC synthesis by deleting the CHO2 gene resulted in VRCs with wider diameters than those in wild-type cells; however, BMV replication was significantly inhibited, highlighting the critical role of PC in VRC formation and viral replication. We further show that enhanced PC levels also accumulate at the replication sites of hepatitis C virus and poliovirus, revealing a conserved feature among a group of positive-strand RNA viruses. Our work also highlights a potential broad-spectrum antiviral strategy that would disrupt PC synthesis at the sites of viral replication but would not alter cellular processes.All positive-strand RNA viruses [(+)RNA viruses], which include numerous important human, animal, and plant pathogens, share similar strategies for genomic replication. A highly conserved and indispensable feature of their replication is the proliferation and reorganization of host cellular membranes to assemble viral replication complexes (VRCs). Despite this central importance, it is largely unknown how cellular membranes are rearranged by the viral replication proteins and how cellular lipid metabolism is modulated to accommodate membrane proliferation and remodeling.Brome mosaic virus (BMV) serves as a model for understanding VRC formation of (+)RNA viruses (1). BMV is the type member of the plant virus family Bromoviridae and a representative member of the alphavirus-like superfamily, which includes many human, animal, and plant-infecting viruses (2). BMV encodes two replication proteins, 1a and 2a^pol. 2a^pol serves as the replicase, whereas 1a has an N-terminal methyltransferase domain (3, 4) and a C-terminal ATPase/helicase-like domain (5). Together, 1a and 2a^pol are necessary and sufficient for BMV replication. BMV induces vesicular structures in its surrogate host, the yeast Saccharomyces cerevisiae, and its natural host, barley (6, 7). These structures, termed spherules, have been shown to be the VRCs in yeast as 1a, 2a^pol, and nascent viral RNAs reside in the interior of these compartments. Spherules are invaginations of the outer perinuclear endoplasmic reticulum (ER) membrane into the ER lumen and are about 60–80 nm in diameter (6). Remarkably, expression of 1a alone in yeast induces spherule formation (6), which requires 1a’s amphipathic α-helix (1a amino acids 392–407) (8), helix A, and 1a–1a interactions (9, 10). In addition, several host proteins, including membrane-shaping reticulons (RTNs) (11) and an ESCRT (endosomal sorting complex required for transport) component, Snf7p (sucrose nonfermenting7) (12), are recruited by 1a to form spherules.Similar to other (+)RNA viruses, BMV promotes host lipid synthesis and requires balanced lipids for the formation and activity of VRCs. Expression of 1a in yeast induces a ∼30% increase in total fatty acids (FAs) per cell (13). BMV also requires a high level of unsaturated FA (UFA) because a ∼12% decrease of UFAs in the yeast ole1w mutant blocks its replication more than 20-fold (13). In addition, deleting host ACB1 (Acyl-CoA-binding 1) gene results in formation of spherules that are smaller in size but are in greater number than in wild-type (WT) cells (14). ACB1 encodes acyl-CoA binding protein, which binds long-chain fatty acyl-CoAs and is involved in maintaining lipid homeostasis. Supplemented long-chain UFAs largely complement the BMV replication defects in cells lacking ACB1, indicating that the altered lipid composition is primarily responsible for BMV replication defects (14).Cellular membranes are mainly composed of phospholipids, and in particular, phosphatidylcholine (PC) constitutes ∼50% of total phospholipids (15). PC is synthesized via the CDP–DAG (cytidine diphosphate–diacylglycerol) and Kennedy pathways in eukaryotes (16). PC synthesis is significantly enhanced during infection of Dengue virus (DENV) (17), Flock House virus (FHV) (18), and poliovirus (19, 20). A 70% and 35% increase of total PC levels was recorded in DENV-infected mosquito cells (17) and FHV-infected Drosophila cells (18), respectively. At the peak time of poliovirus replication in HeLa cells, a ∼37% increase of PC content was observed after a 30-min chase (19). It was further found that poliovirus promotes the import of FAs, which were subsequently channeled to the viral replication sites. In addition, FAs were mainly incorporated into PCs (20).One critical question based on the aforementioned research is whether the enhanced PC is synthesized in association with the VRCs or elsewhere in cells and subsequently transported into the VRCs. If PC is produced in association with the VRCs, what key enzymes are recruited? We report here that several (+)RNA viruses, including BMV, hepatitis C virus (HCV), and poliovirus, promote significantly enhanced accumulation of PC content at the viral replication sites, revealing a common feature of viral replication among a group of (+)RNA viruses. We further demonstrate that BMV 1a interacts with and redistributes the host enzyme, Cho2p (choline requiring 2), to the viral replication sites. As Cho2p converts phosphatidylethanolamine (PE) to PC in the CDP–DAG pathway, the relocalization of Cho2p suggests the VRC-localized PC synthesis. Deleting CHO2 inhibits BMV replication up to 30-fold and results in formation of spherules that are larger than those of WT cells. This work highlights the importance of PC in VRC formation and the possibility of developing a novel and broad-spectrum antiviral strategy by specifically disrupting PC synthesis at the viral replication sites but not general PC synthesis.     

High-resolution asymmetric structure of a Fab–virus complex reveals overlap with the receptor binding site

Canine parvovirus is an important pathogen causing severe diseases in dogs, including acute hemorrhagic enteritis, myocarditis, and cerebellar disease. Overlap on the surface of parvovirus capsids between the antigenic epitope and the receptor binding site has contributed to cross-species transmission, giving rise to closely related variants. It has been shown that Mab 14 strongly binds and neutralizes canine but not feline parvovirus, suggesting this antigenic site also controls species-specific receptor binding. To visualize the conformational epitope at high resolution, we solved the cryogenic electron microscopy (cryo-EM) structure of the Fab–virus complex. We also created custom software, Icosahedral Subparticle Extraction and Correlated Classification, to solve a Fab–virus complex with only a few Fab bound per capsid and visualize local structures of the Fab-bound and -unbound antigenic sites extracted from the same complex map. Our results identified the antigenic epitope that had significant overlap with the receptor binding site, and the structures revealed that binding of Fab induced conformational changes to the virus. We were also able to assign the order and position of attached Fabs to allow assessment of complementarity between the Fabs bound to different positions. This approach therefore provides a method for using cryo-EM to investigate complementarity of antibody binding.

Canine parvovirus (CPV) emerged as a host range variant virus in the mid-1970s, subsequently causing a pandemic of disease in dogs during 1978 (1, 2). Since that time, multiple variants have emerged with additional mutations in the viral capsid (3, 4). Extensive genetic and biochemical studies have shown that specific mutations displayed on or near the capsid surface alter binding to the host receptor, transferrin receptor type-1 (TfR). Since the specific host ranges of canine and feline parvoviruses are primarily controlled by the ability of the virus to bind TfR, changes in the binding site alter the ability of the virus to infect different hosts (3, 5, 6).The virus capsid is highly antigenic, and an infection elicits many different host antibodies, which recognize specific structures on the surface of the virus that are primarily displayed as conformational epitopes. Most antibodies efficiently neutralize virus as IgGs, whereas they vary in their neutralization abilities when tested as Fabs (7, 8). In a number of cases, selection of antibody escape mutations by antibodies also selects for host range variation in the viruses, and conversely, selection for host range variation alters the antigenic structure recognized by specific antibodies (6, 9, 10). Although some of these changes appear to result from overlap of the receptor and antibody binding sites, it is still not clear how different selections operate in the natural evolution of the viruses. Understanding the mechanisms of host recognition and the dynamics of the binding by antibodies and receptors would provide insight into the connections between antigenic and host range variation, enabling us to predict the ability of a given virus capsid to change hosts or to escape host immunity. The coordinated overlap between antibody and receptor binding has also been seen in other viruses, including SARS-CoV-2 and influenza viruses (11–13).CPV has a small, 26-nm diameter, T = 1 icosahedral capsid that packages a single-stranded DNA (ssDNA) genome of about 5,000 bases. The capsid shell is composed of VP2 (∼90%) and VP1 (∼10%), which are generated by differential messenger RNA splicing events so that the entire sequence of VP2 is also contained within VP1. Both proteins fold into the same eight-stranded, antiparallel β-barrel structure, where the β-strands are connected by loops that make up the surface features of the capsid. A raised region known as the threefold spike surrounds each icosahedral threefold axis and contains most of the antigenic structures recognized by different antibodies (14, 15). MAb 14 is a mouse monoclonal antibody generated against CPV capsids that has particularly interesting properties. MAb 14 binding, hemagglutination inhibition properties, and neutralization are all virus-strain specific, and it bound with significantly higher affinity to CPV capsids than to the closely related but host range variant–virus that infected cats, feline panleukopenia virus (FPV) (16–18). The virus-specific binding of MAb is controlled by the capsid surface residue 93, which is Lys in FPV and Asn in CPV (19–21). In addition to antibody recognition, residue 93 also controls canine host range, since Asn93 allows binding to canine TfR and infection of canine cells, whereas Lys93 in the equivalent position on the FPV capsid prevents both of these processes (20).Despite the central role of antibodies in protecting animals against virus infections and allowing recovery from disease, in many cases, we still lack a detailed understanding of epitope characteristics, the dynamics of binding processes, and the viral neutralization mechanisms. Previous X-ray crystallography and cryo-electron microscopy (cryo-EM) structures include the Fab of MAb 14 (Fab 14), CPV and FPV capsids, and a Fab 14–CPV capsid complex at moderate resolution of 12.5 Å (Protein Data Bank [PDB] IDs: 2CAS, 1C8F, 3IY0, and 3GK8) (18, 22, 23). Crystal structures of Fab 14, CPV, and FPV fitted into the cryo-EM map of Fab–virus complex have allowed us to predict protein interactions in the binding interface (18). Although this was the most rigorous approach at the time, the resulting pseudoatomic structure based on the fitting did not explain why variation in residue 93 controlled Fab binding, identify likely mechanisms of antibody neutralization, or explain how the minor changes in the site also affect TfR binding. Recent technological advances in cryo-EM now allow us to solve Fab–virus structures at high enough resolution to build atomic models directly into the density map for identifying interactions unambiguously.The binding and occupancy of Fabs on the CPV capsid have also been determined previously using charge detection mass spectrometry, which revealed that some of the tested monoclonal antibody–derived Fabs, including Fab 14, could fully occupy all 60 epitopes of the capsid but with some differences in the kinetics of attachment (24). Incubating with excess Fab molecules to occupy all icosahedrally equivalent sites on capsids has long been the preferred cryo-EM structural approach since this allows icosahedral symmetry averaging to be imposed during the reconstruction process for maximizing resolution (18, 25, 26). However, there are few other studies confirming the occupancies of Fabs on viral capsids, and the larger IgG likely does not saturate the entire surface of the capsid so that an undersaturated capsid (with fewer than 60 bound Fab in the case of parvoviruses) would more closely mimic the physiologically relevant complexes. Solving such an asymmetric structure at atomic resolution is now possible due to advances in cryo-EM and the reconstruction approaches.Here, we define an atomic model of Fab 14 bound to the capsid of CPV based on cryo-EM of the complex and examine the functional mechanisms that affect binding by testing antibody mutants. Of the two data sets used to reconstruct Fab–virus complex maps, one had close to complete occupancy of the 60 capsid epitopes, whereas the other had an average of 10 Fabs bound per capsid. These data were used initially to solve the icosahedrally averaged structures of fully Fab-occupied and partially Fab-occupied complexes to resolutions of 3.2 and 2.3 Å, respectively. An asymmetric, partially Fab-occupied virus map calculated with local reconstruction approaches attained 2.4-Å global resolution and revealed the Fab-occupied and unoccupied sites on the same virus capsid. These structures allowed unambiguous identification of residues and side chains involved in the Fab–virus binding interface and also revealed local conformational changes in the antibody binding site induced by Fab binding. The partial occupancy of the capsids by Fab provided an opportunity to develop an innovative algorithm to test for complementarity of Fab binding to different positions on the capsid. Notably, it was the asymmetric approach and not the traditional icosahedrally averaged reconstruction that revealed the details of antibody binding.     

Cellular Lipids—Hijacked Victims of Viruses

Over the millions of years-long co-evolution with their hosts, viruses have evolved plenty of mechanisms through which they are able to escape cellular anti-viral defenses and utilize cellular pathways and organelles for replication and production of infectious virions. In recent years, it has become clear that lipids play an important role during viral replication. Viruses use cellular lipids in a variety of ways throughout their life cycle. They not only physically interact with cellular membranes but also alter cellular lipid metabolic pathways and lipid composition to create an optimal replication environment. This review focuses on examples of how different viruses exploit cellular lipids in different cellular compartments during their life cycles.     

Structure of a herpesvirus nuclear egress complex subunit reveals an interaction groove that is essential for viral replication

Herpesviruses require a nuclear egress complex (NEC) for efficient transit of nucleocapsids from the nucleus to the cytoplasm. The NEC orchestrates multiple steps during herpesvirus nuclear egress, including disruption of nuclear lamina and particle budding through the inner nuclear membrane. In the important human pathogen human cytomegalovirus (HCMV), this complex consists of nuclear membrane protein UL50, and nucleoplasmic protein UL53, which is recruited to the nuclear membrane through its interaction with UL50. Here, we present an NMR-determined solution-state structure of the murine CMV homolog of UL50 (M50; residues 1–168) with a strikingly intricate protein fold that is matched by no other known protein folds in its entirety. Using NMR methods, we mapped the interaction of M50 with a highly conserved UL53-derived peptide, corresponding to a segment that is required for heterodimerization. The UL53 peptide binding site mapped onto an M50 surface groove, which harbors a large cavity. Point mutations of UL50 residues corresponding to surface residues in the characterized M50 heterodimerization interface substantially decreased UL50–UL53 binding in vitro, eliminated UL50–UL53 colocalization, prevented disruption of nuclear lamina, and halted productive virus replication in HCMV-infected cells. Our results provide detailed structural information on a key protein–protein interaction involved in nuclear egress and suggest that NEC subunit interactions can be an attractive drug target.Herpesviruses encompass a large family of infectious agents, including important veterinary and human pathogens (1). Among the latter is human cytomegalovirus (HCMV), which can cause serious disease, particularly in immunocompromised individuals and newborns (2). Despite the importance of HCMV in these medically vulnerable populations, currently available treatment options suffer from issues with toxicities, drug resistance, and/or pharmacokinetics (2, 3), motivating the identification of new drug targets.All herpesviruses of mammals, birds, and reptiles undergo a remarkable process known as nuclear egress as part of the viral lifecycle. It is generally accepted that, after assembly in the nucleus, the viral nucleocapsid undergoes envelopment to cross the inner nuclear membrane (INM) followed by deenvelopment to cross the outer nuclear membrane, resulting in release into the cytoplasm for continuation of the virion maturation process (4). Nuclear egress is orchestrated by a highly conserved, heterodimeric nuclear egress complex (NEC), which recruits one or more protein kinases to disrupt the nuclear lamina, permitting access of nucleocapsids to the INM, where the NEC induces budding of the nucleocapsid into the perinuclear space (5–13). In HCMV, the NEC is comprised of UL50, which is an INM protein, and UL53, which is a nucleoplasmic protein that is brought to the INM by its interaction with UL50. These two proteins and their murine CMV (MCMV) homologues, M50 and M53, are essential for replication and nuclear egress (8, 14–17) of their respective viruses. Although a process similar to herpesvirus nuclear egress was recently described for movement of ribonucleoprotein particles during Drosophila myogenesis (18), no host cell homolog of the NEC that would serve as mediator of this mechanism has yet been identified. Furthermore, no structural information currently exists for any NEC subunit across the Herpesviridae family.To gain a better molecular understanding of herpesvirus nuclear egress, we used NMR methods to solve the structure of the conserved half of MCMV M50 and map residues on the surface of M50 that are involved in interactions with the other NEC subunit. We then tested the importance of several of these residues for heterodimerization of both the MCMV and HCMV NECs by looking at the effect of single-alanine mutations in both M50 and UL50 on binding affinity and replication of HCMV by looking at the effect of mutations in the context of NEC localization, nuclear lamina disruption, and virus production. Our results identified a subunit interaction interface with features that suggest that it could be an attractive antiviral drug target.     

The VP3 Protein of Bluetongue Virus Associates with the MAVS Complex and Interferes with the RIG-I-Signaling Pathway

Bluetongue virus (BTV), an arbovirus transmitted by Culicoides biting midges, is a major concern of wild and domestic ruminants. While BTV induces type I interferon (alpha/beta interferon [IFN-α/β]) production in infected cells, several reports have described evasion strategies elaborated by this virus to dampen this intrinsic, innate response. In the present study, we suggest that BTV VP3 is a new viral antagonist of the IFN-β synthesis. Indeed, using split luciferase and coprecipitation assays, we report an interaction between VP3 and both the mitochondrial adapter protein MAVS and the IRF3-kinase IKKε. Overall, this study describes a putative role for the BTV structural protein VP3 in the control of the antiviral response.     

The retromer is co-opted to deliver lipid enzymes for the biogenesis of lipid-enriched tombusviral replication organelles

Biogenesis of viral replication organelles (VROs) is critical for replication of positive-strand RNA viruses. In this work, we demonstrate that tomato bushy stunt virus (TBSV) and the closely related carnation Italian ringspot virus (CIRV) hijack the retromer to facilitate building VROs in the surrogate host yeast and in plants. Depletion of retromer proteins, which are needed for biogenesis of endosomal tubular transport carriers, strongly inhibits the peroxisome-associated TBSV and the mitochondria-associated CIRV replication in yeast and in planta. In vitro reconstitution revealed the need for the retromer for the full activity of the viral replicase. The viral p33 replication protein interacts with the retromer complex, including Vps26, Vps29, and Vps35. We demonstrate that TBSV p33-driven retargeting of the retromer into VROs results in delivery of critical retromer cargoes, such as 1) Psd2 phosphatidylserine decarboxylase, 2) Vps34 phosphatidylinositol 3-kinase (PI3K), and 3) phosphatidylinositol 4-kinase (PI4Kα-like). The recruitment of these cellular enzymes by the co-opted retromer is critical for de novo production and enrichment of phosphatidylethanolamine phospholipid, phosphatidylinositol-3-phosphate [PI(3)P], and phosphatidylinositol-4-phosphate [PI(4)P] phosphoinositides within the VROs. Co-opting cellular enzymes required for lipid biosynthesis and lipid modifications suggest that tombusviruses could create an optimized lipid/membrane microenvironment for efficient VRO assembly and protection of the viral RNAs during virus replication. We propose that compartmentalization of these lipid enzymes within VROs helps tombusviruses replicate in an efficient milieu. In summary, tombusviruses target a major crossroad in the secretory and recycling pathways via coopting the retromer complex and the tubular endosomal network to build VROs in infected cells.

Viruses are intracellular parasites which co-opt cellular resources to produce abundant viral progeny. Positive-strand (+)RNA viruses replicate on subcellular membranes by forming viral replication organelles (VROs) (1–5). VROs sequester the viral proteins and viral RNAs together with co-opted host factors to provide an optimal subcellular environment for the assembly of numerous viral replicase complexes (VRCs), which are then responsible for robust viral RNA replication. VROs also spatially and temporally organize viral replication. Importantly, the VROs hide the viral RNAs from cellular defense mechanisms as well (5, 6). VROs consist of extensively remodeled membranes with unique lipid composition. How viruses achieve these membrane remodeling and lipid modifications and lipid enrichment is incompletely understood. Therefore, currently, there is a major ongoing effort to dissect the VRC assembly process and to understand the roles of viral and host factors in driving the biogenesis of VROs (1, 3, 7).Tomato bushy stunt virus (TBSV), a plant-infecting tombusvirus, has been shown to induce complex rearrangements of cellular membranes and alterations in lipid and other metabolic processes during infections (8–10). The VROs formed during TBSV infections include extensive membrane contact sites (vMCSs) and harbor numerous spherules (containing VRCs), which are vesicle-like invaginations in the peroxisomal membranes (8, 11–13). A major gap in our understanding of the biogenesis of VROs, including vMCSs and VRCs, is how the cellular lipid-modifying enzymes are recruited to the sites of viral replication.Tombusviruses belong to the large Flavivirus-like supergroup that includes important human, animal, and plant pathogens. Tombusviruses have a small single-component (+)RNA genome of ∼4.8 kb that codes for five proteins. Among those, there are two essential replication proteins, namely p33 and p92^pol, the latter of which is the RdRp protein and it is translated from the genomic RNA via readthrough of the translational stop codon in p33 open reading frame (14). The smaller p33 replication protein is an RNA chaperone, which mediates the selection of the viral (+)RNA for replication (14–16). Altogether, p33 is the master regulator of VRO biogenesis (3). We utilize a TBSV replicon (rep)RNA, which contains four noncontiguous segments from the genomic RNA, and it can efficiently replicate in yeast and plant cells expressing p33 and p92^pol (14, 17).Tombusviruses hijack various cellular compartments and pathways for VRO biogenesis (18). These include peroxisomes by TBSV or mitochondria (in the case of the closely related carnation Italian ringspot virus [CIRV]), the endoplasmic reticulum (ER) network, Rab1-positive COPII vesicles, and the Rab5-positive endosomes (8, 19–23). Tombusviruses also induce membrane proliferation, new lipid synthesis, and enrichment of lipids, most importantly phosphatidylethanolamine (PE), sterols, phosphatidylinositol-4-phosphate [PI(4)P], and phosphatidylinositol-3-phosphate [PI(3)P] phosphoinositides in peroxisomal or mitochondrial membranes for different tombusviruses (13, 24–27). This raised the question that how TBSV could hijack lipid synthesis enzymes from other subcellular locations that leads to enrichment of critical lipids in the large VROs in model yeast and plant hosts.The endosomal network (i.e., early, late, and recycling endosomes) is a collection of pleomorphic organelles which sort membrane-bound proteins and lipids either for vacuolar/lysosomal degradation or recycling to other organelles. With the help of the so-called retromer complex, tubular transport carriers formed from the endosomes recycle cargoes to the Golgi and ER or to the plasma membrane (28–31). The core retromer complex consists of three conserved proteins, Vps26, Vps29, and Vps35, which are involved in cargo sorting and selection. The retromer complex affects several cellular processes, including autophagy through the maturation of lysosomes (32), neurodegenerative diseases (33), plant root hair growth (34), and plant immunity (35).The cellular retromer is important for several pathogen–host interactions. For example, the retromer is targeted by Brucella, Salmonella, and Legionella bacteria (36–39) and the rice blast fungus (40). The retromer is also involved in the intracellular transport of the Shigella and Cholera toxins and the plant ricin toxin. The NS5A replication protein of hepatitis C virus (HCV) interacts with Vps35 and this interaction is important for HCV replication in human cells (41). The cytoplasmic tail of the Env protein of HIV-1 binds to the retromer components Vps35 and Vps26, which is required for Env trafficking and infectious HIV-1 morphogenesis (42). Moreover, the retromer complex affects the morphogenesis of vaccinia virus (43) and HPV16 human papillomavirus entry and delivery to the trans-Golgi network (44). Despite the importance of the retromer in pathogen–host interactions, the mechanistic insights are far from complete.In the case of tombusviruses, enrichment of PE and PI(3)P within VROs is facilitated by co-opting the endosomal Rab5 small GTPase and Vps34 PI3K (20, 24), suggesting that the endosome-mediated trafficking pathway might be involved in viral replication in host cells. However, the actual mechanism of how tombusviruses exploit the endosomal/endocytic pathway and induce lipid enrichment within VROs is not yet dissected. Therefore, in this work, we targeted the retromer complex, based on previous genome-wide screens using yeast gene-deletion libraries, which led to the identification of VPS29 and VPS35 as host genes affecting TBSV replication and recombination, respectively (45, 46). These proteins are components of the retromer complex (28–31). We found TBSV and the closely related CIRV co-opt the retromer complex for the biogenesis of VROs in yeast and plants. We observed that depletion of retromer proteins strongly inhibited TBSV and CIRV replication. The recruitment of the retromer is driven by the viral p33 replication protein, which interacts with Vps26, Vps29, and Vps35 retromer proteins. We show that the retromer helps delivering critical cargo proteins, such as Psd2 phosphatidylserine decarboxylase, Vps34 phosphatidylinositol 3-kinase (PI3K), and Stt4 phosphatidylinositol 4-kinase (PI4Kα-like). These co-opted cellular enzymes are then involved in de novo production and enrichment of PE phospholipid, PI(3)P, and PI(4)P phosphoinositides within the VROs. Altogether, these virus-driven activities create an optimized membrane microenvironment within VROs to support efficient tombusvirus replication.     

Characterization of SARS-CoV-2 Evasion: Interferon Pathway and Therapeutic Options

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for the current COVID-19 pandemic. SARS-CoV-2 is characterized by an important capacity to circumvent the innate immune response. The early interferon (IFN) response is necessary to establish a robust antiviral state. However, this response is weak and delayed in COVID-19 patients, along with massive pro-inflammatory cytokine production. This dysregulated innate immune response contributes to pathogenicity and in some individuals leads to a critical state. Characterizing the interplay between viral factors and host innate immunity is crucial to better understand how to manage the disease. Moreover, the constant emergence of new SARS-CoV-2 variants challenges the efficacy of existing vaccines. Thus, to control this virus and readjust the antiviral therapy currently used to treat COVID-19, studies should constantly be re-evaluated to further decipher the mechanisms leading to SARS-CoV-2 pathogenesis. Regarding the role of the IFN response in SARS-CoV-2 infection, in this review we summarize the mechanisms by which SARS-CoV-2 evades innate immune recognition. More specifically, we explain how this virus inhibits IFN signaling pathways (IFN-I/IFN-III) and controls interferon-stimulated gene (ISG) expression. We also discuss the development and use of IFNs and potential drugs controlling the innate immune response to SARS-CoV-2, helping to clear the infection.     

Cell cycle deregulation by a poxvirus partial mimic of anaphase-promoting complex subunit 11

The anaphase-promoting complex (APC), or cyclosome, is a ubiquitin ligase with major roles in cell cycle regulation. It is required for mitotic exit, but must be deactivated for the G₁/S phase transition to occur. APC consists of at least 12 subunits with the catalytic core formed by a scaffold protein, APC2, and a RING-H2 protein, APC11. APC11 facilitates ubiquitin chain formation by recruiting ubiquitin-charged conjugating enzymes through its RING-H2 domain. We report that a small number of poxviruses encode RING-H2 proteins with sequence similarities to APC11. We show that a representative of these viral proteins mimics APC11 in its interactions with APC, but unlike APC11, the viral protein fails to promote ubiquitin chain formation. This absence of ubiquitin ligase activity is linked to a distinctive sequence variation within its RING-H2 domain. Expression of the viral protein led to cell cycle deregulation and the accumulation of APC substrates in a manner consistent with impaired APC function. Our data characterize this protein as a regulator of APC activity, and consequently, we have called it PACR (poxvirus APC/cyclosome regulator). Deletion of the PACR gene substantially reduced viral replication. Here, we report a viral mimic of an APC component and reveal an intriguing mechanism by which viruses can manipulate cell cycle progression and, thereby, promote their own replication.     

Manipulation of Host Microtubule Networks by Viral Microtubule-Associated Proteins

Diverse DNA and RNA viruses utilize cytoskeletal networks to efficiently enter, replicate, and exit the host cell, while evading host immune responses. It is well established that the microtubule (MT) network is commonly hijacked by viruses to traffic to sites of replication after entry and to promote egress from the cell. However, mounting evidence suggests that the MT network is also a key regulator of host immune responses to infection. At the same time, viruses have acquired mechanisms to manipulate and/or usurp MT networks to evade these immune responses. Central to most interactions of viruses with the MT network are virally encoded microtubule-associated proteins (MAPs) that bind to MTs directly or indirectly. These MAPs associate with MTs and other viral or cellular MAPs to regulate various aspects of the MT network, including MT dynamics, MT-dependent transport via motor proteins such as kinesins and dyneins, and MT-dependent regulation of innate immune responses. In this review, we examine how viral MAP interactions with the MT network facilitate viral replication and immune evasion.