Journal of Virology

Equine infectious anemia virus (EIAV) and HIV-1 are both members of the Lentivirus genus and are similar in virological characters. EIAV is of great concern in the equine industry. Lentiviruses establish a complex interaction with the host cell to counteract the antiviral responses. There are various pattern recognition receptors in the host, for instance, the cytosolic RNA helicases interact with viral RNA to activate the mitochondrial antiviral signaling protein (MAVS) and subsequent interferon (IFN) response. However, viruses also exploit multiple strategies to resist host immunity by targeting MAVS, but the mechanism by which lentiviruses are able to target MAVS has remained unclear. In this study, we found that EIAV infection induced MAVS degradation, and that EIAV Gag protein recruited the E3 ubiquitin ligase Smurf1 to polyubiquitinate and degrade MAVS. The CARD domain of MAVS and the WW domain of Smurf1 are responsible for the interaction with Gag. EIAV Gag is a precursor polyprotein of the membrane-interacting matrix p15, the capsid p26, and the RNA-binding nucleocapsid proteins p11 and p9. Therefore, we analyzed which protein domain of Gag could interact with MAVS and Smurf1. We found that p15 and p26, but not p11 or p9, target MAVS for degradation. Moreover, we identified the key amino acid residues that support the interactions between p15 or p26 and MAVS or Smurf1. The present study describes a novel role of the EIAV structural protein Gag in targeting MAVS to counteract innate immunity, and reveals the mechanism by which the equine lentivirus can antagonize against MAVS. IMPORTANCE Host anti-RNA virus innate immunity relies mainly on the recognition by retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5), and subsequently initiates downstream signaling through interaction with mitochondrial antiviral signaling protein (MAVS). However, viruses have developed various strategies to counteract MAVS-mediated signaling, although the method of antagonism of MAVS by lentiviruses is still unknown. In this article, we demonstrate that the precursor (Pr55gag) polyprotein of EIAV and its protein domains p15 and p26 target MAVS for ubiquitin-mediated degradation through E3 ubiquitin ligase Smurf1. MAVS degradation leads to the inhibition of the downstream IFN-β pathway. This is the first time that lentiviral structural protein has been found to have antagonistic effects on MAVS pathway. Overall, our study reveals a novel mechanism by which equine lentiviruses can evade host innate immunity, and provides insight into potential therapeutic strategies for the control of lentivirus infection.

The papillomavirus (PV) E2 protein is highly conserved, consisting of an N-terminal transactivation domain linked to a C-terminal DNA binding and dimerization domain (DBD) by a flexible hinge region. The E2 DBD exhibits a helix-turn-helix structure that dimerizes into a beta barrel prior to binding DNA; the first helix, α1, is responsible for recognition of the palindromic E2 binding site. The DNA recognition helix consists of a tract of basic amino acids with a highly conserved central cysteine residue. Previous mutational analysis studies on this conserved cysteine have found that it is not required for viral replication or DNA binding. To investigate the function of this conserved cysteine in vitro and in vivo , we generated point mutations in MmuPV1 E2 at cysteine 307. We report here that this cysteine in the DNA recognition helix is required for transient viral replication and transactivation of proximal promoters, but C307 point mutants are still capable of enhancing the activation of distant upstream promoters in vitro . MmuPV1 genomes with the C307 mutation failed to produce warts when injected into mice, suggesting that the DNA recognition cysteine is required for viral replication in vivo . IMPORTANCE Papillomaviruses are the etiological agents of cancers of the oropharynx and anogenital tract. Understanding the mechanisms underlying PV pathogenesis is complicated by the strict species tropism displayed by the virus. The research presented here is significant because it links in vitro and in vivo models investigating the role of a conserved cysteine in the MmuPV1 E2 protein. This work elucidates the molecular mechanisms that regulate PV transcription and DNA replication and how these contribute to disease progression.

Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic disease that causes severe disease in both domestic and wild ungulates and humans, making it a significant threat to livestock and public health. The RVFV genome consists of three single-stranded, negative-sense RNA segments differing in size: small (S), medium (M), and large (L). Segment S encodes the virus nucleoprotein N and the virulence-associated factor non-structural (NSs) protein in opposite orientations, separated by an intergenic region (IGR). To overcome the current need to use secondary techniques to detect the presence of RVFV in infected cells, we used T7-driven polymerase plasmid-based reverse genetics to generate replication-competent recombinant (r)RVFV expressing Nanoluciferase (Nluc) or Venus fluorescent proteins. These reporter genes were used as valid surrogates to track the presence of RVFV in mammalian and insect cells. Notably, we explored the genome plasticity of RVFV and compared four different strategies by modifying the viral segment S to introduce the reporter gene foreign sequences. The reporter-expressing rRVFV were stable and able to replicate in cultured mammalian and insect cells, although to a lesser extent than the recombinant wild-type (WT) counterpart. Moreover, rRVFV-expressing reporter genes were validated to identify neutralizing antibodies or compounds with antiviral activity. In vivo , all mice infected with the reporter-expressing rRVFV displayed an attenuated phenotype, although at different levels. These rRVFV-expressing reporter genes provide a novel approach to better understand the biology and pathogenesis of RVFV and represent an excellent biotechnological tool for developing new therapeutics against RVFV infections. IMPORTANCE Rift Valley fever virus (RVFV) is a mosquito-borne virus and zoonotic agent threat that can be deadly to domestic or wild ungulates, and humans. In this work, we used reverse genetics approaches to explore the genome plasticity of RVFV by generating a set of recombinant (r)RVFV that express fluorescent or luminescent proteins to track viral infection. All the generated reporter-expressing rRVFVs were able to propagate in mammalian or insect cells and a mouse model of infection. Our studies may contribute to advances in research on RVFV and other bunyaviruses and pave the way for the development of novel vaccines and the identification of new antivirals for the prophylactic and therapeutic treatment, respectively, of RVFV infections.

Anellovirus infections are ubiquitous in mammals but lack any clear disease association, suggesting a commensal virus-host relationship. Although anelloviruses have been identified in numerous mammalian hosts, their presence in members of the family Delphinidae has yet to be reported. Here, using a metagenomic approach, we characterize complete anellovirus genomes ( n = 69) from four Delphinidae host species: short-finned pilot whale ( Globicephala macrorhynchus , n = 19), killer whale ( Orcinus orca , n = 9), false killer whale ( Pseudorca crassidens , n = 6), and pantropical spotted dolphin ( Steno attenuatus , n = 1). Sequence comparison of the open reading frame 1 (ORF1) encoding the capsid protein, the only conserved gene shared by all anelloviruses, shows that the Delphinidae anelloviruses form a novel genus-level clade that encompasses 22 unique species-level groupings. We provide evidence that different Delphinidae species can be co-infected by multiple anelloviruses belonging to distinct species groupings. Notably, the ORF1 protein of the Delphinidae anelloviruses is considerably larger than those encoded by all previously described anelloviruses from other hosts (spanning 14 vertebrate orders and including 27 families). Comprehensive analysis of the ORF1 sequences and predicted protein structures showed that the increased size of these proteins results from divergent elaborations within the capsid-distal P2 subdomain and elongation of the C-terminal domain of ORF1. Comparative structural and phylogenetic analyses suggest that acquisition of the P2 subdomain and its diversification occurred convergently in the anelloviruses associated with primate and Delphinidae hosts. Collectively, our results further the appreciation of diversity and evolution of the ubiquitous and enigmatic viruses in the family Anelloviridae . IMPORTANCE Anelloviruses are ubiquitous in mammals, but their infection has not yet been linked to any disease, suggesting a commensal virus-host relationship. Here, we describe the first anelloviruses associated with diverse species of dolphins. The dolphinid anelloviruses represent a new genus (tentatively named “Qoptorquevirus”) and encode open reading frame 1 (ORF1) (capsid) proteins that are considerably larger than those encoded by previously described anelloviruses from other hosts. Comprehensive analysis of the ORF1 sequences and predicted protein structures revealed the underlying structural basis for such an extravagant ORF1 size and suggested that ORF1 size increased convergently in the anelloviruses associated with primate and Delphinidae hosts, respectively. Collectively, our results provide insights into the diversity and evolution of Anelloviridae . Further exploration of the anellovirus diversity, especially in the host species that have not yet been sampled, is expected to further clarify their evolutionary trajectory and explain the unusual virus-host commensal relationship.

The human cytomegalovirus (HCMV) encoded chemokine receptor US28 plays a critical role in viral pathogenesis, mediating several processes such as cellular migration, differentiation, transformation, and viral latency and reactivation. Despite significant research examining the signal transduction pathways utilized by US28, the precise mechanism by which US28 activates these pathways remains unclear. We performed a mutational analysis of US28 to identify signaling domains that are critical for functional activities. Our results indicate that specific residues within the third intracellular loop (ICL3) of US28 are major determinants of G-protein coupling and downstream signaling activity. Alanine substitutions at positions S218, K223, and R225 attenuated US28-mediated activation of MAPK and RhoA signal transduction pathways. Furthermore, we show that mutations at positions S218, K223, or R225 result in impaired coupling to multiple Gα isoforms. However, these substitutions did not affect US28 plasma membrane localization or the receptor internalization rate. Utilizing CD34 ⁺ HPC models, we demonstrate that attenuation of US28 signaling via mutation of residues within the ICL3 region results in an inability of the virus to efficiently reactivate from latency. These results were recapitulated in vivo , utilizing a humanized mouse model of HCMV infection. Together, our results provide new insights into the mechanism by which US28 manipulates host signaling networks to mediate viral latency and reactivation. The results reported here will guide the development of targeted therapies to prevent HCMV-associated disease. IMPORTANCE Human cytomegalovirus (HCMV) is a β-herpesvirus that infects between 44% and 100% of the world population. Primary infection is typically asymptomatic and results in the establishment of latent infection within CD34 ⁺ hematopoietic progenitor cells (HPCs). However, reactivation from latent infection remains a significant cause of morbidity and mortality in immunocompromised individuals. The viral chemokine receptor US28 influences various cellular processes crucial for viral latency and reactivation, yet the precise mechanism by which US28 functions remains unclear. Through mutational analysis, we identified key residues within the third intracellular loop (ICL3) of US28 that govern G-protein coupling, downstream signaling, and viral reactivation in vitro and in vivo . These findings offer novel insights into how US28 manipulates host signaling networks to regulate HCMV latency and reactivation and expand our understanding of HCMV pathogenesis.

Enteroviral 3C protease (3Cpro) is an essential enzyme for viral replication and is responsible for combating the host anti-viral immune response by targeting cellular proteins for cleavage. The identification and characterization of 3Cpro substrates will contribute to our understanding of viral pathogenesis. In this study, we performed a motif search for 3Cpro substrates in the human protein database using FIMO, which refers to a common cleavage sequence of 3Cpro. We identified and characterized NEDD4-binding protein 1 (N4BP1), a key negative regulator of the NF-κB pathway, as a novel 3Cpro substrate. N4BP1 is cleaved at residue Q816 by 3Cpro from several human enteroviruses, resulting in the loss of its ability to regulate tumor necrosis factor alpha-activated NF-κB signaling. In addition, we found that mouse N4BP1, which has a threonine at the P1′ site, is resistant to human enteroviral 3Cpro cleavage. However, rodent enteroviral 3Cpro derived from encephalomyocarditis virus (EMCV) can cleave both human and mouse N4BP1 at a species-specific site. By combining bioinformatic, biochemical, and cell biological approaches, we identified and characterized N4BP1 as a novel substrate of enteroviral 3Cpro. These findings provide valuable insights into the interplay between 3Cpro, its substrates, and viral pathogenesis. IMPORTANCE Targeting cellular proteins for cleavage by enteroviral 3Cpro is a conserved strategy used by enteroviruses to promote viral replication. While the cleavage of certain host proteins by 3Cpro may not affect viral replication, it is strongly associated with the pathogenesis of viral infection. In this study, we identified and characterized N4BP1, which plays such a role, using a combination of bioinformatic, biochemical, and cell biological approaches. Our data show that multiple 3Cpros cleave N4BP1 at residue Q816 and that cleavage of endogenous N4BP1 can occur during viral infection. N4BP1 has no effect on coxsackievirus B3 replication, but 3Cpro-induced N4BP1 cleavage abolishes its regulatory function in NF-κB signaling. We also show that mouse N4bp1 resists human enteroviral 3Cpro cleavage. In contrast, rodent enteroviral EMCV 3Cpro can target human and mouse N4BP1 for cleavage at different residues, which indicates that future investigations are needed to elucidate the potential mechanisms involved.

The Vpx protein encoded by HIV-2/simian immunodeficiency virus (SIV) can antagonize the restriction of the host intrinsic restriction factor, SAMHD1, in nondividing cells by promoting its polyubiquitination and subsequent degradation, thereby facilitating viral replication and immune evasion. However, the role of deubiquitinating enzymes (DUBs) in the dynamics of virus and host remains poorly understood. Here, we demonstrate that DUB USP37 significantly reverses the Vpx-mediated degradation of SAMHD1 in various HIV-2/SIV subtypes by interacting with SAMHD1 and removing its ubiquitin chains. Notably, USP37 deubiquitinates SAMHD1 by directly recognizing SAMHD1 rather than by targeting the E3 ubiquitin ligase. The deubiquitinase activity of USP37 and its ubiquitin interacting motifs are essential for the deubiquitination of SAMHD1, whereas the phosphorylation state of USP37 does not influence its activity. Additionally, USP37 enhances the suppression of the retrotransposition of LINE-1 elements by SAMHD1 via stabilizing SAMHD1. Our findings provide important evidence that enhancing the deubiquitinating activity of some DUBs results in the stability of the host restriction factor and might be a viable strategy against HIV/SIV infections. IMPORTANCE SAMHD1 is a multifunctional protein, including restricting virus replication, maintaining genomic integrity through DNA repair, modulating the immune response by influencing the production of type I interferons and other cytokines, and affecting cancer cell proliferation and sensitivity to chemotherapy. However, HIV-2/simian immunodeficiency virus (SIV)-encoded Vpx and the host E3 ligase TRIM21 can induce the degradation of SAMHD1 via the ubiquitin–proteasome pathway. Therefore, it is necessary to find the strategy to stabilize SAMHD1. Our study demonstrates that the deubiquitinase USP37 reverses Vpx- and TRIM21-mediated degradation of SAMHD1, thereby inhibiting SIV replication and LINE-1 activity by stabilizing SAMHD1. Thus, we report a novel role of USP37, which represents a potentially useful target for the development of new drugs.

Polymycoviridae is a relatively new viral family that was established nearly 5 years ago, but their viral morphologies (naked or encapsidated) remain controversial since only one member namely, Colletotrichum camelliae filamentous virus 1 (CcFV1), was identified as being encapsidated in filamentous virions. Here, three novel double-stranded RNA (dsRNA) viruses belonging to the family Polymycoviridae were identified in three phytopathogenic fungal strains and tentatively named Pseudopestalotiopsis camelliae -sinensis polymycovirus 1 (PcsPmV1), and Phyllosticta capitalensis polymycovirus 1 and 2 (PhcPmV1 and 2), respectively. PcsPmV1 and PhcPmVs have five or six genomic dsRNAs, ranging from 1,055 to 2,405 bp, encoding five or seven putative open reading frames (ORFs), of which ORF1 encodes an RNA-dependent RNA polymerase, ORF5 encodes a prolein-alanine-serine-rich (P-A-S-rich) protein behaving as coat protein (CP); and dsRNAs 4 and 6 encode putative proteins with unknown functions and share no detectable identities with known viral sequences. Upon examination under transmission electron microscopy after purification from fungal mycelia, PcsPmV1 and PhcPmVs were found to be encapsidated in filamentous particles, as was a known polymycovirus, Botryosphaeria dothidea RNA virus 1 (BdRV1), which was previously assumed to likely have no conventional virions. The morphology of PcsPmV1 was further supported by the observation that its particles could be decorated by polyclonal antibodies against its CP and bound by immuno-gold particles conjugated to the specific CP antibody. Together with CcFV1, BdRV1, PcsPmV1, and PhcPmVs, these provide strong evidence to support the notion that polymycoviruses are encapsidated in filamentous virions constituted by P-A-S-rich CPs. Moreover, their biological effects on their fungal hosts were assessed, suggesting that PcsPmV1 infection could enhance growth and virulence. IMPORTANCE Polymycoviridae , a recently established viral family, has raised questions about encapsidation. Here, we identify and characterize three novel polymycoviral double-stranded RNA (dsRNA) viruses in phytopathogenic fungal strains, tentatively named Pseudopestalotiopsis camelliae -sinensis polymycovirus 1, and Phyllosticta capitalensis polymycovirus 1 and 2, respectively. These polymycoviruses possess five or six genomic dsRNAs, ranging from 1,055 to 2,405 bp, with two encoding putative proteins of unknown functions and sharing no detectable identities with known viral sequences. Their morphologies indicate filamentous virions constituted by proline-alanine-serine-rich coat proteins, observed using immunosorbent electron microscopy combined with immune-gold labeling techniques. Additionally, Botryosphaeria dothidea RNA virus 1, previously assumed to lack conventional virions, is also shown to be encapsidated in filamentous particles. This study provides new evidence supporting the encapsidation of polymycoviruses into elongated and flexuous virions, significantly contributing to our understanding of the evolutionary particle architecture within the virosphere and expanding our knowledge of viral diversity and evolution. Moreover, this is the first report of a polymycovirus enhancing the virulence and growth of a phytopathogenic fungus.

Human cytomegalovirus (HCMV) is a betaherpesvirus capable of infecting numerous cell types and persisting throughout an infected individual’s life. Disease usually occurs in individuals with compromised or underdeveloped immune systems. Several antivirals exist but have limitations relating to toxicity and resistance. HCMV replication involves upregulation of host proteasomal activities, which play important roles in the temporal stages of replication. Here, we defined the impact on replication kinetics of the proteasome inhibitor, bortezomib. We demonstrate that bortezomib significantly reduces levels of viral genomes and infectious virions produced from a population of cells. Inhibition reduced expression of viral proteins that are influenced by genome synthesis. When added prior to 24 hpi, we observe decreases in PCNA and Cdk1 while increases in p21 whose regulations contribute to efficient replication. This response synergized with an antiviral, maribavir. Since some replication occurred, we tested the hypothesis that a subset of infected cells might break through inhibition. Initially, we simulated bortezomib activities using a mechanistic computational model of late-lytic replication. Upon reducing multiplicity of infection (MOI) in silico , we observed near-identical simulated results compared to experimental data. Next, we analyzed replication using live-cell imaging. This revealed treated cultures do contain a population of cells with fully developed late-stage cytoplasmic assembly compartments but at significantly lower numbers. We refer to this as the effective MOI. Overall, our studies support a hypothesis in which 20S proteasome inhibition disrupts HCMV replication by reducing the MOI to an effective MOI, defined by a fraction of infected cells capable of progressing to fulminant infection. IMPORTANCE Human cytomegalovirus (HCMV) infection and reactivation continues to contribute to morbidity and mortality around the world. Antiviral compounds are available but have limitations. Here, we have defined the impact of the proteasome inhibitor bortezomib on HCMV replication. Proteasomal activities play a critical role in temporal changes required for replication. We demonstrate that disrupting these activities inhibits viral replication while likely supporting increased antiviral activity of the anti-HCMV agent, maribavir. Using a combination of live-cell imaging and computational tools, we discover that a subset of infected cells progresses to fulminant infection, which we define as the effective multiplicity of infection, and this subset would otherwise be missed when analyzing the average of the population.

Alongshan virus (ALSV) is a newly discovered pathogen in the Flaviviridae family, characterized by a unique multi-segmented genome that is distantly related to the canonical flaviviruses. Understanding the pathogenic mechanism of this emerging segmented flavivirus is crucial for the development of effective intervention strategies. In this study, we demonstrate that ALSV can infect various mammalian cells and induce the expression of antiviral genes. Furthermore, ALSV is sensitive to IFN-β, but it has developed strategies to counteract the host’s type I IFN response. Mechanistically, ALSV’s nonstructural protein NSP1 interacts with and degrades human STAT2 through an autophagy pathway, with species-dependent effects. This degradation directly inhibits the expression of interferon-stimulated genes (ISGs). Additionally, NSP1-mediated degradation of STAT2 disrupts mitochondrial dynamics, leading to mitophagy and inhibition of mitochondrial biogenesis. This, in turn, suppresses the host’s innate immune response. Interestingly, we found that inhibiting mitophagy using 3-methyladenine and enhancing mitochondrial biogenesis with the PPARγ agonist pioglitazone can reverse NSP1-mediated inhibition of ISGs, suggesting that promoting mitochondrial mass could serve as an effective antiviral strategy. Specifically, the NSP1 methyltransferase domain binds to the key sites of F175/R176 located in the coiled-coil domain of STAT2. Our findings provide valuable insights into the intricate regulatory cross talk between ALSV and the host’s innate immune response, shedding light on the pathogenesis of this emerging segmented flavivirus and offering potential intervention strategies. IMPORTANCE Alongshan virus (ALSV), a segmented flavivirus belonging to the Flaviviridae family, was first identified in individuals who had been bitten by ticks in Northeastern China. ALSV infection is responsible for causing Alongshan fever, a condition characterized by various clinical symptoms, including fever, headache, skin rash, myalgia, arthralgia, depression, and coma. There is an urgent need for effective antiviral therapies. Here, we demonstrate that ALSV is susceptible to IFN-β but has developed mechanisms to counteract the host’s innate immune response. Specifically, the ALSV nonstructural protein NSP1 interacts with STAT2, leading to its degradation via an autophagy pathway that exhibits species-dependent effects. Additionally, NSP1 disrupts mitochondrial dynamics and suppresses mitochondrial biogenesis, resulting in a reduction in mitochondrial mass, which ultimately contributes to the inhibition of the host’s innate immune response. Interestingly, we found that inhibiting mitophagy and promoting mitochondrial biogenesis can reverse NSP1-mediated suppression of innate immune response by increasing mitochondrial mass. These findings provide valuable insights into the molecular mechanisms of ALSV pathogenesis and suggest potential therapeutic targets against ALSV infection.

Viruses represent a diverse pool of obligate parasites that infect virtually every known organism, as such, their study is incredibly valuable for a range of fields including public health, medicine, agriculture, and ecology, and the development of biomedical technologies. Having evolved over millions of years, each virus has a unique and often complicated biology, that must be characterized on a case-by-case basis, even between strains of the same taxon. Owing to its nanoscale spatial resolution, atomic force microscopy (AFM) represents a powerful tool for exploring virus biology, including structural features, kinetics of binding to host cell ligands, virion self-assembly, and budding behaviors. Through the availability of numerous chemistries and advances in imaging modes, AFM is able to explore the complex web of host-virus interactions and life-cycle at a single virus level, exploring features at the level of individual bonds and molecules. Due to the wide array of techniques developed and data analysis approaches available, AFM can provide information that cannot be furnished by other modalities, especially at a single virus level. Here, we highlight the unique methods and information that can be obtained through the use of AFM, demonstrating both its utility and versatility in the study of viruses. As the technology continues to rapidly evolve, AFM is likely to remain an integral part of research, providing unique and important insight into many aspects of virology.

Following reactivation of a latent alphaherpesvirus infection, viral particles are assembled in neuronal cell bodies, trafficked anterogradely within axons to nerve termini, and spread to adjacent epithelial cells. The virally encoded membrane proteins US9p and the glycoprotein heterodimer gE/gI of pseudorabies virus (PRV) and herpes simplex virus type 1 (HSV-1) play critical roles in anterograde spread, likely as a tripartite gE/gI-US9p complex. Two kinesin motors, kinesin-1 and kinesin-3, are implicated in the egress of these viruses, but how gE/gI-US9p coordinates their activities is poorly understood. Here, we report that PRV, in addition to associating with the kinesin-3 motor KIF1A, recruits the neuronal kinesin-1 isoforms KIF5A and KIF5C, but not the broadly expressed isoform KIF5B, during egress from differentiated CAD neurons. Similarly, in the axons of dorsal root ganglia (DRG)-derived sensory neurons, PRV colocalized with KIF5C but not KIF5B. In differentiated CAD cells, the association of KIF1A with egressing PRV was dependent upon US9p, whereas the recruitment of KIF5 isoforms required gE/gI. Consistent with these findings, the number of PRV particles trafficking within CAD neurites and the axons of DRG neurons increased when kinesin-1 motor activity was upregulated by hyperacetylating microtubules using trichostatin A (TSA) or tubacin, and this enhanced trafficking depended upon the presence of gE/gI. We propose that, following its recruitment by US9p, KIF1A delivers PRV particles to a location where KIF5 motors are subsequently added by a gE/gI-dependent mechanism. KIF5A/C isoforms then serve to traffic viral particles along axons, resulting in characteristic recrudescent infection. IMPORTANCE Alphaherpesviruses include important human and veterinary pathogens that share a unique propensity to establish life-long latent infections in the peripheral nervous system. Upon reactivation, these viruses navigate back to body surfaces and transmit to new hosts. In this study, we demonstrate that the virus gE/gI-US9p membrane complex routes virus particles down this complex neuronal egress pathway by coordinating their association with multiple kinesin microtubule motors.

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to cause substantial economic losses to the pig industry worldwide. Previous studies from other groups showed that CD163 is required for PRRSV uncoating and genome release. However, CD163 does not interact with nucleocapsid (N) protein. In this study, the neonatal Fc receptor (FcRn) was demonstrated to be irreplaceable for PRRSV infection by knockdown, overexpression, antibodies or IgG blocking, knockout, and replenishment assays. FcRn was further revealed to be involved in PRRSV uncoating for the first time rather than viral attachment and internalization. In detail, FcRn was determined to colocalize with CD163 and PRRSV virions in early endosomes and specially interact with N protein in early endosomes. Taken together, these results provide evidence that FcRn is an essential cellular factor for PRRSV uncoating, which will be a promising target to interfere with the viral infection. IMPORTANCE PRRSV infection results in a severe swine disease affecting pig farming in the world. Although CD163 has been implicated as the uncoating receptor for PRRSV but the uncoating mechanism of PRRSV remains unclear. Here, we identified that FcRn facilitated virion uncoating via interaction with viral N protein in early endosomes. Our work actually expands the knowledge of PRRSV infection and provides an attractive therapeutic target for the prevention and control of PRRS.

The attack and defense of infected cells and cytotoxic CD8 T cells occur in germinal centers in lymphoid tissue in chronic persistent HIV/SIV infection. Latently infected cells, the therapeutic target of HIV infection, accumulate in follicular helper T (Tfh) cells in lymphoid tissue; the impact of HIV-specific follicular CD8 (fCD8) T cells in lymphoid tissue on the latently infected cells remains unknown. We infected 15 cynomolgus macaques with SIVmac239 and examined the contribution of SIV-Gag-specific fCD8 T cells, defined by activation-induced markers (AIMs), to SIV-infected cells. Eight out of the 15 infected macaques served as progressors; a chronic phase combination antiretroviral therapy (cART) model was established for the eight macaques (progressors) with chronic persistent infection status, wherein cART was started in the chronic phase and discontinued after 27 weeks. Seven macaques that naturally controlled the viremia served as natural controllers. The frequency of SIV-Gag-specific fCD8 T cells was inversely correlated with the amount of cell-associated SIV- gag RNA in the Tfh only under cART or in the controllers but not in untreated progressors. scRNA-seq of SIV-Gag-specific fCD8 T cells in various conditions revealed that the gene expression pattern of SIV-Gag-specific fCD8 T cells in the controllers was closer to that of those under cART than the untreated progressors. Comparing the SIV-Gag-specific fCD8 T cells of those under cART to the controllers revealed their more exhausted and immunosenescent nature under cART. Improving the HIV/SIV-specific fCD8 T cells under cART by targeting those pathways might contribute to the development of potential curative strategies. IMPORTANCE We infected cynomolgus macaques with SIVmac239 to establish an SIV-chronically infected cART model. We performed an in-depth characterization of Tfh and fCD8 T cells in three conditions—chronic stage of untreated, cART-treated, and natural controller cynomolgus macaques—by combining tissue section analysis and single-cell analyses of sorted cells. We revealed the inverse relationship between Tfh infection and SIV-Gag-specific fCD8 T cell frequencies as observed in HIV-infected individuals, thereby establishing the cynomolgus macaque as a relevant animal model to study the determinants of HIV/SIV persistence in lymphoid tissue. Additionally, scRNA-seq analysis of SIV-Gag-specific fCD8 T cells revealed an enrichment of exhausted or senescent transcriptomic signatures under cART. These data will provide the basic insights into virus-host CD8 T cell interactions, particularly within the follicular region, during latent HIV infection under ART.

Subacute sclerosing panencephalitis (SSPE) is a lethal neurological disorder occurring several years after measles. Reconstruction of the evolution of the measles virus (MeV) genome in an SSPE case suggested that the matrix (M) protein mutation M-F50S, when added to other mutations, drove neuropathogenesis. However, whether and how M-F50S would promote spread independently from other mutations was in question. We investigated here the cell specificity of MeV spread in this brain and documented that both neurons and astrocytes were heavily infected. We then generated recombinant MeV with individual mutations in the three proteins of the viral membrane fusion apparatus, M, fusion (F), and hemagglutinin (H). These viruses reached similar titers as the parental wild-type virus, kept the respective mutations upon passage, and infected cells expressing the tissue-specific MeV receptors SLAM and nectin-4 with similar efficiencies. However, after inoculation of receptor-negative neurons and astrocytes differentiated from human induced pluripotent stem cells, only MeV M-F50S spread with moderate efficiency; the parental virus and its derivatives coding for a hyperfusogenic F protein, or for a cytoplasmic tail-mutated H protein, did not spread. When delivered to primary mouse neurons by cell-mediated neurite overlay, MeV M-F50S frequently reached the cell bodies and occasionally formed small infectious centers, while the other MeV reached the cell bodies only sporadically. These results demonstrate that, in neuronal cell cultures, M-F50S can enable receptor-independent spread in the absence of other mutations, and validate the inference that this single amino acid change initiated ubiquitous MeV brain spread. IMPORTANCE Measles virus (MeV), a non-integrating negative-strand RNA virus, rarely causes subacute sclerosing panencephalitis (SSPE) several years after acute infection. During brain adaptation, the MeV genome acquires multiple mutations reducing the dependence of its membrane fusion apparatus (MFA) from an activating receptor. It was proposed that one of these mutations, matrix protein F50S, drove neuropathogenesis in an SSPE case. We report here that, in two types of neuronal cultures, a recombinant MeV with only this mutation gained receptor-independent spread, whereas viruses expressing MFA proteins with other mutations acquired during brain adaptation did not. Our results validate the inference that M-F50S initiated ubiquitous MeV brain spread resulting in lethal disease. They also prompt studies of the impact of analogous amino acid changes of the M proteins of other nonsegmented negative-strand RNA viruses on their interactions with membrane lipids and cytoskeletal components.

Swine enteric coronaviruses pose a significant challenge to the global pig industry, inflicting severe diarrhea and high mortality rates among piglets, and resulting in substantial economic losses. In our clinical practice, we observed that the addition of potassium molybdate (PM) to the feed could dramatically reduce diarrhea and diarrhea-related mortality in piglets. However, the underlying mechanisms remain elusive and merit further investigation. In this study, we revealed that PM effectively inhibited the infection of both aminopeptidase N (APN)-dependent coronaviruses, transmissible gastroenteritis virus (TGEV), and porcine respiratory coronavirus (PRCV), both in vitro and ex vivo . Specifically, PM was found to block TGEV and PRCV penetration by degrading the cell receptor APN through the upregulation of phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3) expression. In addition, knockdown and knockout of PIK3C3 resulted in the attenuation of PM-induced autophagy, thereby rescuing APN expression and viral infection. Correspondingly, replenishment of PIK3C3 in PIK3C3-null ST cells restored PM-mediated APN degradation and successfully blocked viral entry. Furthermore, our findings demonstrated that PM promoted the assembly of the PIK3C3-BECN1-ATG14 complex, leading to induced autophagic degradation by upregulating PIK3C3 Ser249 phosphorylation. In vivo experiments further confirmed that PM-induced PIK3C3-mediated autophagic degradation of APN, thereby limiting the pathogenicity of TGEV. In summary, our study for the first time identified the mechanism by which PM blocked TGEV and PRCV internalization by degrading the cell receptor APN via PIK3C3-mediated autophagy. This study provides valuable insights and potential strategies for preventing APN-restricted coronavirus infection. IMPORTANCE Aminopeptidase N (APN) is one of the most important host receptors of coronavirus. Modulating APN expression can represent a novel approach for controlling APN-dependent coronaviruses and their variants infection. Here we found that a chemical compound potassium molybdate (PM) negatively regulates APN expression by inducing phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3)-mediated autophagy against APN-dependent coronavirus internalization, including transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV). Furthermore, PM can promote PIK3C3-BECN1-ATG14 complex assembly to induce autophagic degradation of APN by upregulating PIK3C3 Ser249 phosphorylation. Lastly, results from pig experiments also confirmed that PM can trigger PIK3C3-mediated autophagic degradation of APN to restrict TGEV pathogenicity in vivo without toxicity. Our findings underscore the promising potential of PM as an effective agent against APN-dependent coronavirus and potentially emerging viral disease entry.

Understanding how immune history influences influenza immunity is essential for developing effective vaccines and therapeutic strategies. This study examines the antigenic imprinting of influenza hemagglutinin (HA) and neuraminidase (NA) using a mouse model with sequential infections by H1N1 virus strains exhibiting substantial antigenic differences in HA. In our pre-2009 influenza infection model, we observed that mice with more extensive infection histories produced higher levels of functional NA-inhibiting antibodies (NAI). However, following further infection with the 2009 pandemic H1N1 strain, these mice demonstrated a reduced NAI to the challenged virus. Interestingly, prior exposure to older strains resulted in a lower HA antibody response (neutralization and HAI) to the challenged virus in both pre- and post-2009 scenarios, potentially due to faster viral clearance facilitated by immune memory recall. Overall, our findings reveal distinct trajectories in HA and NA immune responses, suggesting that immune imprinting can differentially impact these proteins based on the extent of antigenic variation in influenza viruses. IMPORTANCE Influenza viruses continue to pose a significant threat to human health, with vaccine effectiveness remaining a persistent challenge. Individual immune history is a crucial factor that can influence antibody responses to subsequent influenza exposures. While many studies have explored how pre-existing antibodies shape the induction of anti-HA antibodies following influenza virus infections or vaccinations, the impact on anti-NA antibodies has been less extensively studied. Using a mouse model, our study demonstrates that within pre-2009 H1N1 strains, an extensive immune history negatively impacted anti-HA antibody responses but enhanced anti-NA antibody responses. However, in response to the 2009 pandemic H1N1 strain, which experienced an antigenic shift, both anti-HA and anti-NA antibody responses were hindered by antibodies from prior pre-2009 H1N1 virus infections. These findings provide important insights into how antigenic imprinting affects both anti-HA and anti-NA antibody responses and underscore the need to consider immune history in developing more effective influenza vaccination strategies.

Kaposi’s sarcoma herpesvirus (KSHV) is the causative agent of Kaposi’s sarcoma and is associated with primary effusion lymphoma (PEL), multicentric Castleman’s disease, and two inflammatory diseases. KSHV-associated cancers are primarily associated with genes expressed during latency, while other pathologies are associated with lytic gene expression. The major lytic switch of the virus, Replication and Transcription Activator (RTA), interacts with cellular machinery to co-opt the host ubiquitin proteasome system to evade the immune response as well as activate the program of lytic replication. Through stable isotope labeling using amino acids in cell culture (SILAC) labeling, ubiquitin remnant enrichment, and mass spectrometry, we have analyzed the RTA-dependent ubiquitin-modified proteome. We identified RTA-dependent changes in the populations of polyubiquitin chains, as well as changes in ubiquitinated proteins in both cells expressing RTA and naturally infected cells following lytic reactivation. We observed an enrichment of proteins that are also reported to be SUMOylated, suggesting that RTA, a small ubiquitin-like modifier (SUMO) targeting ubiquitin ligase, may function to alleviate a SUMO-dependent block to lytic reactivation. RTA targeted substrates directly through a ubiquitin ligase domain-dependent mechanism as well as indirectly through cellular ubiquitin ligase RAUL. Our ubiquitome analysis revealed an RTA-dependent mechanism of immune evasion. We provide evidence of inhibition of transporter associated with antigen processing (TAP)-dependent peptide transport, resulting in decreased human leukocyte antigen (HLA) complex stability. The results of this analysis increase our understanding of mechanisms governing the latent to lytic transition in addition to the identification of a novel RTA-dependent mechanism of immune evasion. IMPORTANCE Kaposi’s sarcoma herpesvirus, an AIDS-associated pathogen, is associated with multiple cancers and inflammatory syndromes. This virus has a latent and lytic lifecycle, each associated with pathogenesis and oncogenesis. Here, we identify proteins that display differential abundance in different phases of the lifecycle. We provide evidence supporting a new model of viral immune evasion. These findings increase our understanding of how the virus manipulates the host cell and provides new targets for intervention.

Herpesviruses carry an assortment of proteins in the interstitial space between the capsid and membrane envelope, collectively referred to as the tegument. Upon virion fusion with a cell, envelope integrity is disrupted, and many tegument constituents disperse into the cytosol to carry out individual effector functions, while others direct transport of the capsid to the nucleus. To gain insight into the tegument dynamics that occur with disruption of envelope integrity, we used a combination of single-particle fluorescence and biochemical approaches that leveraged the previously established use of n-ethylmaleimide to inhibit virion dynamics. We document that the large tegument protein (pUL36), which is stably bound to the capsid surface at its C-terminus, is also conditionally bound to the capsid via its N-terminal deubiquitinase (DUB) domain. The DUB is released, while remaining tethered to the capsid by the pUL36 C-terminus, by a mechanism dependent on reactive cysteines. Mutation of these cysteines locks the DUB in a capsid bound state and suppresses enzymatic activity. IMPORTANCE Neuroinvasive alphaherpesviruses, such as herpes simplex virus and pseudorabies virus, cause a broad range of diseases in humans and other animals. Novel strategies to interfere with the virion structural rearrangements required for infectivity could prove valuable to treat infections, yet critical aspects of the virion architecture and its metastability remain poorly defined. In this study, we document that the pUL36 tegument protein exhibits conditional capsid binding in its N-terminal deubiquitinase domain that regulates enzymatic activity during infection.

Enterovirus 71 (EV71) infection is usually accompanied by neurological damage, which is the leading cause of death in children with hand-foot-mouth disease. In this study, we demonstrated that EV71 infection can cause pathological damage in the nervous system, such as neuronal vacuolar degeneration, shrinkage of some neurons, edema of brain tissues in the hippocampus, and a decreased number of Nissl bodies in the infarction area. Also, EV71 infection caused apparent structural damage to Schwann cells, including a decreased number of cytoplasmic organelles and severe damage of rough endoplasmic reticulum and mitochondria. However, the pathological damage was alleviated with the decrease of EV71 viral load. The cell experiment in vitro showed that EV71 infection significantly reduced ATP levels and promoted Schwann cell apoptosis, thus inhibiting cell growth. The extended infection time and the decreased viral load resulted in the gradual improvement of cell growth status. Meanwhile, EV71 inhibited the expression of miR-29b and promoted the expression of PMP22 in a time-dependent manner at both mRNA and protein levels, with the most significant change at 36 h of infection. Subsequently, the expression of miR-29b and PMP22 was gradually restored with the reduction of EV71 viral load. In addition, EV71 regulated the expression of hsa_circ_0069335, which could bind and co-localize with miR-29b. Therefore, EV71 infection can cause significant damage to the nervous system and may be related to hsa_circ_0069335/miR-29b/PMP22 pathway. The present study provides a new therapeutic target for neurological damage induced by EV71 infection. IMPORTANCE EV71 can cause severe neurological damage and even death, but the mechanism remains unclear. In this study, we exhibited the pathological changes of nervous system in EV71 infection and revealed that the damage degree was consistent with the EV71 viral load. From the molecular perspective, EV71 infection up-regulated the PMP22 expression in Schwann cells, which is accompanied by apparent structural damage of Schwann cells and myelin sheaths. Furthermore, EV71 promoted the expression of PMP22 and inhibited the expression of miR-29b in a time-dependent manner, with the most significant change at 36 h of infection. Otherwise, the hsa_circ_0069335, which binds and co-localizes with miR-29b, was also regulated by EV71 infection. The hsa_circ_0069335/miR-29b/PMP22 axis may be a potential molecular mechanism involved in EV71 infection-induced fatal neuronal damage. Drug development targeting this pathway may bring clinical improvement of EV71-infected patients.

Infection by human astrovirus (HAstV), a small, positive-strand RNA virus, is a major cause of gastroenteritis and has been implicated in an increasing number of severe, sometimes fatal, neurological diseases since 2008. Currently, there are no vaccines or antiviral treatments available to treat HAstV infection. An attractive target for antiviral therapeutics is the viral protease due to its essential functions throughout infection. However, the molecular mechanisms of the HAstV protease, nonstructural protein 1a/3 (nsp1a/3), are poorly understood. In fact, the specific residues within the cleavage junctions that are targeted by nsp1a/3 during polyprotein processing have yet to be experimentally identified. To identify the junctions between viral proteins, we performed mass spectrometry and site-directed mutagenesis using epitope-tagged viral polyprotein expression plasmids. Using these strategies, we identified a consensus motif that is found throughout the polyprotein near previously proposed junctions. We found that cleavage occurs after a hydrophobic residue – X – Gln motif. Further mutagenesis of surrounding sequence identified the importance of basic residues following the motif for efficient processing. Cleavage at each junction was determined to be essential for the production of progeny virions. However, abolishing nsp1a/4-VPg cleavage allowed efficient replication, suggesting that VPg can function in an intermediate form. Overall, our results identify a conserved cleavage motif that is recognized by the nsp1a/3 protease within the viral polyprotein, and cleavage at this motif was found to be essential for the recovery of progeny virions. These findings will be instrumental in further understanding the basic functions of HAstV polyprotein processing during infection. IMPORTANCE Human astroviruses (HAstVs) are a leading cause of non-bacterial gastroenteritis in children, elderly individuals, and immunocompromised patients. However, infection by divergent strains of HAstV is now recognized as a causative agent of severe neurological diseases, which can have fatal outcomes. Despite the global prevalence of HAstV, we currently have a limited understanding of the biology of these viruses. Translation of the viral genome leads to the production of polyproteins that are processed by viral and host proteases into functional proteins. In this study, we identified a conserved recognition sequence targeted by the viral protease for cleavage. Importantly, these findings elucidate the N- and C-termini of the nonstructural proteins within the HAstV polyprotein, offering valuable information for future studies on the function of individual viral proteins. Similar to other positive-sense RNA viruses, the necessity of proteolytic processing for the HAstV polyprotein highlights the viral protease as a promising target for antiviral development.

H5 highly pathogenic avian influenza (HPAI) viruses of the A/Goose/Guangdong/1/96 (Gs/Gd) lineage continue to evolve and cause outbreaks in domestic poultry and wild birds, with sporadic spillover infections in mammals. The global spread of clade 2.3.4.4b viruses via migratory birds since 2020 has facilitated the introduction of novel reassortants to China, where avian influenza of various subtypes have been epizootic or enzootic among domestic birds. To determine the impact of clade 2.3.4.4b re-introduction on local HPAI dynamics, we analyzed the genetic diversity of H5N6 and H5N8 detected from monthly poultry market surveillance in Guangdong, China, between 2020 and 2022. Our findings reveal that H5N6 viruses clustered in clades 2.3.4.4b and 2.3.4.4h, while H5N8 viruses were exclusively clustered in clade 2.3.4.4b. After 2020, the re-introduced clade 2.3.4.4b viruses replaced the clade 2.3.4.4h viruses detected in 2020. The N6 genes were divided into two clusters, distinguished by an 11 amino acid deletion in the stalk region, while the N8 genes clustered with clade 2.3.4.4 H5N8 viruses circulating among wild birds. Genomic analysis identified 10 transient genotypes. H5N6, which was more prevalently detected, was also clustered into more genotypes than H5N8. Specifically, H5N6 isolates contained genes derived from HPAI H5Nx viruses and low pathogenic avian influenza in China, while the H5N8 isolates contained genes derived from HPAI A(H5N8) 2.3.4.4b and A(H5N1) 2.3.2.1c. No positive selection on amino acid residues associated with mammalian adaptation was found. Our results suggest expanded genetic diversity of H5Nx viruses in China since 2021 with increasing challenges for pandemic preparedness. IMPORTANCE Since 2016/2017, clade 2.3.4.4b H5Nx viruses have spread via migratory birds to all continents except Oceania. Here, we evaluated the impact of the re-introduction of clade of 2.3.4.4b on highly pathogenic avian influenza (HPAI) virus genetic diversity in China. Twenty-two H5N6 and H5N8 HPAI isolated from monthly surveillance in two poultry markets in Guangdong between 2020 and 2022 were characterized. Our findings showed that clade 2.3.4.4h, detected in 2020, was replaced by clade 2.3.4.4b in 2021–2022. H5N6 ( n = 18) were clustered into more genotypes than H5N8 ( n = 4), suggesting that H5N6 may possess better replication fitness in poultry. Conversely, the H5N8 genotypes are largely derived from the clade 2.3.4.4b wild bird isolates. As clade 2.3.4.4b continues to spread via migratory birds, it is anticipated that the genetic diversity of H5N6 viruses circulating in China may continue to expand in the coming years. Continuous efforts in surveillance, genetic analysis, and risk assessment are therefore crucial for pandemic preparedness.