Journal of Virology

Fusion-associated small transmembrane (FAST) proteins are viral nonstructural proteins that mediate cell-cell fusion to form multinucleated syncytia. We previously reported that human species B rotavirus NSP1-1 is a FAST protein that induces syncytia in primate epithelial cells but not rodent fibroblasts. We hypothesized that the NSP1-1 proteins of other rotavirus species could also mediate cell-cell fusion and that fusion activity might be limited to cell types derived from homologous hosts. To test this hypothesis, we predicted the structure and domain organization of NSP1-1 proteins of species B rotavirus from a human, goat, and pig, species G rotavirus from a pigeon and turkey, and species I rotavirus from a dog and cat. We cloned these sequences into plasmids and transiently expressed the NSP1-1 proteins in avian, canine, hamster, human, porcine, and simian cells. Regardless of host origin of the virus, each NSP1-1 protein induced syncytia in primate cells, while few induced syncytia in other cell types. To identify the domains that determined cell-specific fusion activity for human species B rotavirus NSP1-1, we engineered chimeric proteins containing domain exchanges with the p10 FAST protein from Nelson Bay orthoreovirus. Using the chimeric proteins, we found that the N-terminal and transmembrane domains determined the cell type specificity of fusion activity. Although the species and cell type criteria for fusion activity remain unclear, these findings suggest that rotavirus species B, G, and I NSP1-1 are functional FAST proteins whose N termini play a role in specifying the cells in which they mediate syncytia formation. IMPORTANCEMechanisms of membrane fusion and determinants of host range for pathogens remain poorly understood. Improved understanding of these concepts could open new areas for therapeutic development and shed light on virus epidemiology. Our analyses of NSP1-1 proteins from species B, G, and I rotaviruses provide insights into the diversity of domain features tolerated by functional FAST proteins. Further, the observation that all putative FAST proteins tested can induce syncytia formation in at least some cell types provides evidence that rotaviruses that encode NSP1-1 proteins are fusogenic viruses. Finally, although the criteria for their specificity remain unclear, our observations regarding fusion capacities of different NSP1-1 proteins and of chimeric FAST proteins suggest a potential role for rotavirus FAST proteins in determining the efficiency of viral replication within a given host or cell type.

Viral myocarditis is a major cause of heart damage, sudden death, and heart failure. Some strains of Mammalian Orthoreoviruses (REOV) cause myocarditis in newborn mice. This study examines the role of the REOV {sigma}3 protein, encoded by the S4 gene, in modulating the virulence and myocarditic potential of a highly virulent T1L/T3DM2 reassortant virus that contains the M2 gene from the Type 3 Dearing (T3D) strain in the Type 1 Lang background. We introduced single-point mutations in the double-stranded RNA-binding region of {sigma}3 in the T1L/T3DM2 background. Our findings show that the K287T mutation in {sigma}3 prevents the myocarditic phenotype and significantly reduces the virulence of T1L/T3DM2. Unlike the parental reassortant virus and a control reassortant mutant S4(R296T), infection of neonatal mice with the T1L-S4(K287T)/T3DM2 virus resulted in 100% survival, lower viral titers, particularly in the heart and spleen, and no gross or severe histological signs of myocarditis. This attenuation, despite similar in vitro growth and in vivo dissemination, indicates a tissue-specific replication deficit and highlights a key role for {sigma}3 in the development of myocardial disease. The K287T mutation, unlike R296T, eliminates {sigma}3s capacity to inhibit protein kinase R (PKR) activation and to suppress NF-{kappa}B-driven transcription, leading to a strong innate immune response that likely controls viral replication and reduces cardiac damage. These results underscore the crucial role of the {sigma}3 protein in modulating host innate immune responses and in influencing the outcome of REOV infection and myocarditis development. IMPORTANCEViral myocarditis is a serious disease with life-threatening consequences, particularly in neonates and young animals. Infection of neonatal mice with certain strains of mammalian reovirus induces myocarditis. A reassortant virus containing the M2 gene from the Type 3 Dearing Strain in an otherwise Type 1 Lang background causes severe myocarditis in 100% of neonatal mice. Here, we show that a single point mutation in the S4 gene, which abolishes the capacity of the encoded {sigma}3 protein to inhibit protein kinase R (PKR) and to suppress transcription of NF-{kappa}B-dependent genes, significantly attenuates the lethality of the T1L/T3DM2 reassortant and significantly reduces the severity of myocarditis. These findings highlight the importance of viral innate immune suppression in the induction of myocarditis.

Herpes simplex virus 2 (HSV-2) establishes latent infection in dorsal root ganglion (DRG) neurons after productive (lytic) infection in peripheral tissues. A neuron-specific microRNA, miR-138, favors HSV-1 latency by repressing viral ICP0, and host Oct-1 and Foxc1 genes, yet the role of miR-138 in HSV-2 infection was unknown. The ICP0 mRNAs of HSV-1, HSV-2 and chimpanzee herpesvirus each have one to two canonical miR-138 binding sites. The sites are 100% conserved in 308 HSV-1 and 300 HSV-2 published sequences of clinical isolates. In co-transfection assays, miR-138 repressed HSV-2 ICP0 expression through the seed region and surrounding interactions that are different from HSV-1. An HSV-2 mutant with disrupted miR-138 binding sites on ICP0 showed increased ICP0 expression in Neuro-2a cells. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation confirmed miR-138 binding to HSV-2 ICP0, and identified UL19 and UL20 as additional targets, whose expression was repressed by miR-138 during co-transfection. In Neuro-2a cells, transfected miR-138 and its antagomir decreased and increased HSV-2 replication, respectively, and knockout experiment showed that miR-138s host targets OCT-1 and FOXC1 were important for HSV-2 replication. In primary mouse DRG neurons, both ICP0 and FOXC1 positively regulated HSV-2 replication, but both overexpressed and endogenous miR-138 suppressed HSV-2 replication primarily by repressing ICP0 expression. Thus, miR-138 can suppress HSV-2 neuronal replication through multiple viral and host pathways. These results reveal functional similarities and mechanistic differences in how miR-138 regulates HSV-1 and HSV-2 infection and indicate an evolutionary advantage of using miR-138 to repress lytic infection in neurons. ImportanceHerpes simplex virus 1 (HSV-1) and HSV-2 are closely related viruses with major differences. Both viruses establish latency in neurons from which they reactivate to cause disease. A key aspect of HSV latency is repression of productive infection in neurons. Based on previous work with HSV-1, we investigated the role of a neuron-specific microRNA, miR-138, in HSV-2 infection, and established it as a repressor of HSV-2 productive infection in neuronal cells. This repression is mediated mainly by targeting viral ICP0 and host Foxc1 mRNAs, but other pathways also contribute. Despite functional conservation of the role of miR-138 between HSV-1 and HSV-2, many molecular mechanisms differ including how miR-138 represses ICP0 expression and miR-138 targeting of HSV-2 but not HSV-1 UL19 and UL20. To our knowledge, this study provides the first example of host microRNA regulation of HSV-2 infection.

Antibodies inhibit human immunodeficiency virus type 1 (HIV-1) infection by targeting the envelope glycoprotein (Env). Env cleavage is a key determinant of antibody binding, as cleavage reduces Env flexibility and alters glycosylation. Given the greater abundance of uncleaved Env on the cell surface compared to virions, we investigated whether Env endocytosis, initiated through a membrane-proximal tyrosine motif in its cytoplasmic tail, regulates the abundance of cleaved Env at the cell surface. We hypothesize that such a shift would alter the cleavage and glycosylation profile of cell surface Env, affecting the sensitivity of cell-to-cell infection to neutralizing antibodies. To address this, we generated an endocytic mutant (ASPI-Env) and compared its antigenic properties to a cleavage-site mutant (SEKS-Env). Immunoprecipitation and ratiometric antibody binding studies of cell surface Env demonstrated that the ASPI mutation increases the amount of uncleaved Env on the cell surface. Consequently, ASPI-Env in cell-free infection was more sensitive to NAbs recognizing uncleaved Env. Notably, only b12, which can engage both cleaved and uncleaved Env but has a higher affinity for uncleaved Env, showed increased inhibition of ASPI-Env during cell-to-cell infection. This mirrors the enhanced neutralization of SEKS-Env during the cell-to-cell transfer assay and supports a model in which uncleaved Env participates in CD4-dependent virion transfer. Finally, the ASPI mutation altered lectin binding and differentially affected cell-to-cell and cell-free infections. Together, these findings indicate that Env endocytosis modulates the abundance of cleaved Env at the cell surface, thereby influencing antibody neutralization and lectin recognition in distinct modes of HIV-1 transmission. IMPORTANCEWe find that Env endocytosis modulates the antigenicity of Env on both the cell surface and virions. Blocking internalization increased uncleaved Env on the cell surface, thereby reshaping HIV-1 neutralization due to enhanced binding of antibodies that preferentially recognize uncleaved Env. We further show that uncleaved Env on the cell surface can initiate CD4-dependent transfer of virions across virological synapses. These findings demonstrate that Env endocytosis shapes cleavage-associated antigenicity in a manner that can differentially impact the recognition and neutralization of cells and viruses. The impact of uncleaved Env on neutralization indicates that a broader spectrum of cleaved and uncleaved Env conformations may be relevant when designing vaccines and cure strategies that must contend with a diverse antigenic landscape.

Equid alphaherpesvirus 1 (EHV1) is a DNA virus that causes severe disease outcomes in equids. Some EHV1 strains are neurotropic and cause disease in the central nervous system, whereas others are non-neurotropic and can cause negative reproductive outcomes. The molecular mechanisms that govern pathotype of individual EHV1 strains are not understood. However, EHV1 replication in the presence of epigenetic inhibitors suggests that neurotropic and non-neurotropic EHV1 are differentially susceptible to epigenetic silencing. Aside from this evidence, little is known about EHV1 chromatin or its regulation. Here, we used fluorescence recovery after photobleaching to characterize EHV1 lytic chromatin dynamics. Infection with neurotropic or non-neurotropic EHV1 mobilized all histones. Canonical (H2A, H2B, H3.1, H4) or variant (H2A.B, H2A.Z, H2A.X, macroH2A, H3.3) core and linker H1.2 histones were equally mobilized by either strain. Thus, there were no vast differences in histone mobility during neurotropic or non-neurotropic EHV1 infection. All histones except for H2A.B were more mobile within EHV1 replication compartments (RCs) than the surrounding infected-cell chromatin. The differential mobility of histones within domains enriched for viral or cellular chromatin is consistent with distinct mechanisms to assemble and regulate the chromatin associated with viral or host DNA. Histones were further mobilized within RCs in cells in which infection had further progressed. Such mobilization indicates that increased levels of EHV1 transcription, DNA replication, or protein expression directly or indirectly mobilize histones. The high histone mobility within EHV1 RCs is consistent with assembly of EHV1 genomes in very dynamic and unstable nucleosomes. These data support a model in which EHV1 limits genome silencing by preventing stable chromatin assembly, or destabilizing the chromatin assembled, with viral genomes during lytic infection. We propose that manipulation of histone dynamics represents a novel mechanism of epigenetic regulation adopted by alphaherpesviruses to maintain genome accessibility and prevent gene silencing. Author summaryDNA viruses are subjected to epigenetic regulation that silences or promotes gene expression. Multiple epigenetic mechanisms contribute to stabilize chromatin to silence gene expression or destabilize it to promote gene expression. Knowledge of the mechanisms whereby viruses prevent or overcome genome silencing and promote expression of their genes is important to understand how viruses, including alphaherpesviruses, take over the host cell to establish productive infection. Here we show that EHV1 broadly mobilizes histones within nuclear domains enriched in viral chromatin. Histone mobilization destabilizes chromatin and is consistent with the assembly of EHV1 genomes in dynamic, unstable nucleosomes. The manipulation of histone mobility is a phenomenon first described for the alphaherpesvirus herpes simplex virus 1 (HSV1). The conserved approach to dysregulate chromatin dynamics and mobilize histones represents a unique means whereby herpesviruses destabilize chromatin. Understanding the mechanisms that mobilize histones during infection will increase our general understanding of epigenetic regulation, which is important in the pathogenesis of infectious diseases and also of developmental or genetic ones. Moreover, knowledge of the processes whereby herpesviruses destabilize chromatin will support the development of novel therapeutics to maintain viral genomes in stable, silenced chromatin to prevent productive infection and development of associated diseases.

The mature human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer, which consists of non-covalently associated gp120 exterior and gp41 transmembrane subunits, mediates virus entry into cells. The pretriggered (State-1) Env conformation is the major target for broadly neutralizing antibodies (bNAbs), whereas receptor-induced downstream Env conformations elicit immunodominant, poorly neutralizing antibody (pNAb) responses. To examine the contribution of membrane anchorage to the maintenance of the metastable pretriggered Env conformation, we compared wild-type and State-1-stabilized Envs solubilized in detergents or in styrene-maleic acid (SMA) copolymers. SMA directly incorporates membrane lipids and resident membrane proteins into lipid nanodiscs (SMALPs). The integrity of the Env trimer in SMALPs was maintained at both 4{degrees}C and room temperature. By contrast, Envs solubilized in Cymal-5, a non-ionic detergent, were unstable at room temperature, although their stability was improved at 4{degrees}C and after incubation with the entry inhibitor BMS-806. Envs solubilized in ionic detergents were relatively unstable at either temperature. Comparison of Envs solubilized in Cymal-5 and SMA at 4{degrees}C revealed subtle differences in bNAb binding to the gp41 membrane-proximal external region (MPER), consistent with these distinct modes of Env solubilization. Otherwise, the antigenicity of the Cymal-5- and SMA- solubilized Envs was remarkably similar, both in the absence and presence of BMS-806. However, both solubilized Envs were recognized differently from the mature membrane Env by specific bNAbs and pNAbs. Thus, detergent-based and detergent-free solubilization at 4{degrees}C alters the pretriggered membrane Env conformation in consistent ways, indicating that loss of Env association with the membrane results in default state(s). IMPORTANCEThe human immunodeficiency virus (HIV-1) envelope glycoproteins (Envs) in the viral membrane mediate virus entry into the host cell and are targeted by neutralizing antibodies elicited by natural infection or vaccines. Detailed studies of membrane proteins rely on purification procedures that allow the proteins to maintain their natural conformation. In this study, we show that a styrene-maleic acid (SMA) copolymer can extract HIV-1 Env from a membrane without the use of detergents. The Env in SMA is more stable at room temperature than Env in detergents. The purified Env in SMA maintains many but not all of the characteristics expected of the natural membrane Env. Our results underscore the importance of the membrane environment to the native conformation of HIV-1 Env. Purification methods that bypass the need for detergents could be useful tools for future studies of HIV-1 Env structure and its interaction with receptors and antibodies.

The reovirus {sigma}1 attachment protein mediates virus interaction with host cell receptors that is critical for cell entry. Reovirus tropism is controlled by properties of {sigma}1. {sigma}1 is present as trimers that are held within turrets at the icosahedral vertices of reovirus virions. However, because {sigma}1 has not been visualized on reovirus virions in high resolution structures and because the fulllength structure of purified {sigma}1 protein has not been solved, it is not clear how {sigma}1 is presented on virions. What properties of {sigma}1 are essential for its incorporation on virions is also not known. In this study, we used ColabFold to model the structure of reovirus serotype 1 (T1) and serotype 3 (T3) {sigma}1 proteins. We find that these proteins fold into similar structures with regions of flexibility between the head and body domains of {sigma}1. We also predicted the structures of chimeric {sigma}1 proteins comprised of domain swaps between T1 and T3 {sigma}1 proteins. Our analyses indicate that chimeric proteins with mismatched body and head domain have increased flexibility in this region. Characterization of particles expressing such chimeric {sigma}1 proteins demonstrated that deviation from the flexibility of parental {sigma}1 leads to a reduction in {sigma}1 incorporation on to the virion. Further, we find that even when incorporation is not affected, virus attachment to host cell receptors is influenced by altered {sigma}1 flexibility. Finally, our work demonstrates that 1 protein impacts the encapsidation pattern and receptor engagement properties of {sigma}1 and that this effect is influenced by properties of the N-terminal portion of {sigma}1. ImportanceAttachment to host cell receptors is a critical step in initiation of virus infection. Some viruses attach to cellular receptors via dedicated viral proteins. Both the number of attachment factors present on the virus and whether they are present on the virus particle in the correct form can influence cell attachment. Here, using reovirus as a model, we use a protein structure prediction algorithm to model the as yet unknown structure of full-length reovirus attachment protein {sigma}1. We find predicted regions of flexibility in the protein and identify how this flexibility is regulated. We find that the flexibility of {sigma}1 independently regulates whether it is stably incorporated into particles and if can efficiently interact with host receptors.

The envelope glycoprotein (Env) trimer on the surface of human immunodeficiency virus type I (HIV-1) mediates viral entry into host CD4+ T cells and is the sole target of neutralizing antibodies. Broadly neutralizing antibodies (bnAbs) that target gp120 V3-glycan of HIV-1 Env trimer are potent and block the entry of diverse HIV-1 strains. Most V3-glycan bnAbs interact, to a different extent, with a glycan attached to N332 but Asn at this position is not absolutely conserved or required for HIV-1 entry based on prevalence of N332 in different circulating HIV-1 strains from diverse clades. Here, we studied the effects of amino acid changes at position 332 of HIV-1AD8 Envs on HIV-1 sensitivity to antibodies, cold exposure, and soluble CD4. We further investigated how these changes affect Env function and HIV-1 infectivity in vitro. Our results suggest robust tolerability of HIV-1 AD8 Env N332 to changes with specific changes that resulted in extended exposure of gp120 V3 loop, which is typically concealed in most primary HIV-1 isolates. Viral evolution leading to Asn at position 332 of HIVAD8 Envs is supported by the selection advantage of high levels of cell-cell fusion, transmission, and infectivity even though cell surface expression levels are lower than most N332 variants. Thus, tolerance of HIV-1AD8 Envs to different amino acids at position 332 provides increased flexibility to respond to changing conditions/environments and to evade the immune system. Modeling studies of the distance between N332 glycan and specific bnAbs was in agreement with N332 glycan dependency on bnAb neutralization. Overall, our studies provide insights into the contribution of specific amino acids at position 332 to Env antigenicity, stability on ice, and conformational states.

Reovirus strains differ in their capacity to modulate host antiviral signaling, but the viral determinants underlying these differences have remained unclear. In this study, we identify the outer capsid protein {sigma}3 as the viral determinant responsible for inducing the loss of the NF-{kappa}B kinase, IKK{beta}. We demonstrate that introduction of the {sigma}3 encoding gene segment of strain T3A in the background of strain T3DL recapitulates IKK loss observed during T3A infection. Our results indicate that de novo expression of T3A {sigma}3 during infection is necessary to mediate the loss of IKK. Further, the presence of T3A {sigma}3 alone is sufficient. We show that T3A {sigma}3 interacts with the IKK complex and targets IKK{beta} for degradation through a Cullin-RING ligase-proteasome dependent mechanism. Loss of IKK{beta} results in impaired NF-{kappa}B activation and reduced IFN-{beta} expression. Consequently, the T3DL/T3A S4 monoreassortant exhibits a replication advantage compared to T3DL. Collectively, these findings uncover a previously uncharacterized mechanism by which reovirus {sigma}3 suppresses innate antiviral signaling through targeted degradation of IKK{beta}, providing new insight into reovirus mediated immune modulation. IMPORTANCESuccessful viral infection and replication requires that the virus suppress host immune defenses, particularly innate immunity, which relies on signaling cascades to produce antiviral cytokines. Many viruses encode proteins to target key signaling molecules, such as those in the NF-{kappa}B pathway, but the exact mechanisms of evasion vary across viral families and even between closely related strains. In this study, we uncover a novel function of the reovirus outer capsid protein {sigma}3 to promote IKK{beta} degradation. Our findings reveal that {sigma}3 associates with a complex of proteins that includes IKK{beta}, a central kinase in the NF-{kappa}B pathway, to mediate its degradation via the proteasomal pathway. {sigma}3 mediated IKK{beta} loss decreases type I IFN expression during infection and enhances viral replication. Coupled with previous observations that {sigma}3 from other reovirus strains antagonizes NF-{kappa}B by altering its transcriptional activity, this work demonstrates that this conserved viral protein adopts strain-specific strategies to subvert host defenses.

Viruses use miRNAs to enable efficient replication, control host defense mechanisms, and regulate latent infection. Herpes simplex virus 1 (HSV-1) expresses multiple miRNAs, whose functions are largely unknown. The evolutionary conservation of many HSV-1 miRNAs in the closely related HSV-2 suggests their functional importance. miRNAs, similar to other transcripts, can undergo various posttranscriptional modifications that may affect their biogenesis, stability and targeting. To investigate whether editing occurs in HSV-1 miRNAs, we sequenced samples from latently infected human ganglia. We show that one of the six HSV-1 miRNAs (miR-H2 to -H8) that define HSV-1 latency, miR-H2, exhibits A-to-I hyperediting within the miRNA seed sequence. We observed the same specific miR-H2 hyperediting phenomenon in miRNAs isolated from the ganglia of latently infected mice and, to a lesser extent, during productive infection in cultured cells. Curiously, we found no evidence of editing of the encoded HSV-2 homolog in latently infected mice or in cultured cells. The efficient loading of the edited miRNAs onto the RISC complex, indicates their ability to function as miRNAs. To investigate the potential of the edited miRNA to alter mRNA targeting, we predicted the host and viral targets for the modified miRNAs. Nucleotide substitution in the seed region significantly increased the number of potential host and viral targets. Most notably, ICP4, an essential viral protein, was predicted to be an additional target. Using transfection assays, we demonstrated that edited miRNAs have the potential to regulate ICP4 in addition to the previously identified target ICP0. Our study identifies a specific hyperedited HSV-1 mRNA, miR-H2, and highlights how the virus can use a single miRNA to target multiple transcripts during persistent, latent infection. ImportanceHerpes simplex virus 1 is an important human pathogen and intensively studied for many decades. Nevertheless, the molecular mechanisms regulating its establishment, maintenance, and reactivation from latency are poorly understood. Here, we show that HSV- 1 encoded miR-H2 is post-transcriptionally edited in latently infected human tissues. Hyperediting of viral miRNAs increases the targeting potential of these miRNAs and may play an important role in regulating latency. We show that the edited miR-H2 (miR-H2-e) can target ICP4, an essential viral protein. Interestingly, we found no evidence of hyperdating of its homolog, miR-H2, which is expressed by the closely related virus HSV-2. The discovery of posttranslational modifications of viral miRNA in the latency phase suggests that these processes may also be important for other non-coding viral RNA in the latency phase, including the intron LAT, which in turn may be crucial for understanding the biology of this virus.

New methods for deep sequence analysis provide an opportunity to follow the emergence and dynamics of virus mutations in real time. Although viruses are commonly grown in cell culture for research and for vaccine development, the cells used to grow the virus are often not derived from the same tissue or even the same host that the virus naturally replicates in. The selective pressures of culturing virus in vitro are still only partially understood. MDCK cells are the standard cell for growing influenza viruses yet are derived from the epithelium of the canine kidney and are also heterogenous. We passaged human H3N2, H1N1 pandemic, and canine H3N2 influenza A viruses (IAV) in different lineages of MDCK cells, as well as lines engineered to express variant Sia receptors, including 2,3- and 2,6-linkages or N-glycolylneuraminic acid (Neu5Gc) or N-acetylneuraminic acid (Neu5Ac) forms. MDCK-Type II cells had lower infection efficiency and virus production, and infection appeared more dependent on protease activation of the virus. When viruses were passaged in the different cells, they exhibited only small numbers of consensus-level mutations, and most were within the HA gene. Both human IAVs showed selection for single nucleotide minority variants in the HA stem across cell types, as well as low frequency variants in the HA receptor binding site of virus passaged in cells expressing Neu5Gc. Canine H3N2 also showed minority variants near the receptor-binding site in cells expressing Neu5Gc and also in those expressing 2,6-linkages. IMPORTANCEThe genetic variation and adaptability of viruses are fundamental properties that allow their evolutionary success in the face of differing host environments and immune responses. The growth of viruses in cell culture is widely used for their study and for preparing vaccines. However, the selection pressures that cell passaging imposes on viruses are often poorly understood. We used deep sequence analysis to define, in detail, how three different influenza A viruses respond to passaging in different lineages of canine MDCK cells that are commonly used for their growth, as well as in variant cells engineered to express different forms of their cell surface receptor, sialic acid. This analysis revealed that most mutations occur in the HA gene and few sequence changes in the virus population reached high proportions. This is relevant for understanding the selective pressures of virus growth in cell culture and how it shapes evolutionary patterns.

Binding to the receptor, CD4, drives the pretriggered, "closed" (State-1) conformation of the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer into more "open" conformations (States 2 and 3). Broadly neutralizing antibodies, which are elicited inefficiently, mostly recognize the State-1 Env conformation, whereas the more commonly elicited poorly neutralizing antibodies recognize States 2/3. HIV-1 Env metastability has created challenges for defining the State-1 structure and developing immunogens mimicking this labile conformation. The availability of functional State-1 Envs that can be efficiently crosslinked at lysine and/or acidic amino acid residues might assist these endeavors. To that end, we modified HIV-1AD8 Env, which exhibits an intermediate level of triggerability by CD4. We introduced lysine/acidic residues at positions that exhibit such polymorphisms in natural HIV-1 strains. Env changes that were tolerated with respect to gp120-gp41 processing, subunit association and virus entry were further combined. Two common polymorphisms, Q114E and Q567K, as well as a known variant, A582T, additively rendered pseudoviruses resistant to cold, soluble CD4 and a CD4-mimetic compound, phenotypes indicative of stabilization of the pretriggered State-1 Env conformation. Combining these changes resulted in two lysine-rich HIV-1AD8 Env variants (E.2 and AE.2) with neutralization- and cold-resistant phenotypes comparable to those of natural, less triggerable Tier 2/3 HIV-1 isolates. Compared with these and the parental Envs, the E.2 and AE.2 Envs were cleaved more efficiently and exhibited stronger gp120-trimer association in detergent lysates. These highly crosslinkable Envs enriched in a pretriggered conformation should assist characterization of the structure and immunogenicity of this labile state. IMPORTANCEThe development of an efficient vaccine is critical for combating HIV-1 infection worldwide. However, the instability of the pretriggered shape (State 1) of the viral envelope glycoprotein (Env) makes it difficult to raise neutralizing antibodies against HIV-1. Here, by introducing multiple changes in Env, we derived two HIV-1 Env variants that are enriched in State 1 and can be efficiently crosslinked to maintain this shape. These Env complexes are more stable in detergent, assisting their purification. Thus, our study provides a path to a better characterization of the native pretriggered Env, which should assist vaccine development.

Triple-negative breast cancer (TNBC) constitutes 12% of all breast cancer and is associated with worse prognosis compared to other subtypes of breast cancer. Current therapies are limited to cytotoxic chemotherapy, radiation, and surgery, leaving a need for targeted therapeutics to improve outcomes for TNBC patients. Mammalian orthoreovirus (reovirus) is a nonenveloped, segmented, dsRNA virus in the Reoviridae family. Reovirus preferentially kills transformed cells and is in clinical trials to assess its efficacy against several types of cancer. We previously engineered a reassortant reovirus, r2Reovirus, that infects TNBC cells more efficiently and induces cell death with faster kinetics than parental reoviruses. In this study, we sought to understand the mechanisms by which r2Reovirus induces cell death in TNBC cells. We show that r2Reovirus infection of TNBC cells of a mesenchymal-stem like (MSL) lineage downregulates the MAPK/ERK pathway and induces non-conventional cell death that is caspase dependent, but caspase 3-independent. Infection of different MSL lineage TNBC cells with r2Reovirus results in caspase 3-dependent cell death. We map the enhanced oncolytic properties of r2Reovirus in TNBC to epistatic interactions between the Type 3 Dearing M2 gene segment and Type 1 Lang genes. These findings suggest that the genetic composition of the host cell impacts the mechanism of reovirus-induced cell death in TNBC. Together, our data show that understanding host and virus determinants of cell death can identify novel properties and interactions between host and viral gene products that can be exploited for the development of improved viral oncolytics. ImportanceTriple negative breast cancer (TNBC) is unresponsive to hormone therapies, leaving patients afflicted with this disease with limited treatment options. We previously engineered an oncolytic reovirus (r2Reovirus) with enhanced infective and cytotoxic properties in TNBC cells. However, how r2Reovirus promotes TNBC cell death is not known. In this study, we show that reassortant r2Reovirus can promote non-conventional caspase-dependent but caspase 3-independent cell death and that the mechanism of cell death depends on the genetic composition of the host cell. We also map the enhanced oncolytic properties of r2Reovirus in TNBC to interactions between a Type 3 M2 gene segment and Type 1 genes. Our data show that understanding the interplay between the host cell environment and the genetic composition of oncolytic viruses is crucial for the development of efficacious viral oncolytics.

Herpes simplex virus 1 (HSV-1) transcription is restricted in latently infected neurons and the genomes are in mostly silenced chromatin, whereas all viral genes are transcribed in lytically infected cells, in which the genomes are dynamically chromatinized. Epigenetic regulation modulates HSV-1 transcription during lytic, latent, and reactivating infections, but the precise mechanisms are not fully defined. Nucleosomes are dynamic; they slide, breathe, assemble and disassemble. We and others have proposed that the most dynamic HSV-1 chromatin is transcriptionally competent whereas the least dynamic is silenced. However, the mechanisms yielding the unusually dynamic viral chromatin remain unknown. Histone variants affect nucleosome dynamics. The dynamics of H2A, H2A.X and macroH2A were enhanced in infected cells, whereas those of H2A.B uniquely decreased. We constructed stably transduced cells expressing tagged histone H2A, H2A.B, macroH2A, or H2B, which assembles the H2A/H2B nucleosome dimers with all H2A variants. All H2A variants, ectopic, and endogenous H2B, were assembled into HSV-1 chromatin evenly throughout the genome, but canonical H2A was relatively depleted from the viral chromatin whereas H2A.B was enriched in the most dynamic viral chromatin. When viral transcription was restricted, H2A.B became as depleted from the viral chromatin through the entire genome as H2A. We propose that lytic HSV-1 nucleosomes are enriched in the dynamic variant H2A.B/H2B dimers to promote HSV-1 chromatin dynamics and transcriptional competency, and conclude that the dynamics of HSV-1 chromatin are determined in part by the H2A variants. ImportanceHSV-1 transcription is epigenetically regulated during latent and lytic infections, and epigenetic inhibitors have been proposed as potential antiviral drugs to modulate latency and reactivation. However, the detailed mechanisms of regulation of HSV-1 transcription by epigenetics have not been fully characterized and may differ from those regulating cellular transcription. In particular, the lytic HSV-1 chromatin is unusually dynamic, whereas the latent silenced one is not, but the mechanisms resulting in the unique dynamics of the lytic chromatin remain unknown. Here we identify the enrichment on the highly dynamic histone 2A variant H2A in the most dynamic viral chromatin, which provides a mechanistic understanding for its unique dynamics. Future work to identify the mechanisms of enrichment in H2A.B on the viral chromatin may identify novel druggable epigenetic regulators that modulate HSV-1 latency and reactivation.

The dead box helicase DHX29 plays a critical role in the translation of mRNAs containing complex RNA secondary structure in their 5 untranslated regions. The human cytomegalovirus (HCMV) genome has a high GC content, suggesting the 5UTRs of viral mRNAs may contain significant secondary structure and require DHX29 for their efficient translation initiation. We found that depleting DHX29 from primary human fibroblasts prior to infection reduced viral mRNA and protein levels and decreased HCMV replication. The defect in HCMV replication correlated with decreased expression of the HCMV immediate early proteins IE1 and IE2, which are necessary for the establishment of lytic infection. Analysis of polysome associated mRNAs revealed that the defect in IE1 and IE2 expression is due to decreased mRNA translation efficiency. We found that DHX29 depletion led to reduced levels of the eIF4F translation initiation complex, resulting from decreased translation of the eIF4G mRNA. However, in line with our previous results showing a minimal role for the eIF4F complex in HCMV mRNA translation, we found that depleting eIF4G prior to infection did not impact IE1 and IE2 translation. Together our results define a new role for DHX29 in regulating eIF4F-dependent translation and identify a critical role for DHX29 in the translation of HCMV mRNAs. SignificanceExpression of the HCMV immediate early proteins IE1 and IE2 is critical for the establishment of lytic replication and the reactivation of latent HCMV infections. Defining the mechanisms controlling HCMV IE1 and IE2 protein expression has the potential to identify new strategies for therapeutic interventions that can limit HCMV disease in immune naive and immune compromised individuals. Our finding that the cellular DHX29 helicase is necessary for the efficient translation of mRNAs encoding IE1 and IE2 suggests that therapies that inhibit DHX29 could potentially be useful in treating HCMV disease and adds to the growing body of literature suggesting DHX29 activity is a disease driver in multiple indications including viral disease, inflammation and cancer.

HSV-1 employs cellular motor proteins and modulates kinase pathways to facilitate intracellular virion capsid transport. Previously, we and others have shown that the Akt inhibitor miltefosine inhibited virus entry. Herein, we show that the protein kinase C inhibitors staurosporine (STS) and gouml inhibited HSV-1 entry into Vero cells, and that miltefosine prevents HSV-1 capsid transport toward the nucleus. We have reported that the HSV-1 UL37 tegument protein interacts with the dynein motor complex during virus entry and virion egress, while others have shown that the UL37/UL36 protein complex binds dynein and kinesin causing a saltatory movement of capsids in neuronal axons. Co-immoprecipitation experiments confirmed previous findings from our laboratory that the UL37 protein interacted with the dynein intermediate chain (DIC) at early times post infection. This UL37-DIC interaction was concurrent with DIC phosphorylation in infected, but not mock-infected cells. Miltefosine inhibited dynein phosphorylation when added before, but not after virus entry. Inhibition of motor accessory protein dynactins (DCTN2, DCTN3), the adaptor proteins EB1 and the Bicaudal D homolog 2 (BICD2) expression using lentiviruses expressing specific shRNAs, inhibited intracellular transport of virion capsids toward the nucleus of human neuroblastoma (SK-N-SH) cells. Co-immunoprecipitation experiments revealed that the major capsid protein Vp5 interacted with dynactins (DCTN1/p150 and DCTN4/p62) and the end-binding protein (EB1) at early times post infection. These results show that Akt and kinase C are involved in virus entry and intracellular transport of virion capsids, but not in dynein activation via phosphorylation. Importantly, both the UL37 and Vp5 viral proteins are involved in dynein-dependent transport of virion capsids to the nuclei of infected cells. ImportanceHerpes simplex virus type-1 enter either via fusion at the plasma membranes or endocytosis depositing the virion capsids into the cytoplasm of infected cells. The viral capsids utilize the dynein motor complex to move toward the nuclei of infected cells using the microtubular network. This work shows that inhibitors of the Akt kinase and kinase C inhibit not only viral entry into cells but also virion capsid transport toward the nucleus. In addition, the work reveals that the virion protein ICP5 (VP5) interacts with the dynein cofactor dynactin, while the UL37 protein interacts with the dynein intermediate chain (DIC). Importantly, neither Akt nor Kinase C was found to be responsible for phosphorylation/activation of dynein indicating that other cellular or viral kinases may be involved.

The current model for human papillomavirus (HPV) replication is comprised of three modes of replication. Following infectious delivery, the viral genome is amplified during the establishment phase to reach up to some hundred copies per cell. HPV genome copy number remains constant during the maintenance stage. Differentiation of infected cells induces HPV genome amplification. Using highly sensitive in situ hybridization (DNAscope) and freshly HPV16-infected as well as established HPV16-positive cell lines, we observed that viral genome is amplified in each S phase of undifferentiated keratinocytes cultured as monolayers. Nuclear viral genome copy number is reset to pre-S phase levels during mitosis. The majority of viral genome fails to tether to host chromosomes and is lost to the cytosol. Cytosolic viral genome gradually decreases during cell cycle progression. Loss of cytosolic genome is blocked in presence of NH4Cl or other drugs interfering with lysosomal acidification, suggesting the involvement of autophagy in viral genome degradation. These observations were also made with HPV31 cell lines obtained from patient samples. Cytosolic viral genome was not detected in UMSCC47 cells carrying integrated HPV16 DNA. Analyses of organotypic raft cultures derived from keratinocytes harboring episomal HPV16 revealed the presence of cytosolic viral genome as well. We conclude that HPV maintains viral genome copy number by balancing viral genome amplification during S phase with loss of viral genome that is lost to the cytosol during mitosis. It seems plausible that restrictions to viral genome tethering to mitotic chromosomes resets genome copy number in each cell cycle. IMPORTANCEHPV genome maintenance is currently being thought to be achieved by regulating expression and activity of viral replication factors E1 and E2. In addition, the E8^E2 repressor has been shown to be important for restricting genome copy number by competing with E1 and E2 for binding to the viral origin of replication and by recruitment of repressor complexes. Herein, we demonstrate that HPV viral genome is amplified in each S phase. Nuclear genome copy number is reset during mitosis by a failure of the majority of genomes to tether to mitotic chromosomes. Rather, they accumulate in the cytoplasm of freshly divided cells. Cytosolic viral DNA is quickly degraded in G1 in a lysosome dependent manner contributing to the genome copy reset. Our data imply that the mode of replication during establishment and maintenance is the same and further suggest that restrictions to genome tethering significantly contributes to viral genome maintenance.

Infection with Human Cytomegalovirus (HCMV) can result in a significant burden of disease in those that are immunocompromised or immunonaive. HCMV encodes a repertoire of glycoproteins that facilitate its extensive viral tropism, some of which remain to be characterized. Currently, there is no effective vaccine or cure for HCMV, therefore emphasizing the need to identify viral proteins of critical function. UL14 was selected as an open reading frame of interest due to its high scoring on an in-silico prediction algorithm, as well as its conservation amongst CMVs. Our goal was to elucidate the function of this uncharacterized viral open reading frame. We hypothesized that UL14 functions in the establishment of infection in epithelial cells, due to its predicted structural similarity to UL141. This study demonstrates that HCMV UL14 is a glycosylated viral protein packaged with the virion. Importantly, the deletion of UL14 resulted in a significant reduction of viral growth in epithelial cells, whereas no growth defect was observed in fibroblasts. Mechanistically, we found this defect to be a result of post entry, pre-IE transcription in the establishment of infection, consistent with a defect endosomal escape. Taken together, our results suggest that UL14 functions in the establishment of infection in an epithelial cell-specific manner and may be a novel target for future vaccines or antiviral therapies. Author SummaryHCMV is found in a wide variety of human cells during the course of viral infection. As such, HCMV encodes several glycoprotein complexes that dictate tropism. In this work we report the identification of a novel glycoprotein, UL14, that is involved in establishing productive infections of epithelial cells, a common site of HCMV induced disease. We report that deletion of UL14 from the viral genome impacts its ability to infect ARPE19 cells at a stage indicative of viral events post viral entry but prior to viral transcriptional activation. Further, trans complementation of UL14 by expansion of mutant virus in cells expressing the viral glycoprotein, restore viral infectivity suggesting that UL14 mediates events early in viral infection. Importantly, the characterization of this viral envelope protein provides key insights into viral tropism and identifies a novel target for vaccine design and antiviral therapies.

Viral protein genome-linked (VPg) protein plays an essential role in protein-primed replication of plus stranded RNA viruses. VPg is covalently linked to the 5 end of the viral RNA genome via a phosphodiester bond typically at a conserved amino acid. Whereas most viruses have a single VPg, some viruses encode multiple VPgs that are proposed to have redundant yet undefined roles in viral replication. Here, we use the dicistrovirus, cricket paralysis virus (CrPV), which encodes four non-identical copies of VPg, as a model to characterize the role of VPg copies in infection. Dicistroviruses encode two main open reading frames (ORFs) that are driven by distinct IRESs. We systematically generated single and combinatorial deletions and mutations of VPg1-4 within the CrPV infectious clone and monitored viral yield in Drosophila S2 cells. Deletion of one to three VPg copies progressively decreased viral yield and delayed viral replication, suggesting a threshold number of VPgs for productive infection. Mass spectrometry analysis of CrPV VPg-linked RNAs revealed viral RNA linkage to either a serine or threonine in VPg, from which mutations in all VPgs attenuated infection. Mutating serine 4 in a single VPg abolished viral infection, indicating a dominant-negative effect. Using viral minigenome reporters that monitor dicistrovirus 5 untranslated (UTR) and intergenic internal ribosome entry site (IRES) translation revealed a relationship between VPg copy number and the ratio of distinct IRES translation. We uncover a novel viral strategy whereby VPg copies in dicistrovirus genomes compensate for the relative IRES translation efficiencies to promote infection. ImportanceGenetic duplication is exceedingly rare in small RNA viral genomes as there is selective pressure to prevent RNA genomes from expanding. However, some small RNA viruses encode multiple copies of a viral protein, most notably an unusual viral protein that is linked to the viral RNA genome. Here, we investigate a family of viruses that contains multiple viral protein genome-linked proteins and reveal a novel viral strategy whereby viral protein copy number counterbalances differences in viral protein synthesis mechanisms.

The HIV-1 epidemic originated from a cross-species transmission of a primate lentivirus from chimpanzees to humans near the turn of the 18th century. Simian immunodeficiency viruses have been jumping between old world monkeys in West/Central Africa for thousands of years. So why did HIV-1 only emerge in the past century? This study examined the replicative fitness, transmission, restriction, and cytopathogenicity of 26 primate lentiviruses. Pairwise competitions of these primate lentiviruses revealed that SIVcpz had the highest replicative fitness in human or chimpanzee peripheral blood mononuclear cells, even higher fitness than HIV-1 group M strains responsible for 37 million infections worldwide. In contrast the "HIV-2 lineage" (SIVsmm, SIVmac, SIVagm, and HIV-2) had the lowest replicative fitness. SIVcpz strains were less inhibited by human restriction factors than the "HIV-2 lineage" strains, a restriction that was inversely correlated with replicative fitness. SIVcpz from the chimpanzee subspecies Pan troglodytes troglodytes (Ptt) was slightly more fit in human cells than the strains from Pt schweinfurthii (Pts). However, unlike all other primate lentiviruses (including the HIV-2 lineage), SIVcpz was nonpathogenic in human tonsillar tissue and did not deplete CD4+ T-cells, consistent with the slow or nonpathogenic disease observed in chimpanzees. Despite the close phylogenetic relationship between SIVcpz_Ptt and HIV-1, this epidemic was either caused by cross species transmission of a rare, undiscovered SIVcpz strain of higher virulence or higher virulence differentially evolved among HIV-1 subtypes during the human epidemic. Author summaryInvasion of wild animal habitats by humans can have devastating consequences for the human population as evident by the HIV-1 and SARS-CoV-2 epidemics. With SARS-CoV-2, a recent zoonotic jump, likely from bats, will help to identify a coronavirus progenitor. In contrast, simian immunodeficiency virus (SIV) jumped into humans over 100 years ago from a possibly extinct sub-species of chimpanzees and/or extinct lineage of SIV. We examined replicative fitness and pathogenesis of 26 different primate lentiviruses in human and chimpanzee primary lymphoid cells from blood and within tonsils. SIV from a specific chimpanzee species and lowland gorillas were the most capable of infecting and replicating in human and chimp lymphoid cells but they did not result in the pathogenesis related to disease in humans. In contrast, SIV from other old world monkeys were pathogenic but could not replicate efficiently in human cells. We propose the main HIV-1 is derived from a distinct jump of a very rare SIV strain in chimps leading to AIDS pandemic.