An Epstein-Barr virus protein interaction map reveals NLRP3 inflammasome evasion via MAVS UFMylation.

Epstein-Barr virus (EBV) causes infectious mononucleosis, triggers multiple sclerosis, and is associated with 200,000 cancers/year. EBV colonizes the human B cell compartment and periodically reactivates, inducing expression of 80 viral proteins. However, much remains unknown about how EBV remodels host cells and dismantles key antiviral responses. We therefore created a map of EBV-host and EBV-EBV interactions in B cells undergoing EBV replication, uncovering conserved herpesvirus versus EBV-specific host cell targets. The EBV-encoded G-protein-coupled receptor BILF1 associated with MAVS and the UFM1 E3 ligase UFL1. Although UFMylation of 14-3-3 proteins drives RIG-I/MAVS signaling, BILF1-directed MAVS UFMylation instead triggered MAVS packaging into mitochondrial-derived vesicles and lysosomal proteolysis. In the absence of BILF1, EBV replication activated the NLRP3 inflammasome, which impaired viral replication and triggered pyroptosis. Our results provide a viral protein interaction network resource, reveal a UFM1-dependent pathway for selective degradation of mitochondrial cargo, and highlight BILF1 as a novel therapeutic target.

Similarities and Dissimilarities of COVID-19 and Other Coronavirus Diseases.

In less than two decades, three deadly zoonotic coronaviruses, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2, have emerged in humans, causing SARS, MERS, and coronavirus disease 2019 (COVID-19), respectively. The current COVID-19 pandemic poses an unprecedented crisis in health care and social and economic development. It reinforces the cruel fact that CoVs are constantly evolving, possessing the genetic malleability to become highly pathogenic in humans. In this review, we start with an overview of CoV diseases and the molecular virology of CoVs, focusing on similarities and differences between SARS-CoV-2 and its highly pathogenic as well as low-pathogenic counterparts. We then discuss mechanisms underlying pathogenesis and virus-host interactions of SARS-CoV-2 and other CoVs, emphasizing the host immune response. Finally, we summarize strategies adopted for the prevention and treatment of CoV diseases and discuss approaches to develop effective antivirals and vaccines.

Human FAM111A inhibits vaccinia virus replication by degrading viral protein I3 and is antagonized by poxvirus host range factor SPI-1.

Zoonotic poxviruses such as mpox virus (MPXV) continue to threaten public health safety since the eradication of smallpox. Vaccinia virus (VACV), the prototypic poxvirus used as the vaccine strain for smallpox eradication, is the best-characterized member of the poxvirus family. VACV encodes a serine protease inhibitor 1 (SPI-1) conserved in all orthopoxviruses, which has been recognized as a host range factor for modified VACV Ankara (MVA), an approved smallpox vaccine and a promising vaccine vector. FAM111A (family with sequence similarity 111 member A), a nuclear protein that regulates host DNA replication, was shown to restrict the replication of a VACV SPI-1 deletion mutant (VACV-ΔSPI-1) in human cells. Nevertheless, the detailed antiviral mechanisms of FAM111A were unresolved. Here, we show that FAM111A is a potent restriction factor for VACV-ΔSPI-1 and MVA. Deletion of FAM111A rescued the replication of MVA and VACV-ΔSPI-1 and overexpression of FAM111A significantly reduced viral DNA replication and virus titers but did not affect viral early gene expression. The antiviral effect of FAM111A necessitated its trypsin-like protease domain and DNA-binding domain but not the PCNA-interacting motif. We further identified that FAM111A translocated into the cytoplasm upon VACV infection by degrading the nuclear pore complex via its protease activity, interacted with VACV DNA-binding protein I3, and promoted I3 degradation through autophagy. Moreover, SPI-1 from VACV, MPXV, or lumpy skin disease virus was able to antagonize FAM111A by prohibiting its nuclear export. Our findings reveal the detailed mechanism by which FAM111A inhibits VACV and provide explanations for the immune evasive function of VACV SPI-1.

Host factor MxA restricts Dabie bandavirus infection by targeting the viral NP protein to inhibit NP-RdRp interaction and ribonucleoprotein activity.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with high case mortality rates, which is caused by Dabie bandavirus (DBV), a novel pathogen also termed as SFTS virus (SFTSV). Currently, no specific therapeutic drugs or vaccines are available for SFTS. Myxovirus resistance protein A (MxA) has been shown to inhibit multiple viral pathogens; however, the role of MxA in DBV infection is unknown. Here, we demonstrated that DBV stimulates MxA expression which, in turn, restricts DBV infection. Mechanistic target analysis revealed that MxA specifically interacts with the viral nucleocapsid protein (NP) in a manner independent of RNA. Minigenome reporter assay showed that in agreement with its targeting of NP, MxA inhibits DBV ribonucleoprotein (RNP) activity. In detail, MxA interacts with the NP N-terminal and disrupts the interaction of NP with the viral RNA-dependent RNA polymerase (RdRp) but not NP multimerization, the critical activities of NP for RNP formation and function. Furthermore, MxA N-terminal domain was identified as the functional domain inhibiting DBV infection, and, consistently, then was shown to interact with NP and obstruct the NP-RdRp interaction. Additionally, threonine 103 within the N-terminal domain is important for MxA inhibition to DBV, and its mutation (T103A) attenuates MxA binding to NP and obstruction of the NP-RdRp interaction. This study uncovers MxA inhibition of DBV with a series of functional and mechanistical analyses, providing insights into the virus-host interactions and probably helping inform the development of antiviral agents in the future.IMPORTANCEDBV/SFTSV is an emerging high-pathogenic virus. Since its first identification in China in 2009, cases of DBV infection have been reported in many other countries, posing a significant threat to public health. Uncovering the mechanisms of DBV-host interactions is necessary to understand the viral pathogenesis and host response and may advance the development of antiviral therapeutics. Here, we found that host factor MxA whose expression is induced by DBV restricts the virus infection. Mechanistically, MxA specifically interacts with the viral NP and blocks the NP-RdRp interaction, inhibiting the viral RNP activity. Further studies identified the key domain and amino acid residue required for MxA inhibition to DBV. Consistently, they were then shown to be important for MxA targeting of NP and obstruction of the NP-RdRp association. These findings unravel the restrictive role of MxA in DBV infection and the underlying mechanism, expanding our knowledge of the virus-host interactions.

Hepatitis E virus ORF3 protein hijacking thioredoxin domain-containing protein 5 (TXNDC5) for its stability to promote viral particle release.

Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide, responsible for approximately 20 million infections annually. Among the three open reading frames (ORFs) of the HEV genome, the ORF3 protein is involved in virus release. However, the host proteins involved in HEV release need to be clarified. In this study, a host protein, thioredoxin domain-containing protein 5 (TXNDC5), interacted with the non-palmitoylated ORF3 protein by co-immunoprecipitation analysis. We determined that the overexpression or knockdown of TXNDC5 positively regulated HEV release from the host cells. The 17FCL19 mutation of the ORF3 protein lost the ability to interact with TXNDC5. The releasing amounts of HEV with the ORF3 mutation (FCL17-19SSP) were decreased compared with wild-type HEV. The overexpression of TXNDC5 can stabilize and increase ORF3 protein amounts, but not the TXNDC5 mutant with amino acids 1-88 deletion. Meanwhile, we determined that the function of TXNDC5 on the stabilization of ORF3 protein is independent of the Trx-like domains. Knockdown of TXNDC5 could lead to the degradation of ORF3 protein by the endoplasmic reticulum (ER)-associated protein degradation-proteasome system. However, the ORF3 protein cannot be degraded in the knockout-TXNDC5 stable cells, suggesting that it may hijack other proteins for its stabilization. Subsequently, we found that the other members of protein disulfide isomerase (PDI), including PDIA1, PDIA3, PDIA4, and PDIA6, can increase ORF3 protein amounts, and PDIA3 and PDIA6 interact with ORF3 protein. Collectively, our study suggested that HEV ORF3 protein can utilize TXNDC5 for its stability in ER to facilitate viral release. IMPORTANCE: Hepatitis E virus (HEV) infection is the leading cause of acute viral hepatitis worldwide. After the synthesis and modification in the cells, the mature ORF3 protein is essential for HEV release. However, the host protein involved in this process has yet to be determined. Here, we reported a novel host protein, thioredoxin domain-containing protein 5 (TXNDC5), as a chaperone, contributing to HEV release by facilitating ORF3 protein stability in the endoplasmic reticulum through interacting with non-palmitoylated ORF3 protein. However, we also found that in the knockout-TXNDC5 stable cell lines, the HEV ORF3 protein may hijack other proteins for its stabilization. For the first time, our study demonstrated the involvement of TXNDC5 in viral particle release. These findings provide some new insights into the process of the HEV life cycle, the interaction between HEV and host factors, and a new direction for antiviral design.

CCR4-NOT differentially controls host versus virus poly(a)-tail length and regulates HCMV infection.

Unlike most RNA and DNA viruses that broadly stimulate mRNA decay and interfere with host gene expression, human cytomegalovirus (HCMV) extensively remodels the host translatome without producing an mRNA decay enzyme. By performing a targeted loss-of-function screen in primary human fibroblasts, we here identify the host CCR4-NOT deadenylase complex members CNOT1 and CNOT3 as unexpected pro-viral host factors that selectively regulate HCMV reproduction. We find that the scaffold subunit CNOT1 is specifically required for late viral gene expression and genome-wide host responses in CCR4-NOT-disrupted cells. By profiling poly(A)-tail lengths of individual HCMV and host mRNAs using nanopore direct RNA sequencing, we reveal poly(A)-tails of viral messages to be markedly longer than those of cellular mRNAs and significantly less sensitive to CCR4-NOT disruption. Our data establish that mRNA deadenylation by host CCR4-NOT is critical for productive HCMV replication and define a new mechanism whereby herpesvirus infection subverts cellular mRNA metabolism to remodel the gene expression landscape of the infected cell. Moreover, we expose an unanticipated host factor with potential to become a therapeutic anti-HCMV target.

A New Cellular Interactome of SARS-CoV-2 Nucleocapsid Protein and Its Biological Implications.

There is still much to uncover regarding the molecular details of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. As the most abundant protein, coronavirus nucleocapsid (N) protein encapsidates viral RNAs, serving as the structural component of ribonucleoprotein and virion, and participates in transcription, replication, and host regulations. Virus-host interaction might give clues to better understand how the virus affects or is affected by its host during infection and identify promising therapeutic candidates. Considering the critical roles of N, we here established a new cellular interactome of SARS-CoV-2 N by using a high-specific affinity purification (S-pulldown) assay coupled with quantitative mass spectrometry and immunoblotting validations, uncovering many N-interacting host proteins unreported previously. Bioinformatics analysis revealed that these host factors are mainly involved in translation regulations, viral transcription, RNA processes, stress responses, protein folding and modification, and inflammatory/immune signaling pathways, in line with the supposed actions of N in viral infection. Existing pharmacological cellular targets and the directing drugs were then mined, generating a drug-host protein network. Accordingly, we experimentally identified several small-molecule compounds as novel inhibitors against SARS-CoV-2 replication. Furthermore, a newly identified host factor, DDX1, was verified to interact and colocalize with N mainly by binding to the N-terminal domain of the viral protein. Importantly, loss/gain/reconstitution-of-function experiments showed that DDX1 acts as a potent anti-SARS-CoV-2 host factor, inhibiting the viral replication and protein expression. The N-targeting and anti-SARS-CoV-2 abilities of DDX1 are consistently independent of its ATPase/helicase activity. Further mechanism studies revealed that DDX1 impedes multiple activities of N, including the N-N interaction, N oligomerization, and N-viral RNA binding, thus likely inhibiting viral propagation. These data provide new clues to better depiction of the N-cell interactions and SARS-CoV-2 infection and may help inform the development of new therapeutic candidates.

Transcriptome profiling in swine macrophages infected with African swine fever virus at single-cell resolution.

African swine fever virus (ASFV) is the causative agent of African swine fever, a highly contagious and usually fatal disease in pigs. The pathogenesis of ASFV infection has not been clearly elucidated. Here, we used single-cell RNA-sequencing technology to survey the transcriptomic landscape of ASFV-infected primary porcine alveolar macrophages. The temporal dynamic analysis of viral genes revealed increased expression of viral transmembrane genes. Molecular characteristics in the ASFV-exposed cells exhibited the activation of antiviral signaling pathways with increased expression levels of interferon-stimulated genes and inflammatory- and cytokine-related genes. By comparing infected cells with unexposed cells, we showed that the unfolded protein response (UPR) pathway was activated in low viral load cells, while the expression level of UPR-related genes in high viral load cells was less than that in unexposed cells. Cells infected with various viral loads showed signature transcriptomic changes at the median progression of infection. Within the infected cells, differential expression analysis and coregulated virus­host analysis both demonstrated that ASFV promoted metabolic pathways but inhibited interferon and UPR signaling, implying the regulation pathway of viral replication in host cells. Furthermore, our results revealed that the cell apoptosis pathway was activated upon ASFV infection. Mechanistically, the production of tumor necrosis factor alpha (TNF-α) induced by ASFV infection is necessary for cell apoptosis, highlighting the importance of TNF-α in ASFV pathogenesis. Collectively, the data provide insights into the comprehensive host responses and complex virus­host interactions during ASFV infection, which may instruct future research on antiviral strategies.

HIV-1 hypermethylated guanosine cap licenses specialized translation unaffected by mTOR.

Appended to the 5' end of nascent RNA polymerase II transcripts is 7-methyl guanosine (m7G-cap) that engages nuclear cap-binding complex (CBC) to facilitate messenger RNA (mRNA) maturation. Mature mRNAs exchange CBC for eIF4E, the rate-limiting translation factor that is controlled through mTOR. Experiments in immune cells have now documented HIV-1 incompletely processed transcripts exhibited hypermethylated m7G-cap and that the down-regulation of the trimethylguanosine synthetase-1-reduced HIV-1 infectivity and virion protein synthesis by several orders of magnitude. HIV-1 cap hypermethylation required nuclear RNA helicase A (RHA)/DHX9 interaction with the shape of the 5' untranslated region (UTR) primer binding site (PBS) segment. Down-regulation of RHA or the anomalous shape of the PBS segment abrogated hypermethylated caps and derepressed eIF4E binding for virion protein translation during global down-regulation of host translation. mTOR inhibition was detrimental to HIV-1 proliferation and attenuated Tat, Rev, and Nef synthesis. This study identified mutually exclusive translation pathways and the calibration of virion structural/accessory protein synthesis with de novo synthesis of the viral regulatory proteins. The hypermethylation of select, viral mRNA resulted in CBC exchange to heterodimeric CBP80/NCBP3 that expanded the functional capacity of HIV-1 in immune cells.

CP204L Is a Multifunctional Protein of African Swine Fever Virus That Interacts with the VPS39 Subunit of the Homotypic Fusion and Vacuole Protein Sorting Complex and Promotes Lysosome Clustering.

Virus replication depends on a complex interplay between viral and host proteins. In the case of African swine fever virus (ASFV), a large DNA virus, only a few virus-host protein-protein interactions have been identified to date. In this study, we demonstrate that the ASFV protein CP204L interacts with the cellular homotypic fusion and protein sorting (HOPS) protein VPS39, blocking its association with the lysosomal HOPS complex, which modulates endolysosomal trafficking and promotes lysosome clustering. Instead, CP204L and VPS39 are targeted to virus factories and localized at the periphery of the virus DNA replication sites. Furthermore, we show that loss of VPS39 reduces the levels of virus proteins synthesized in the early phase of infection and delays ASFV replication but does not completely inhibit it. Collectively, these results identify a novel virus-host protein interaction that modulates host membrane rearrangement during infection and provide evidence that CP204L is a multifunctional protein engaged in distinct steps of the ASFV life cycle. IMPORTANCE African swine fever virus (ASFV) was first identified over a hundred years ago. Since then, much effort has been made to understand the pathogenesis of ASFV. However, the specific roles of many individual ASFV proteins during the infection remain enigmatic. This study provides evidence that CP204L, one of the most abundant ASFV proteins, modulates endosomal trafficking during virus infection. Through protein-protein interaction, CP204L prevents the recruitment of VPS39 to the endosomal and lysosomal membranes, resulting in their accumulation. Consequently, CP204L and VPS39 become sequestered in the ASFV replication and assembly site, known as the virus factory. These results uncover a novel function of viral protein CP204L and extend our understanding of complex interaction between virus and host.

Genome-Scale Analysis of Cellular Restriction Factors That Inhibit Transgene Expression from Adeno-Associated Virus Vectors.

Adeno-associated virus (AAV) vectors are one of the leading platforms for gene delivery for the treatment of human genetic diseases, but the antiviral cellular mechanisms that interfere with optimal transgene expression are incompletely understood. Here, we performed two genome-scale CRISPR screens to identify cellular factors that restrict transgene expression from recombinant AAV vectors. Our screens revealed several components linked to DNA damage response, chromatin remodeling, and transcriptional regulation. Inactivation of the Fanconi anemia gene FANCA; the human silencing hub (HUSH)-associated methyltransferase SETDB1; and the gyrase, Hsp90, histidine kinase, and MutL (GHKL)-type ATPase MORC3 led to increased transgene expression. Moreover, SETDB1 and MORC3 knockout improved transgene levels of several AAV serotypes as well as other viral vectors, such as lentivirus and adenovirus. Finally, we demonstrated that the inhibition of FANCA, SETDB1, or MORC3 also enhanced transgene expression in human primary cells, suggesting that they could be physiologically relevant pathways that restrict AAV transgene levels in therapeutic settings. IMPORTANCE Recombinant AAV (rAAV) vectors have been successfully developed for the treatment of genetic diseases. The therapeutic strategy often involves the replacement of a defective gene by the expression of a functional copy from the rAAV vector genome. However, cells possess antiviral mechanisms that recognize and silence foreign DNA elements thereby limiting transgene expression and its therapeutic effect. Here, we utilize a functional genomics approach to uncover a comprehensive set of cellular restriction factors that inhibit rAAV-based transgene expression. Genetic inactivation of selected restriction factors increased rAAV transgene expression. Hence, modulation of identified restriction factors has the potential to enhance AAV gene replacement therapies.

Co-opted cytosolic proteins form condensate substructures within membranous replication organelles of a positive-strand RNA virus.

Positive-strand RNA viruses co-opt organellar membranes for biogenesis of viral replication organelles (VROs). Tombusviruses also co-opt pro-viral cytosolic proteins to VROs. It is currently not known what type of molecular organization keeps co-opted proteins sequestered within membranous VROs. In this study, we employed tomato bushy stunt virus (TBSV) and carnation Italian ringspot virus (CIRV) - Nicotiana benthamiana pathosystems to identify biomolecular condensate formation in VROs. We show that TBSV p33 and the CIRV p36 replication proteins sequester glycolytic and fermentation enzymes in unique condensate substructures associated with membranous VROs. We find that p33 and p36 form droplets in vitro driven by intrinsically disordered region. The replication protein organizes partitioning of co-opted host proteins into droplets. VRO-associated condensates are critical for local adenosine triphosphate production to support energy for virus replication. We find that co-opted endoplasmic reticulum membranes and actin filaments form meshworks within and around VRO condensates, contributing to unique composition and structure. We propose that p33/p36 organize liquid-liquid phase separation of co-opted concentrated host proteins in condensate substructures within membranous VROs. Overall, we demonstrate that subverted membranes and condensate substructures co-exist and are critical for VRO functions. The replication proteins induce and connect the two substructures within VROs.

SARS-CoV-2 NSP12 utilizes various host splicing factors for replication and splicing regulation.

The RNA-dependent RNA polymerase (RdRp) is a crucial element in the replication and transcription of RNA viruses. Although the RdRps of lethal human coronaviruses severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV) have been extensively studied, the molecular mechanism of the catalytic subunit NSP12, which is involved in pathogenesis, remains unclear. In this study, the biochemical and cell biological results demonstrate the interactions between SARS-CoV-2 NSP12 and seven host proteins, including three splicing factors (SLU7, PPIL3, and AKAP8). The entry efficacy of SARS-CoV-2 considerably decreased when SLU7 or PPIL3 was knocked out, indicating that abnormal splicing of the host genome was responsible for this occurrence. Furthermore, the polymerase activity and stability of SARS-CoV-2 RdRp were affected by the three splicing factors to varying degrees. In addition, NSP12 and its homologues from SARS-CoV and MERS-CoV suppressed the alternative splicing of cellular genes, which were influenced by the three splicing factors. Overall, our research illustrates that SARS-CoV-2 NSP12 can engage with various splicing factors, thereby impacting virus entry, replication, and gene splicing. This not only improves our understanding of how viruses cause diseases but also lays the foundation for the development of antiviral therapies.

Clash of the titans: interferons and SARS-CoV-2.

Interferons are our first line of defense against invading viruses. However, viruses encode effector proteins that can modulate human interferon responses. In this forum article, we highlight important discoveries and discuss outstanding questions that will enable us to better understand the nuances of this evolutionary battle between interferons and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Serine-arginine protein kinases and their targets in viral infection and their inhibition.

Accumulating evidence has consolidated the interaction between viral infection and host alternative splicing. Serine-arginine (SR) proteins are a class of highly conserved splicing factors critical for the spliceosome maturation, alternative splicing and RNA metabolism. Serine-arginine protein kinases (SRPKs) are important kinases that specifically phosphorylate SR proteins to regulate their distribution and activities in the central pre-mRNA splicing and other cellular processes. In addition to the predominant SR proteins, other cytoplasmic proteins containing a serine-arginine repeat domain, including viral proteins, have been identified as substrates of SRPKs. Viral infection triggers a myriad of cellular events in the host and it is therefore not surprising that viruses explore SRPKs-mediated phosphorylation as an important regulatory node in virus-host interactions. In this review, we briefly summarize the regulation and biological function of SRPKs, highlighting their involvement in the infection process of several viruses, such as viral replication, transcription and capsid assembly. In addition, we review the structure-function relationships of currently available inhibitors of SRPKs and discuss their putative use as antivirals against well-characterized viruses or newly emerging viruses. We also highlight the viral proteins and cellular substrates targeted by SRPKs as potential antiviral therapeutic candidates.

The phosphatidylserine receptor TIM1 promotes infection of enveloped hepatitis E virus.

The hepatitis E virus (HEV) is an underestimated RNA virus of which the viral life cycle and pathogenicity remain partially understood and for which specific antivirals are lacking. The virus exists in two forms: nonenveloped HEV that is shed in feces and transmits between hosts; and membrane-associated, quasi-enveloped HEV that circulates in the blood. It is suggested that both forms employ different mechanisms for cellular entry and internalization but little is known about the exact mechanisms. Interestingly, the membrane of enveloped HEV is enriched with phosphatidylserine, a natural ligand for the T-cell immunoglobulin and mucin domain-containing protein 1 (TIM1) during apoptosis and involved in 'apoptotic mimicry', a process by which viruses hijack the apoptosis pathway to promote infection. We here investigated the role of TIM1 in the entry process of HEV. We determined that HEV infection with particles derived from culture supernatant, which are cloaked by host-derived membranes (eHEV), was significantly impaired after knockout of TIM1, whereas infection with intracellular HEV particles (iHEV) was unaffected. eHEV infection was restored upon TIM1 expression; and enhanced after ectopic TIM1 expression. The significance of TIM1 during entry was further confirmed by viral binding assay, and point mutations of the PS-binding pocket diminished eHEV infection. In addition, Annexin V, a PS-binding molecule also significantly reduced infection. Taken together, our findings support a role for TIM1 in eHEV-mediated cell entry, facilitated by the PS present on the viral membrane, a strategy HEV may use to promote viral spread throughout the infected body.

Three picorna-like viruses found associated with the spider mite, Tetranychus truncatus (Acari: Tetranychidae).

Herbivorous arthropods, such as mites and insects, host a variety of microorganisms that significantly influence their ecology and evolution. While insect viruses have been extensively studied, our understanding of the diversity and composition of mite viromes and the interactions with mite hosts remains limited. The Asian spider mite, Tetranychus truncatus Ehara (Acari: Tetranychidae), a major agricultural pest, has not yet been reported to harbor any viruses. Here, using publicly available RNA-Seq data, we identified and characterized three picorna-like viruses associated with T. truncatus: Tetranychus truncatus-associated iflavirus 1 (TtAIV-1), Tetranychus truncatus-associated picorna-like virus 1 (TtAV-1), and Tetranychus truncatus-associated picorna-like virus 2 (TtAV-2). TtAIV-1 has a typical Iflaviridae genome structure with a single ORF, representing the first iflavirus associated with the Tetranychus genus. TtAV-1 and TtAV-2 exhibit bicistronic arrangements similar to dicistroviruses and other picorna-like viruses, with complex secondary structures in their non-coding regions. Phylogenetic analysis places TtAIV-1 within Iflaviridae, possibly as a new species, while TtAV-1 and TtAV-2 form distinct clades within unclassified picorna-like viruses, suggesting new families within Picornavirales. We analyzed in silico the presence and abundance of these viruses in T. truncatus across four bioproject SRAs, mostly finding them co-associated, with viral reads reaching up to 30% of total reads. Their presence and abundance varied by mite treatment and origin, with no significant impact from Wolbachia infection or abamectin exposure, although TtAV-2 was absent in abamectin-treated mites. Temperature influenced virus abundance, and variations were observed among Chinese mite populations based on geography and host plant association. Our findings offer insights into picorna-like virus diversity and dynamics in T. truncatus, revealing potential roles in mite biology and suggesting applications for mite population control, thereby enhancing agricultural productivity and food security.

Dual nature of human ACE2 glycosylation in binding to SARS-CoV-2 spike.

Binding of the spike protein of SARS-CoV-2 to the human angiotensin-converting enzyme 2 (ACE2) receptor triggers translocation of the virus into cells. Both the ACE2 receptor and the spike protein are heavily glycosylated, including at sites near their binding interface. We built fully glycosylated models of the ACE2 receptor bound to the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Using atomistic molecular dynamics (MD) simulations, we found that the glycosylation of the human ACE2 receptor contributes substantially to the binding of the virus. Interestingly, the glycans at two glycosylation sites, N90 and N322, have opposite effects on spike protein binding. The glycan at the N90 site partly covers the binding interface of the spike RBD. Therefore, this glycan can interfere with the binding of the spike protein and protect against docking of the virus to the cell. By contrast, the glycan at the N322 site interacts tightly with the RBD of the ACE2-bound spike protein and strengthens the complex. Remarkably, the N322 glycan binds to a conserved region of the spike protein identified previously as a cryptic epitope for a neutralizing antibody. By mapping the glycan binding sites, our MD simulations aid in the targeted development of neutralizing antibodies and SARS-CoV-2 fusion inhibitors.

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

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

Herpesvirus-mediated stabilization of ICP0 expression neutralizes restriction by TRIM23.

Herpes simplex virus (HSV) infection relies on immediate early proteins that initiate viral replication. Among them, ICP0 is known, for many years, to facilitate the onset of viral gene expression and reactivation from latency. However, how ICP0 itself is regulated remains elusive. Through genetic analyses, we identify that the viral γ134.5 protein, an HSV virulence factor, interacts with and prevents ICP0 from proteasomal degradation. Furthermore, we show that the host E3 ligase TRIM23, recently shown to restrict the replication of HSV-1 (and certain other viruses) by inducing autophagy, triggers the proteasomal degradation of ICP0 via K11- and K48-linked ubiquitination. Functional analyses reveal that the γ134.5 protein binds to and inactivates TRIM23 through blockade of K27-linked TRIM23 autoubiquitination. Deletion of γ134.5 or ICP0 in a recombinant HSV-1 impairs viral replication, whereas ablation of TRIM23 markedly rescues viral growth. Herein, we show that TRIM23, apart from its role in autophagy-mediated HSV-1 restriction, down-regulates ICP0, whereas viral γ134.5 functions to disable TRIM23. Together, these results demonstrate that posttranslational regulation of ICP0 by virus and host factors determines the outcome of HSV-1 infection.