Journal of Virology最新文献

Acidic nuclear phosphoprotein 32 family member A (ANP32A) is an important host factor that supports the efficient replication of avian influenza viruses (AIVs). To develop an antiviral strategy against Gs/Gd-lineage H5 highly pathogenic avian influenza (HPAI) viruses in chickens, we established chicken ANP32-knockout (chANP32A-KO) DF-1 cells and evaluated their antiviral efficacy through in vitro validation. The replication of all HPAI viruses tested in chANP32A-KO cells was significantly lower compared to that of wild-type DF-1 cells. However, when HPAI strains A/mountain hawk-eagle/Kumamoto/1/2007 (H5N1; MHE) and A/chicken/Aichi/2/2011 (H5N1; H5Aichi) were passed in chANP32A-KO cells, mutant viruses were generated, which exhibited comparable replication levels in both chANP32A-KO and wild-type DF-1 cells. Sequence analysis revealed that mammalian-adaptive amino acid mutations PB2_D256G and PA_T97I were present in the MHE mutant virus, and the PB2_E627K mutation was identified in the H5Aichi mutant virus. These mutations have also been reported to enhance the polymerase activity of AIVs in mammalian cells; however, the minigenome assay in the present study showed that the polymerase activity of mutant viruses in chANP32A-KO cells was not restored to levels comparable to those in wild-type DF-1 cells. These findings suggest that ANP32A-independent viral replication may induce amino acid substitutions associated with mammalian adaptation in AIVs. They also imply that the high efficiency of viral replication mediated by these amino acid mutations may not result from enhanced polymerase activity but rather involve other undefined mechanisms.IMPORTANCEDuring the host-switching of avian influenza viruses (AIVs) to mammalian hosts, introducing adaptive mutations into viral proteins is essential to ensure optimal functionality through virus-host protein interactions in mammalian cells. However, the mechanisms leading to adaptive mutations in viral proteins remain unclear. Among several host proteins that promote viral growth, acidic nuclear phosphoprotein 32 family member A (ANP32A) is known to be an important factor for efficient viral replication. Here, we generated mutant highly pathogenic avian influenza viruses capable of ANP32A-independent replication in a chicken-derived cell line. We demonstrated that several amino acid mutations found in the mutant viruses correspond to those associated with the mammalian adaptation of AIVs. These results suggest that ANP32A-independent viral replication is one of the mechanisms for introducing amino acid mutations that are reportedly involved in the mammalian adaptation of AIVs.

Membrane fusion occurs at the early stages of SARS-CoV-2 replication, during entry of the virus, and later during the formation of multinucleated cells called syncytia. Fusion is mediated by the binding of the viral Spike protein to its receptor ACE2. Lipid rafts are dynamic nanodomains enriched in cholesterol and sphingolipids. Rafts can act as platforms for entry of different viruses by localizing virus receptors, and attachment factors to the same membrane domains. Here, we first demonstrate that cholesterol depletion by methyl-beta-cyclodextrin inhibits Spike-mediated fusion and entry. To further study the role of ACE2 lipid raft localization in SARS-CoV-2 fusion and entry, we designed a GPI-anchored ACE2 construct. Both ACE2 and ACE2-GPI proteins were similarly expressed at the plasma membrane. Through membrane flotation assays, we show that in different cell lines, ACE2-GPI localizes predominantly to raft domains of the plasma membrane while ACE2 is non-raft associated. We then compare the ability of ACE2 and ACE2-GPI to permit SARS-CoV-2 entry, replication, and syncytia formation of different viral variants. We find little difference in the two proteins. Our results demonstrate that SARS-CoV-2 entry and fusion are cholesterol-dependent and raft-independent processes.IMPORTANCERafts are often exploited by viruses and used as platforms to enhance their entry into the cell or spread from cell to cell. The membrane localization of ACE2 and the role of lipid rafts in SARS-CoV-2 entry and cell-to-cell spread are poorly understood. The function of lipid rafts in viral fusion is often studied through their disruption by cholesterol-depleting agents. However, this process may have off-target impacts on viral fusion independently of lipid-raft disruption. Therefore, we created an ACE2 construct that localizes to lipid rafts using a GPI anchor. Conversely, wild-type ACE2 was non-raft associated. We find that the localization of ACE2 to lipid rafts does not modify the fusion dynamics of SARS-CoV-2.

N6-methyladenosine (m6A) is a common and dynamic epitranscriptomic modification in eukaryotic RNAs, affecting stability, splicing, translation, and degradation. Recent technological advancements have revealed the complex nature of m6A modifications, highlighting their importance in plant and animal species. The m6A modification is a reversible process, with "writers" depositing methylation, "erasers" demethylating it, and "reader" proteins recognizing m6A and executing various biological functions. Studying the relationship between m6A methylation and viral infection is crucial. Animal viruses, including retroviruses, RNA viruses, and DNA viruses, often employ the host's m6A machinery to replicate or avoid immune responses. In plant viruses, host methyltransferases or demethylases can stabilize or degrade viral RNA, depending on the virus-host interaction. Additionally, viral infections can modify the host's m6A machinery, impacting the viral life cycle. This review examines the role of m6A modifications in plant viral pathogenesis, focussing on RNA viruses infecting crops like alfalfa, turnip, wheat, rice, and potato. Understanding the role of m6A in virus-host interactions can aid in studying plant viral disease development and discovering novel antiviral targets for crop protection. In this review, we summarize current information on m6A in RNA biology, focussing on its function in viral infections and plant-virus interactions.

During infection the autonomous parvovirus minute virus of mice (MVM) generates extensive DNA damage which facilitates virus replication and induces a cellular DNA damage response (DDR) driven by the ataxia telangiectasia mutated (ATM) kinase. Atypically, the ataxia telangiectasia and Rad-3-related (ATR) DDR pathway remains inactive. Upon DNA damage ATR is normally recruited to single-stranded DNA sequences formed at genomic DNA damage sites, and while within a multiprotein complex activates, via phosphorylation, the key DDR regulator checkpoint kinase 1 (Chk1). Inactivation of ATR during MVM infection leads to the accumulation of damaged DNA and enhancement of virus replication. Although ATR is inactivated, we show that during infection, the Chk1 activation pathway downstream of the initial ATR activating events remained functional. Activation of ATR, and consequently of Chk1, requires interaction with TopBP1, which itself is maintained in proximity to ATR by interaction with the phosphorylated S387 residue of Rad9, part of the Rad9-Hus1-Rad1 (911) complex. Both MVM infection and MVM NS1 overexpression inhibited Rad9 S387 phosphorylation and subsequent ATR activation. ATR inactivation during infection was suppressed by expression of Rad9 bearing a phosphomimetic 387 residue, indicating that this site, and the function it served, was the target of NS1 inhibition. NS1 interaction with CK2α and CK2α enzymatic activity was both required to prevent ATR activation, indicating MVM retargeted this kinase's activity during infection. Inhibition of the protein phosphatase 2C (PP2C) prevented Rad9 S387 dephosphorylation and Chk1 inactivation during MVM infection and NS1 overexpression revealing its role in the pathway's suppression.

Importance: Infection by the parvovirus minute virus of mice (MVM) causes significant DNA damage and induces a potent DNA damage response (DDR) which the virus exploits to further its replication. The cell responds to infection with an ATM-regulated DDR; however, atypically, the ATR-regulated DDR pathway is disabled during infection. This prevents Chk1 activation, thus allowing the accumulation of damaged DNA which facilitates virus replication. We describe here how MVM, and specifically the main viral replication protein NS1, inhibits ATR activation. Activation of ATR, and consequently Chk1, requires TopBP1 localization into the activating complex via its interaction with a phosphorylated residue of Rad9. We show that NS1 redirects casein kinase 2 to activate a phosphatase in the PP2C family which causes dephosphorylation of this critical residue, thus inhibiting ATR activation. This work provides mechanistic insight into one of the ways by which parvoviruses modify the host DDR response to facilitate their replication.

During infection, the autonomous parvovirus minute virus of mice (MVM) induces cellular DNA breaks and localizes to such sites, which presumably affords an environment beneficial for genome replication. MVM replication also benefits from the DNA damage response (DDR) mediated by the ataxia-telangiectasia mutated (ATM) kinase, while the ataxia telangiectasia and Rad-3 related (ATR) arm of the DDR is disabled, which prevents activation of its primary target, checkpoint kinase 1 (Chk1). We find here that Chk1 inactivation strongly correlates with dephosphorylation of one of its targets, RAD51, known to play a pivotal role in homologous recombination repair (HRR), thus leading to substantial inhibition of DNA repair in infected cells. We demonstrate colocalization of replicating MVM DNA with cellular double-strand breaks (DSBs) during infection, and show that an agent that exogenously induces cellular DSBs significantly increases viral DNA replication levels, establishing a role for cellular genome damage in facilitating virus DNA replication. Additionally, overexpression of active Chk1 during MVM infection was found to re-establish the activating phosphorylation of RAD51 Thr 309, significantly suppress infection-induced reduction of HRR efficiency with a concomitant increase in cellular genome DSBs, and reduce viral DNA replication levels. Thus, we conclude that during infection, MVM inhibition of Chk1 activation enhances viral replication, at least in part, by inhibiting cellular HRR.IMPORTANCEThe autonomous parvovirus minute virus of mice (MVM) has a compact DNA genome encoding a minimum number of proteins. During infection, it induces cellular DNA damage and both utilizes and modifies the subsequent cellular DNA damage response (DDR) in various ways to facilitate its replication. One of MVM's activities in this regard is to inhibit one of the primary arms of the DDR, the ataxia telangiectasia and Rad-3 related (ATR) pathway, which prevents activation of checkpoint kinase 1 (Chk1), a key protein involved in controlling the cellular DDR and preserving genome integrity. We show that prevention by MVM of Chk1 activation leads to inhibition of homologous recombination repair (HRR) of cellular DNA, which helps sustain viral replication. This work illuminates another way in which autonomous parvoviruses adjust the cellular environment for their replicative advantage.

Viruses are ubiquitous entities that infect organisms across the kingdoms of life. While viruses can infect a range of cells, tissues, and organisms, this aspect is often not explored in cell culture analyses. There is limited information about which infection-induced changes are shared or distinct in different cellular environments. The prevalent pathogen human cytomegalovirus (HCMV) remodels the structure and function of subcellular organelles and their interconnected networks formed by membrane contact sites (MCSs). A large portion of this knowledge has been derived from fibroblasts infected with a lab-adapted HCMV strain. Here, we assess strain- and cell type-specific alterations in MCSs and organelle remodeling induced by HCMV. Integrating quantitative mass spectrometry, super-resolution microscopy, and molecular virology assays, we compare infections with lab-adapted and low-passage HCMV strains in fibroblast and epithelial cells. We determine that, despite baseline proteome disparities between uninfected fibroblast and epithelial cells, infection induces convergent changes and is remarkably similar. We show that hallmarks of HCMV infection in fibroblasts, mitochondria-endoplasmic reticulum (ER) encapsulations and peroxisome proliferation, are also conserved in infected epithelial and macrophage-like cells. Exploring cell type-specific differences, we demonstrate that fibroblasts rely on endosomal cholesterol transport while epithelial cells rely on cholesterol from the Golgi. Despite these mechanistic differences, infections in both cell types result in phenotypically similar cholesterol accumulation at the viral assembly complex. Our findings highlight the adaptability of HCMV, in that infections can be tailored to the initial cell state by inducing both shared and unique proteome alterations, ultimately promoting a unified pro-viral environment.IMPORTANCEHuman cytomegalovirus (HCMV) establishes infections in diverse cell types throughout the body and is connected to a litany of diseases associated with each of these tissues. However, it is still not fully understood how HCMV replication varies in distinct cell types. Here, we compare HCMV replication with lab-adapted and low-passage strains in two primary sites of infection, lung fibroblasts and retinal epithelial cells. We discover that, despite displaying disparate protein compositions prior to infection, these cell types undergo convergent alterations upon HCMV infection, reaching a more similar cellular state late in infection. We find that remodeling of the subcellular landscape is a pervasive feature of HCMV infection, through alterations to both organelle structure-function and the interconnected networks they form via membrane contact sites. Our findings show how HCMV infection in different cell types induces both shared and divergent changes to cellular processes, ultimately leading to a more unified state.

Chloroviruses exhibit a close relationship with their hosts with the phenotypic aspect of their ability to form lytic plaques having primarily guided the taxonomy. However, with the isolation of viruses that are only able to complete their replication cycle in one strain of Chlorella variabilis, systematic challenges emerged. In this study, we described the genomic features of 53 new chlorovirus isolates and used them to elucidate part of the evolutionary history and taxonomy of this clade. Our analysis revealed new chloroviruses with the largest genomes to date (>400 kbp) and indicated that four genomic features are statistically different in the viruses that only infect the Syngen 2-3 strain of C. variabilis (OSy viruses). We found large regions of dissimilarity in the genomes of viruses PBCV-1 and OSy-NE5 when compared with the other genomes. These regions contained genes related to the interaction with the host cell machinery and viral capsid proteins, which provided insights into the evolution of the replicative and structural modules in these giant viruses. Phylogenetic analysis using hallmark genes of Nucleocytoviricota revealed that OSy-viruses evolved from the NC64A-viruses, possibly emerging as a result of the strict relationship with their hosts. Merging phylogenetics and nucleotide identity analyses, we propose strategies to demarcate viral species, resulting in seven new species of chloroviruses. Collectively, our results show how genomic data can be used as lines of evidence to demarcate viral species. Using the chloroviruses as a case study, we expect that similar initiatives will emerge using the basis exhibited here.IMPORTANCEChloroviruses are a group of giant viruses with long dsDNA genomes that infect different species of Chlorella-like green algae. They are host-specific, and some isolates can only replicate within a single strain of Chlorella variabilis. The genomics of these viruses is still poorly explored, and the characterization of new isolates provides important data on their genetic diversity and evolution. In this work, we describe 53 new chlorovirus genomes, including many isolated from alkaline lakes for the first time. Through comparative genomics and molecular phylogeny, we provide evidence of genomic gigantism in chloroviruses and show that a subset of viruses became highly specific for their hosts at a particular point in evolutionary history. We propose criteria to demarcate species of chloroviruses, paving the way for an update in the taxonomy of other groups of viruses. This study is a new and important piece in the complex puzzle of giant algal viruses.

After the ejection of viral DNA into the host cytoplasm, the temperate bacteriophage (phage) lambda integrates a cascade of expressions from various regulatory genes, coupled with DNA replication, to commit to a decision between lysis and lysogeny. Higher multiplicity of infection (MOI) greatly shifts the decision toward the lysogenic pathway. However, how the phage separates the MOI from replicated viral DNA during lysis-lysogeny decision-making is unclear. To quantitatively understand the role of viral DNA replication, we constructed a reporter system facilitating the visualization of individual copies of phage DNA throughout the phage life cycle, along with the lysis-lysogeny reporters. We showed that intracellular viral DNA diverges between the lytic and lysogenic pathways from the early phase of the infection cycle, mostly due to the synchronization and success of DNA injection, as well as the competition for replication resources, rather than the replication rate. Strikingly, we observed two distinct replication patterns during lysogenization and surprisingly heterogeneous integration kinetics, which advances our understanding of temperate phage life cycles. We revealed that the weak repression function of Cro is critical for an optimal replication rate and plays a crucial role in establishing stable lysogens.

Importance: Temperate bacteriophages, such as lambda, incorporate environmental cues including host abundance and nutrient conditions to make optimal decisions between propagation and dormancy. A higher phage-to-host ratio or multiplicity of infection (MOI) during λ infection strongly biases toward lysogeny. However, a comprehensive understanding of this decision-making process and the impact of phage replication prior to the decision is yet to be achieved. Here, we used fluorescence microscopy to quantitatively track the spatiotemporal progression of viral DNA replication in individual cells with different cell fates. The implementation of this fluorescent reporter system and quantitative analysis workflow opens a new avenue for future studies to delve deeper into various types of virus-host interactions at a high resolution.

Type I interferon (IFN-I) and its downstream genes play a profound role in HIV infection. In this study, we found that an IFN-inducible gene, IFI27, was upregulated in HIV-1 infection, which in turn efficiently suppressed HIV-1 replication, specially degraded the viral gag protein, including p24 and p55 subunits. Notably, the anti-HIV-1 activity of IFI27 in Old World monkeys surpassed that in New World monkeys, and IFI27 has a higher potentially inhibitory effect on HIV-1 than simian immunodeficiency virus (SIV). Our initial observations showed that NPM-IFI27, the IFI27 variant in northern pig-tailed macaque (Macaca leonina, NPM), exhibited a strong anti-HIV-1 activity. Further investigation demonstrated that NPM-IFI27 degraded p24 and p55 via the ubiquitin-proteasome pathway, with NPM-IFI27-37-115 interacting with the p24-N domain, and the NPM-IFI27-76-122 domain was closely associated with K48 ubiquitin recruitment. Additionally, Skp2 was identified as the probable E3 ubiquitin ligase responsible for the degradation of p24 and p55. Similarly, human IFI27 (Hu-IFI27) showed a mechanism similar to NPM-IFI27 in HIV-1 inhibition. These findings underscore the pivotal role of NPM-IFI27 in HIV-1 infection and provide a potential strategy for clinical anti-HIV-1 therapy.IMPORTANCEHIV-1 infection can trigger the production of IFN-I, which subsequently activates the expression of various IFN-stimulated genes (ISGs) to antagonize the virus. Therefore, discovering novel host antiviral agents for HIV-1 treatment is crucial. Our previous study revealed that IFI27 can influence HIV-1 replication. In this study, we observed that the NPM-IFI27 complex specifically inhibited HIV-1 by targeting its Gag protein. Further exploration demonstrated that IFI27 interacted with the HIV-1 p24 and p55 proteins, leading to their degradation through the ubiquitin-proteasome pathway. Notably, the NPM-IFI27-37-122 variant exhibited potent anti-HIV-1 activity, comparable to that of SAMHD1. These findings highlight the critical role and inhibitory mechanism of NPM-IFI27 in HIV-1 infection, providing a potential strategy for clinical antiviral therapy.

Swine Alpha-coronaviruses are one of the most destructive pathogens affecting the swine industries across the world. Swine Alpha-coronaviruses include transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus, porcine respiratory coronavirus, and swine acute diarrhea syndrome coronavirus (SADS-CoV). Thus far, swine Alpha-coronaviruses treatment options are very limited. Therefore, the identification of safe and effective treatment of swine Alpha-coronaviruses is urgently needed. In the current study, we screened a library of 240 FDA-approved compounds for antiviral activity against TGEV. Among screening, the 3CL protease inhibitor PF-00835231 was shown to dramatically inhibit TGEV replication in vitro systems. Mechanistically, PF-00835231 inhibits nonstructural protein 5 (Nsp5) protease activity targeting the cleavage at Nsp5-Nsp6 of TGEV. Additionally, PF-00835231 exhibited the potent broad-spectrum swine Alpha-coronaviruses antiviral activity. Treatment of PF-00835231 in mice not only blocks SADS-CoV deadly infection but also dramatically reduces viral copies. Taken together, our study provides evidence that PF-00835231 may control of the current swine Alpha-coronaviruses and emerging swine Alpha-coronaviruses in the future.IMPORTANCEThe COVID-19 pandemic has induced tremendous efforts to develop therapeutic strategies that target Beta-coronavirus including SARS-CoV-2. 3CL protease of Beta-coronavirus has been as a drug target for developing antiviral drugs. However, 3CL protease is not conserved in Alpha-coronavirus and Beta-coronavirus with only 44% amino acid similarity. Therefore, an inhibitor that prevents Alpha-coronaviruses infection is urgently needed. Swine Alpha-coronaviruses are one of the most destructive pathogens affecting the swine industries across the world. Swine herds with coronavirus diarrhea showed a high rate of co-infection between different Alpha-coronavirus. Our study, for the first time, showed that PF-00835231 inhibits swine Alpha-coronavirus infection. At the mechanistic level, we experimentally identified that PF-00835231 inhibits nonstructural protein 5 (Nsp5) protease activity targeting the cleavage at Nsp5-Nsp6 of Alpha-coronaviruses.