Structure of orthoreovirus RNA chaperone σNS, a component of viral replication factories.

The mammalian orthoreovirus (reovirus) σNS protein is required for formation of replication compartments that support viral genome replication and capsid assembly. Despite its functional importance, a mechanistic understanding of σNS is lacking. We conducted structural and biochemical analyses of a σNS mutant that forms dimers instead of the higher-order oligomers formed by wildtype (WT) σNS. The crystal structure shows that dimers interact with each other using N-terminal arms to form a helical assembly resembling WT σNS filaments in complex with RNA observed using cryo-EM. The interior of the helical assembly is of appropriate diameter to bind RNA. The helical assembly is disrupted by bile acids, which bind to the same site as the N-terminal arm. This finding suggests that the N-terminal arm functions in conferring context-dependent oligomeric states of σNS, which is supported by the structure of σNS lacking an N-terminal arm. We further observed that σNS has RNA chaperone activity likely essential for presenting mRNA to the viral polymerase for genome replication. This activity is reduced by bile acids and abolished by N-terminal arm deletion, suggesting that the activity requires formation of σNS oligomers. Our studies provide structural and mechanistic insights into the function of σNS in reovirus replication.

Structure of Orthoreovirus RNA Chaperone σNS, a Component of Viral Replication Factories.

The reovirus σNS RNA-binding protein is required for formation of intracellular compartments during viral infection that support viral genome replication and capsid assembly. Despite its functional importance, a mechanistic understanding of σNS is lacking. We conducted structural and biochemical analyses of an R6A mutant of σNS that forms dimers instead of the higher-order oligomers formed by wildtype (WT) σNS. The crystal structure of selenomethionine-substituted σNS-R6A reveals that the mutant protein forms a stable antiparallel dimer, with each subunit having a well-folded central core and a projecting N-terminal arm. The dimers interact with each other by inserting the N-terminal arms into a hydrophobic pocket of the neighboring dimers on either side to form a helical assembly that resembles filaments of WT σNS in complex with RNA observed using cryo-EM. The interior of the crystallographic helical assembly is positively charged and of appropriate diameter to bind RNA. The helical assembly is disrupted by bile acids, which bind to the same hydrophobic pocket as the N-terminal arm, as demonstrated in the crystal structure of σNS-R6A in complex with bile acid, suggesting that the N-terminal arm functions in conferring context-dependent oligomeric states of σNS. This idea is supported by the structure of σNS lacking the N-terminal arm. We discovered that σNS displays RNA helix destabilizing and annealing activities, likely essential for presenting mRNA to the viral RNA-dependent RNA polymerase for genome replication. The RNA chaperone activity is reduced by bile acids and abolished by N-terminal arm deletion, suggesting that the activity requires formation of σNS oligomers. Our studies provide structural and mechanistic insights into the function of σNS in reovirus replication.

The gut protist Tritrichomonas arnold restrains virus-mediated loss of oral tolerance by modulating dietary antigen-presenting dendritic cells.

Loss of oral tolerance (LOT) to gluten, driven by dendritic cell (DC) priming of gluten-specific T helper 1 (Th1) cell immune responses, is a hallmark of celiac disease (CeD) and can be triggered by enteric viral infections. Whether certain commensals can moderate virus-mediated LOT remains elusive. Here, using a mouse model of virus-mediated LOT, we discovered that the gut-colonizing protist Tritrichomonas (T.) arnold promotes oral tolerance and protects against reovirus- and murine norovirus-mediated LOT, independent of the microbiota. Protection was not attributable to antiviral host responses or T. arnold-mediated innate type 2 immunity. Mechanistically, T. arnold directly restrained the proinflammatory program in dietary antigen-presenting DCs, subsequently limiting Th1 and promoting regulatory T cell responses. Finally, analysis of fecal microbiomes showed that T. arnold-related Parabasalid strains are underrepresented in human CeD patients. Altogether, these findings will motivate further exploration of oral-tolerance-promoting protists in CeD and other immune-mediated food sensitivities.

NgR1 binding to reovirus reveals an unusual bivalent interaction and a new viral attachment protein.

Nogo-66 receptor 1 (NgR1) binds a variety of structurally dissimilar ligands in the adult central nervous system to inhibit axon extension. Disruption of ligand binding to NgR1 and subsequent signaling can improve neuron outgrowth, making NgR1 an important therapeutic target for diverse neurological conditions such as spinal crush injuries and Alzheimer's disease. Human NgR1 serves as a receptor for mammalian orthoreovirus (reovirus), but the mechanism of virus-receptor engagement is unknown. To elucidate how NgR1 mediates cell binding and entry of reovirus, we defined the affinity of interaction between virus and receptor, determined the structure of the virus-receptor complex, and identified residues in the receptor required for virus binding and infection. These studies revealed that central NgR1 surfaces form a bridge between two copies of viral capsid protein σ3, establishing that σ3 serves as a receptor ligand for reovirus. This unusual binding interface produces high-avidity interactions between virus and receptor to prime early entry steps. These studies refine models of reovirus cell-attachment and highlight the evolution of viruses to engage multiple receptors using distinct capsid components.

Paired immunoglobulin-like receptor B is an entry receptor for mammalian orthoreovirus.

Mammalian orthoreovirus (reovirus) infects most mammals and is associated with celiac disease in humans. In mice, reovirus infects the intestine and disseminates systemically to cause serotype-specific patterns of disease in the brain. To identify receptors conferring reovirus serotype-dependent neuropathogenesis, we conducted a genome-wide CRISPRa screen and identified paired immunoglobulin-like receptor B (PirB) as a receptor candidate. Ectopic expression of PirB allowed reovirus binding and infection. PirB extracelluar D3D4 region is required for reovirus attachment and infectivity. Reovirus binds to PirB with nM affinity as determined by single molecule force spectroscopy. Efficient reovirus endocytosis requires PirB signaling motifs. In inoculated mice, PirB is required for maximal replication in the brain and full neuropathogenicity of neurotropic serotype 3 (T3) reovirus. In primary cortical neurons, PirB expression contributes to T3 reovirus infectivity. Thus, PirB is an entry receptor for reovirus and contributes to T3 reovirus replication and pathogenesis in the murine brain.

Neural-Cell-Intrinsic NF-κB Signaling Enhances Reovirus Virulence.

Pathological effects of apoptosis associated with viral infections of the central nervous system are an important cause of morbidity and mortality. Reovirus is a neurotropic virus that causes apoptosis in neurons, leading to lethal encephalitis in newborn mice. Reovirus-induced encephalitis is diminished in mice with germ line ablation of NF-κB subunit p50. It is not known whether the proapoptotic function of NF-κB is mediated by neural-cell-intrinsic (neural-intrinsic) processes, NF-κB-regulated cytokine production by inflammatory cells, or a combination of both. To determine the contribution of cell type-specific NF-κB signaling in reovirus-induced neuronal injury, we established mice that lack NF-κB p65 expression in neural cells using the Cre/loxP recombination system. Following intracranial inoculation of reovirus, 50% of wild-type (WT) mice succumbed to infection, whereas more than 90% of mice lacking neural cell NF-κB p65 (Nsp65-/-) survived. While viral loads in brains of WT and Nsp65-/- mice were comparable, histological analysis revealed that reovirus antigen-positive areas in the brains of WT mice displayed increased immunoreactivity for cleaved caspase-3, a marker of apoptosis, relative to Nsp65-/- mice. These data suggest that neural-intrinsic NF-κB-dependent factors are essential mediators of reovirus neurovirulence. RNA sequencing analysis of reovirus-infected brain cortices of WT and Nsp65-/- mice suggests that NF-κB activation in neuronal cells upregulates genes involved in innate immunity, inflammation, and cell death following reovirus infection. A better understanding of the contribution of cell type-specific NF-κB-dependent signaling to viral neuropathogenesis could inform development of new therapeutics that target and protect highly vulnerable cell populations. IMPORTANCE Viral encephalitis contributes to illness and death in children and adults worldwide and has limited treatment options. Identifying common host factors upregulated by neurotropic viruses can enhance an understanding of virus-induced neuropathogenesis and aid in development of therapeutics. Although many neurotropic viruses activate NF-κB during infection, mechanisms by which NF-κB regulates viral neuropathogenesis and contributes to viral encephalitis are not well understood. We established mice in which NF-κB expression is ablated in neural tissue to study the function of NF-κB in reovirus neurovirulence and identify genes activated by NF-κB in response to reovirus infection in the central nervous system. Encephalitis following reovirus infection was dampened in mice lacking neural cell NF-κB. Reovirus induced a chemokine profile in the brain that was dependent on NF-κB signaling and was similar to chemokine profiles elicited by other neurotropic viruses. These data suggest common underlying mechanisms of encephalitis caused by neurotropic viruses and potentially shared therapeutic targets.

NK cells contribute to reovirus-induced IFN responses and loss of tolerance to dietary antigen.

Celiac disease is an immune-mediated intestinal disorder that results from loss of oral tolerance (LOT) to dietary gluten. Reovirus elicits inflammatory Th1 cells and suppresses Treg responses to dietary antigen in a strain-dependent manner. Strain type 1 Lang (T1L) breaks oral tolerance, while strain type 3 Dearing reassortant virus (T3D-RV) does not. We discovered that intestinal infection by T1L in mice leads to the recruitment and activation of NK cells in mesenteric lymph nodes (MLNs) in a type I IFN-dependent manner. Once activated following infection, NK cells produce type II IFN and contribute to IFN-stimulated gene expression in the MLNs, which in turn induces inflammatory DC and T cell responses. Immune depletion of NK cells impairs T1L-induced LOT to newly introduced food antigen. These studies indicate that NK cells modulate the response to dietary antigen in the presence of a viral infection.

Reovirus Nonstructural Protein σNS Recruits Viral RNA to Replication Organelles.

The function of the mammalian orthoreovirus (reovirus) σNS nonstructural protein is enigmatic. σNS is an RNA-binding protein that forms oligomers and enhances the stability of bound RNAs, but the mechanisms by which it contributes to reovirus replication are unknown. To determine the function of σNS-RNA binding in reovirus replication, we engineered σNS mutants deficient in RNA-binding capacity. We found that alanine substitutions of positively charged residues in a predicted RNA-binding domain decrease RNA-dependent oligomerization. To define steps in reovirus replication facilitated by the RNA-binding property of σNS, we established a complementation system in which wild-type or mutant forms of σNS could be tested for the capacity to overcome inhibition of σNS expression. Mutations in σNS that disrupt RNA binding also diminish viral replication and σNS distribution to viral factories. Moreover, viral mRNAs only incorporate into viral factories or factory-like structures (formed following expression of nonstructural protein µNS) when σNS is present and capable of binding RNA. Collectively, these findings indicate that σNS requires positively charged residues in a putative RNA-binding domain to recruit viral mRNAs to sites of viral replication and establish a function for σNS in reovirus replication. IMPORTANCE Viral replication requires the formation of neoorganelles in infected cells to concentrate essential viral and host components. However, for many viruses, it is unclear how these components coalesce into neoorganelles to form factories for viral replication. We discovered that two mammalian reovirus nonstructural proteins act in concert to form functioning viral factories. Reovirus µNS proteins assemble into exclusive factory scaffolds that require reovirus σNS proteins for efficient viral mRNA incorporation. Our results demonstrate a role for σNS in RNA recruitment to reovirus factories and, more broadly, show how a cytoplasmic non-membrane-enclosed factory is formed by an RNA virus. Understanding the mechanisms of viral factory formation will help identify new targets for antiviral therapeutics that disrupt assembly of these structures and inform the use of nonpathogenic viruses for biotechnological applications.

Cytidine Monophosphate N-Acetylneuraminic Acid Synthetase and Solute Carrier Family 35 Member A1 Are Required for Reovirus Binding and Infection.

Engagement of cell surface receptors by viruses is a critical determinant of viral tropism and disease. The reovirus attachment protein σ1 binds sialylated glycans and proteinaceous receptors to mediate infection, but the specific requirements for different cell types are not entirely known. To identify host factors required for reovirus-induced cell death, we conducted a CRISPR-knockout screen targeting over 20,000 genes in murine microglial BV2 cells. Candidate genes required for reovirus to cause cell death were highly enriched for sialic acid synthesis and transport. Two of the top candidates identified, CMP N-acetylneuraminic acid synthetase (Cmas) and solute carrier family 35 member A1 (Slc35a1), promote sialic acid expression on the cell surface. Two reovirus strains that differ in the capacity to bind sialic acid, T3SA+ and T3SA-, were used to evaluate Cmas and Slc35a1 as potential host genes required for reovirus infection. Following CRISPR-Cas9 disruption of either gene, cell surface expression of sialic acid was diminished. These results correlated with decreased binding of strain T3SA+, which is capable of engaging sialic acid. Disruption of either gene did not alter the low-level binding of T3SA-, which does not engage sialic acid. Furthermore, infectivity of T3SA+ was diminished to levels similar to those of T3SA- in cells lacking Cmas and Slc35a1 by CRISPR ablation. However, exogenous expression of Cmas and Slc35a1 into the respective null cells restored sialic acid expression and T3SA+ binding and infectivity. These results demonstrate that Cmas and Slc35a1, which mediate cell surface expression of sialic acid, are required in murine microglial cells for efficient reovirus binding and infection.IMPORTANCE Attachment factors and receptors are important determinants of dissemination and tropism during reovirus-induced disease. In a CRISPR cell survival screen, we discovered two genes, Cmas and Slc35a1, which encode proteins required for sialic acid expression on the cell surface and mediate reovirus infection of microglial cells. This work elucidates host genes that render microglial cells susceptible to reovirus infection and expands current understanding of the receptors on microglial cells that are engaged by reovirus. Such knowledge may lead to new strategies to selectively target microglial cells for oncolytic applications.

Coping with COVID: How a Research Team Learned To Stay Engaged in This Time of Physical Distancing.

Physical distancing imposed by the COVID-19 pandemic has led to alterations in routines and new responsibilities for much of the research community. We provide some tips for how research teams can cope with physical distancing, some of which require a change in how we define productivity. Importantly, we need to maintain and strengthen social connections in this time when we can't be physically together.

Disruption of Type III Interferon (IFN) Genes Ifnl2 and Ifnl3 Recapitulates Loss of the Type III IFN Receptor in the Mucosal Antiviral Response.

Type III interferon (IFN), or IFN lambda (IFN-λ), is an essential component of the innate immune response to mucosal viral infections. In both the intestine and the lung, signaling via the IFN-λ receptor (IFNLR) controls clinically important viral pathogens, including influenza virus, norovirus, and rotavirus. While it is thought that IFN-λ cytokines are the exclusive ligands for signaling through IFNLR, it is not known whether genetic ablation of these cytokines phenotypically recapitulates disruption of the receptor. Here, we report the serendipitous establishment of Ifnl2-/- Ifnl3-/- mice, which lack all known functional murine IFN-λ cytokines. We demonstrate that, like Ifnlr1-/- mice lacking IFNLR signaling, these mice display defective control of murine norovirus, reovirus, and influenza virus and therefore genocopy Ifnlr1-/- mice. Thus, for regulation of viral infections at mucosal sites of both the intestine and lung, signaling via IFNLR can be fully explained by the activity of known cytokines IFN-λ2 and IFN-λ3. Our results confirm the current understanding of ligand-receptor interactions for type III IFN signaling and highlight the importance of this pathway in regulation of mucosal viral pathogens.IMPORTANCE Type III interferons are potent antiviral cytokines important for regulation of viruses that infect at mucosal surfaces. Studies using mice lacking the Ifnlr1 gene encoding the type III interferon receptor have demonstrated that signaling through this receptor is critical for protection against influenza virus, norovirus, and reovirus. Using a genetic approach to disrupt murine type III interferon cytokine genes Ifnl2 and Ifnl3, we found that mice lacking these cytokines fully recapitulate the impaired control of viruses observed in mice lacking Ifnlr1 Our results support the idea of an exclusive role for known type III interferon cytokines in signaling via IFNLR to mediate antiviral effects at mucosal surfaces. These findings emphasize the importance of type III interferons in regulation of a variety of viral pathogens and provide important genetic evidence to support our understanding of the ligand-receptor interactions in this pathway.

Molecular Analyses in a Rabbit Model of Craniosynostosis: Likely Exclusion of Known Candidate Genes as the Loci of Origin.

OBJECTIVE: Craniosynostosis (CS) involves the premature fusion of one or more cranial sutures. We work with a naturally occurring rabbit model of CS with an undefined etiology. Known causes of coronal CS were evaluated to identify potential associations with CS in the rabbit. DESIGN: Candidate genes were sequenced in control New Zealand White (NZW) rabbits (n = 4) and synostotic NZW rabbits (n = 4). Variants were identified by alignment using Clustal Omega. OUTCOME MEASURES: Single nucleotide variants (SNVs) were classified according to phenotypic associations and predicted impact on protein structure. Human correlates were identified in the database of single nucleotide polymorphisms (dbSNP). RESULTS: A total of 21 SNVs were identified in the 10 genes examined. Variant classification and inheritance patterns are inconsistent with causality. CONCLUSIONS: The genetic basis for disease in the CS rabbit likely involves novel loci and is not associated with known causes of coronal synostosis.

Molecular Analysis of Gli3, Ihh, Rab23, and Jag1 in a Rabbit Model of Craniosynostosis: Likely Exclusion as the Loci of Origin.

OBJECTIVE: Craniosynostosis (CS) involves the premature fusion of one or more cranial sutures. The etiology of CS is complex and mutations in more than 50 distinct genes have been causally linked to the disorder. Many of the genes that have been associated with CS in humans play an essential role in tissue patterning and early craniofacial development. Among these genes are members of the Hedgehog (HH) and Notch signal transduction pathways, including the GLI family member Gli3, Indian Hedgehog ( Ihh), the RAS oncogene family member Rab23, and the Notch ligand JAGGED1 ( Jag1). We have previously described a colony of rabbits with a heritable pattern of coronal suture synostosis, although the genetic basis for synostosis within this model remains unknown. The present study was performed to determine if coding errors in Gli3, Ihh, Rab23, or Jag1 could be causally linked to craniosynostosis in this unique animal model. DESIGN: Sequencing of cDNA templates was performed using samples obtained from wild-type and craniosynostotic rabbits. RESULTS: Several nucleotide polymorphisms were identified in Gli3, Ihh, and Rab23, although these variants failed to segregate by phenotype. No nucleotide polymorphisms were identified in Jag1. CONCLUSIONS: These data indicate that the causal locus for heritable craniosynostosis in this rabbit model is not located within the protein coding regions of Gli3, Ihh, Rab23, or Jag1.

Molecular Analysis of Ephrin A4 and Ephrin B1 in a Rabbit Model of Craniosynostosis: Likely Exclusion as the Loci of Origin.

Craniosynostosis (CS) has a prevalence of approximately 1 in every 2000 live births and is characterized by the premature fusion of one or more cranial sutures. Failure to maintain the cell lineage boundary at the coronal suture is thought to be involved in the pathology of some forms of CS. The Ephrin family of receptor tyrosine kinases consists of membrane-bound receptors and ligands that control cell patterning and the formation of developmental boundaries. Mutations in the ephrin A4 (EFNA4) and ephrin B1 (EFNB1) ligands have been linked to nonsyndromic CS and craniofrontonasal syndrome, respectively, in patient samples. We have previously described a colony of rabbits with a heritable pattern of coronal suture synostosis, although the genetic basis for synostosis within this model remains unknown. The present study was performed to determine if EFNA4 or EFNB1 could be the loci of the causal mutation in this unique animal model. Sequencing of EFNA4 and EFNB1 was performed using templates obtained from wild-type (n = 4) and craniosynostotic (n = 4) rabbits. No structural coding errors were identified in either gene. A single-nucleotide transversion was identified in one wild-type rabbit within the third intron of EFNA4. These data indicate that the causal locus for heritable CS in this rabbit model is not located within the structural coding regions of either EFNA4 or EFNB1.

TLR4 Inactivation in Myeloid Cells Accelerates Bone Healing of a Calvarial Defect Model in Mice.

BACKGROUND: Toll-like receptor 4 (TLR4) has been implicated in inflammation-induced bone destruction in various chronic bone diseases; however, its direct influence on bone healing is not well understood. The authors' previous study showed accelerated bone healing with higher osteoclastogenesis gene expression in toll-like receptor 4 knockout mice (TLR4). This study aimed to further elucidate the underlying cellular mechanisms during fracture healing by generating a myeloid cell-specific toll-like receptor 4 knockout model (Lyz-TLR4 mice). METHODS: Calvarial defects, 1.8 mm in diameter, were created in wild-type, TLR4, and Lyz-TLR4 mice. Bone healing was investigated using micro-computed tomography and histologic, histomorphometric, and immunohistochemistry analyses. Primary bone marrow-derived cells were also isolated from wild-type, TLR4, and Lyz-TLR4 mice to measure their osteoclast differentiation and resorption properties. RESULTS: A similar faster bone healing response, with active intramembranous bone formation, intense osteopontin staining, and more osteoblast infiltration, was observed in TLR4 and Lyz-TLR4 mice. Tartrate-resistant acid phosphatase staining showed more osteoclast infiltration in Lyz-TLR4 mice than in wild-type mice at day 7. Primary bone marrow-derived cells isolated from TLR4 and Lyz-TLR4 mice presented enhanced osteoclastogenesis and resorption activity compared with those from wild-type mice. Comparable M0, M1, and M2 macrophage infiltration was found among all groups at days 1, 4, and 7. CONCLUSIONS: This study revealed that inactivation of toll-like receptor 4 in myeloid cells enhanced osteoclastogenesis and accelerated healing response during skull repair. Together with the role of toll-like receptor 4 in inflammation-mediated bone destruction, it suggests that toll-like receptor 4 might regulate inflammation-induced osteoclastogenesis under different clinical settings.

Enhanced Calvarial Bone Healing in CD11c-TLR4-/- and MyD88-/- Mice.

BACKGROUND: Inflammation is integral to the injury response. The inflammatory response is essential to the host defense against infection and also to tissue regeneration and repair. Toll-like receptors (TLRs) are critical activators of the innate immune response and present attractive therapeutic targets for inflammation-modulated tissue regeneration. The authors' previous study showed that depletion of TLR4 resulted in accelerated skull bone healing concurrent with increased expression of osteoclastogenic genes. As such, in the present study, the authors used various knockout mouse models for TLR4 and its associated signaling mediators as tools to further understand the role of Toll-like receptor-mediated inflammation in calvarial bone healing. METHODS: Calvarial defects (1.8-mm diameter) were created in wild-type, TLR4 knockout (TLR4), TLR2, MyD88, TRIF, TLR4 knockout in myeloid cell (Lyz-TLR4), and TLR4 knockout in dendritic-lineage cell (CD11c-TLR4) mice. Bone healing was examined using micro-computed tomographic, histologic, and histomorphometric analyses. RESULTS: Micro-computed tomographic and histomorphometric analyses revealed that TLR4-deficient mice (TLR4, Lyz-TLR4, and CD11c-TLR4) exhibited a faster intramembraneous healing response at postoperative day 7, whereas MyD88 and CD11c-TLR4 mice showed enhanced bone healing at day 28. CONCLUSIONS: The authors' data suggest a detrimental role for TLR4 in CD11c cells, mediated by Myd88 signaling, during calvarial bone healing. The authors have demonstrated that Toll-like receptor signaling components affect calvarial bone healing, establishing a link between the skeletal and immune systems during craniofacial bone healing. Toll-like receptor signaling components might be used to initiate enhanced healing in bone defects to improve clinical outcomes.

Genome-wide RNAi screen identifies novel host proteins required for alphavirus entry.

The enveloped alphaviruses include important and emerging human pathogens such as Chikungunya virus and Eastern equine encephalitis virus. Alphaviruses enter cells by clathrin-mediated endocytosis, and exit by budding from the plasma membrane. While there has been considerable progress in defining the structure and function of the viral proteins, relatively little is known about the host factors involved in alphavirus infection. We used a genome-wide siRNA screen to identify host factors that promote or inhibit alphavirus infection in human cells. Fuzzy homologue (FUZ), a protein with reported roles in planar cell polarity and cilia biogenesis, was required for the clathrin-dependent internalization of both alphaviruses and the classical endocytic ligand transferrin. The tetraspanin membrane protein TSPAN9 was critical for the efficient fusion of low pH-triggered virus with the endosome membrane. FUZ and TSPAN9 were broadly required for infection by the alphaviruses Sindbis virus, Semliki Forest virus, and Chikungunya virus, but were not required by the structurally-related flavivirus Dengue virus. Our results highlight the unanticipated functions of FUZ and TSPAN9 in distinct steps of alphavirus entry and suggest novel host proteins that may serve as targets for antiviral therapy.

NMR relaxation studies of an RNA-binding segment of the rous sarcoma virus gag polyprotein in free and bound states: a model for autoinhibition of assembly.

Assembly of retrovirus particles is promoted by interaction of the Gag polyprotein with RNA. Nonspecific RNA association with the nucleocapsid domain (NC) of Gag induces the dimerization of Gag through protein-protein contacts in the capsid domain (CA), followed by higher order assembly to form the immature virus particle. NMR relaxation studies were conducted to investigate the initial steps of Rous sarcoma virus (RSV) assembly by examining the association with nucleic acid of a fragment of Gag comprising the C-terminal domain of CA (CTD) postulated to mediate Gag dimerization, the spacer region between CA and NC (SP), and NC. This fragment, CTD-SP-NC (residues 394-577), spans the critical SP region and allows assessment of this key Gag-nucleic acid interaction in the context of the Gag polyprotein rather than the isolated domains. Main-chain amide relaxation of CTD-SP-NC was measured in the absence and presence of (GT)(4), an 8-mer DNA oligonucleotide that binds tightly to the polyprotein but is too short to promote Gag dimerization. The results show that the CTD and NC domains tumble independently. In contrast, the two zinc finger domains within NC are rotationally coupled in both the unbound and bound states, even though only the first zinc finger appears to make direct contact with (GT)(4). In addition, the NMR data indicate that SP and flanking residues undergo a conformational exchange process that is slowed in the presence of (GT)(4). This region around SP where relaxation is strongly affected by (GT)(4) binding is nearly identical to the assembly domain defined previously by mutagenesis studies. Other changes in relaxation induced by (GT)(4) implicate conformational perturbations of helices 1 and 4 in CTD. On the basis of the combined data, we propose a model for the promotion of Gag dimerization by RNA association in which NC-RNA binding disrupts an assembly inhibitory, intramolecular interaction involving SP and CTD. Disruption of this intramolecular interaction is proposed to enhance the accessibility of the Gag dimer contact surface and release the assembly domain to promote intermolecular oligomerization.

Ubiquitin depletion and dominant-negative VPS4 inhibit rhabdovirus budding without affecting alphavirus budding.

The budding reactions of a number of enveloped viruses use the cellular machinery involved in the formation of the luminal vesicles of endosomal multivesicular bodies (MVB). Budding of these viruses is dependent on the presence of specific late-domain motifs in membrane-associated viral proteins. Such budding reactions usually involve ubiquitin and are blocked by expression of an ATPase-deficient form of VPS4, a cellular AAA+ ATPase believed to be required late in the MVB pathway for the disassembly/release of the MVB machinery. Here we examined the role of the MVB pathway in the budding of the late-domain-containing rhabdovirus vesicular stomatitis virus (VSV) and the alphavirus Semliki Forest virus (SFV). We tested early and late steps in the MVB pathway by depleting ubiquitin with the proteasome inhibitor MG-132 and by using cell lines inducibly expressing VPS4A or VPS4B protein. As previously shown, VSV budding was strongly dependent on ubiquitin. In contrast to the findings of previous studies with VPS4A, expression of ATPase-deficient mutants of either VPS4A or VPS4B inhibited VSV budding. Inhibition by VPS4 required the presence of the PPPY late domain on the VSV matrix protein and resulted in the accumulation of nonreleased VSV particles at the plasma membrane. In contrast, SFV budding was independent of both ubiquitin and the activity of VPS4, perhaps reflecting the important role of the highly organized envelope protein lattice during alphavirus budding.

Promotion of retroviral entry in the absence of envelope protein by chlorpromazine.

Retrovirus packaging cell lines that express the Moloney murine leukemia virus gag, pol, and env genes and a retroviral vector genome can produce virus particles that are capable of transducing cells. Normally if the packaging cell line does not produce a functional viral fusion glycoprotein, such as the retroviral envelope protein or a foreign viral glycoprotein, then the viruses will be incapable of transducing cells. We have found that incubating envelope protein-deficient virus particles bound to cells with chlorpromazine leads to transduction. Chlorpromazine (CPZ) is a membrane-active reagent that is commonly used to induce the hemifusion to fusion transition when membrane fusion is mediated by partially defective viral glycoproteins. The concentration and pH dependence of the promotion of transduction by CPZ is consistent with a role for CPZ micelle formation in viral entry. These data indicate that caution is warranted when experiments concerning membrane fusion completion promoted by CPZ are analyzed.