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Background: TASOR, a component of the HUSH repressor epigenetic complex, and SAMHD1, a cellular triphosphohydrolase (dNTPase), are both anti-HIV proteins antagonized by HIV-2/SIVsmm Viral protein X. As a result, the same viral protein is able to relieve two different blocks along the viral life cell cycle, one at the level of reverse transcription, by degrading SAMHD1, the other one at the level of proviral expression, by degrading TASOR. Phosphorylation of SAMHD1 at T592 has been shown to downregulate its antiviral activity. The discovery that T819 in TASOR was lying within a SAMHD1 T592-like motif led us to ask whether TASOR is phosphorylated on this residue and whether this post-translational modification could regulate its repressive activity. Results: Using a specific anti-phospho-antibody, we found that TASOR is phosphorylated at T819, especially in cells arrested in early mitosis by nocodazole. We provide evidence that the phosphorylation is conducted by a Cyclin/CDK1 complex, like that of SAMHD1 at T592. While we could not detect TASOR in quiescent CD4 + T cells, TASOR and its phosphorylated form are present in activated primary CD4 + T lymphocytes. In addition, TASOR phosphorylation appears to be independent from TASOR repressive activity. Indeed, on the one hand, nocodazole barely reactivates HIV-1 in the J-Lat A1 HIV-1 latency model despite TASOR T819 phosphorylation. On the other hand, etoposide, a second cell cycle arresting drug, reactivates latent HIV-1, without concomitant TASOR phosphorylation. Furthermore, overexpression of wt TASOR or T819A or T819E similarly represses gene expression driven by an HIV-1-derived LTR promoter. Finally, while TASOR is degraded by HIV-2 Vpx, TASOR phosphorylation is prevented by HIV-1 Vpr, likely as a consequence of HIV-1 Vpr-mediated-G2 arrest. Conclusions: Altogether, we show that TASOR phosphorylation occurs in vivo on T819. This event does not appear to correlate with TASOR-mediated HIV-1 silencing. We speculate that TASOR phosphorylation is related to a role of TASOR during cell cycle progression. [ABSTRACT FROM AUTHOR]

Flaviviruses are enveloped single positive-stranded RNA viruses. The capsid (C), a structural protein of flavivirus, is dimeric and alpha-helical, with several special structural and functional features. The functions of the C protein go far beyond a structural role in virions. It is not only responsible for encapsidation to protect the viral RNA but also able to interact with various host proteins to promote virus proliferation. Therefore, the C protein plays an important role in infected host cells and the viral life cycle. Flaviviruses have been shown to affect the health of humans and animals. Thus, there is an urgent need to effectively control flavivirus infections. The structure of the flavivirus virion has been determined, but there is relatively little information about the function of the C protein. Hence, a greater understanding of the role of the C protein in viral infections will help to discover novel antiviral strategies and provide a promising starting point for the further development of flavivirus vaccines or therapeutics. [ABSTRACT FROM AUTHOR]

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a newly identified phlebovirus associated with severe hemorrhagic fever in humans. While many viruses subvert the host cell cycle to promote viral growth, it is unknown whether this is a strategy employed by SFTSV. In this study, we investigated how SFTSV manipulates the cell cycle and the effect of the host cell cycle on SFTSV replication. Our results suggest that cells arrest at the G2/M transition following infection with SFTSV. The accumulation of cells at the G2/M transition did not affect virus adsorption and entry but did facilitate viral replication. In addition, we found that SFTSV NSs, a nonstructural protein that forms viroplasm-like structures in the cytoplasm of infected cells and promotes virulence by modulating the interferon response, induces a large number of cells to arrest at the G2/M transition by interacting with CDK1. The interaction between NSs and CDK1, which is inclusion body dependent, inhibits formation and nuclear import of the cyclin B1-CDK1 complex, thereby leading to cell cycle arrest. Expression of a CDK1 loss-of-function mutant reversed the inhibitive effect of NSs on the cell cycle, suggesting that this protein is a potential antiviral target. Our study provides new insight into the role of a specific viral protein in SFTSV replication, indicating that NSs induces G2/M arrest of SFTSV-infected cells, which promotes viral replication. [ABSTRACT FROM AUTHOR]

Persistent infections with high-risk human papillomaviruses (HR-HPVs) are linked to the development of cervical cancer due to a deregulation of the productive viral cycle in the host cell, leading to cell transformation. The E2 viral protein is expressed early during an HPV infection and regulates viral replication and transcription. Other functions have been attributed to E2, such as the promotion of apoptosis that are independent of its role in the regulation of the expression of E6 and E7 viral oncogenes. Moreover, it has been shown that the HPV16 E2 protein has regulatory effects on cellular gene expression, suggesting that it participates in the modulation of different cellular processes. Intratype genomic variations within high-risk HPV types have an impact on the prognosis of HPV-related lesions. Nevertheless, the biological significance of HPV18 E2 intratype variations has not been analysed previously. The aim of this study was to determine whether HPV18 E2 intratype variations differentially modulate gene expression and whether cell-death-related genes are affected by variations in E2. We demonstrate that HPV18 E2 intratype Asian Amerindian (AsAi) and African (Af) variants differentially affect gene expression profiles. Although the E2-AsAi variant was found to modulate a larger number of cellular genes, both E2 variants affected similar cellular processes. Nevertheless, E2-AsAi and E2-Af variants showed differences in their ability to induce apoptosis, where E2-Af had a stronger effect. The differences in gene expression profiles in cells harbouring E2 intratype variants suggest a possible effect on diverse cellular signalling pathways, and this might suggest an approach for identifying biological processes regulated by HPV18 E2 intratype variants. [ABSTRACT FROM AUTHOR]

Geminiviruses are single-stranded DNA (ssDNA) viruses that infect a wide range of plants. To promote viral replication, geminiviruses manipulate the host cell cycle. The viral protein Rep is essential to reprogram the cell cycle and then initiate viral DNA replication by interacting with a plethora of nuclear host factors. Even though many protein domains of Rep have been characterized, little is known about its nuclear targeting. Here, we show that one conserved lysine in the N-terminal part of Rep is pivotal for nuclear localization of the Rep protein from Tomato yellow leaf curl virus (TYLCV), with two other lysines also contributing to its nuclear import. Previous work had identified that these residues are essential for Rep from Tomato golden mosaic virus (TGMV) to interact with the E2 SUMO-conjugating enzyme (SCE1). We here show that mutating these lysines leads to nuclear exclusion of TYLCV Rep without compromising its interaction with SCE1. Moreover, the ability of TYLCV Rep to promote viral DNA replication also depends on this highly conserved lysine independently of its role in nuclear import of Rep. Our data thus reveal that this lysine potentially has a broad role in geminivirus replication, but its role in nuclear import and SCE1 binding differs depending on the Rep protein examined. IMPORTANCE Nuclear activity of the replication initiator protein (Rep) of geminiviruses is essential for viral replication. We now define that one highly conserved lysine is important for nuclear import of Rep from three different begomoviruses. To our knowledge, this is the first time that nuclear localization has been mapped for any geminiviral Rep protein. Our data add another key function to this lysine residue, besides its roles in viral DNA replication and interaction with host factors, such as the SUMO E2-conjugating enzyme. [ABSTRACT FROM AUTHOR]

Herpesviruses alternate between the latent and the lytic life cycle. Switching into the lytic life cycle is important for herpesviral replication and disease pathogenesis. Activation of a transcription factor replication and transcription activator (RTA) has been demonstrated to govern this switch in Kaposi's sarcoma-associated herpesvirus (KSHV). The protein encoded by open reading frame 49 from KSHV (ORF49KSHV) has been shown to upregulate lytic replication in KSHV by enhancing the activities of the RTA. We have solved the crystal structure of the ORF49KSHV protein to a resolution of 2.4 Å. The ORF49KSHV protein has a novel fold consisting of 12 alpha-helices bundled into two pseudodomains. Most notably are distinct charged patches on the protein surface, which are possible protein-protein interaction sites. Homologs of the ORF49KSHV protein in the gammaherpesvirus sub-family have low sequence similarities. Conserved residues are mainly located in the hydrophobic regions, suggesting that they are more likely to play important structural roles than functional ones. Based on the identification and position of three sulfates binding to the positive areas, we performed some initial protein-DNA binding studies by analyzing the thermal stabilization of the protein in the presence of DNA. The ORF49KSHV protein is stabilized in a dose-responsive manner by double-stranded oligonucleotides, suggesting actual DNA interaction and binding. Biolayer interferometry studies also demonstrated that the ORF49KSHV protein binds these oligonucleotides. [ABSTRACT FROM AUTHOR]

Virus entry into host cells relies on interactions between viral and host structures including lipids, carbohydrates and proteins. Particularly, protein–protein interactions between viral surface proteins and host proteins as well as secondary host protein–protein interactions play a pivotal role in coordinating virus binding and uptake. These interactions are dynamic and frequently involve multiprotein complexes. In the past decade mass spectrometry based proteomics methods have reached sensitivities and high throughput compatibilities of genomics methods and now allow the reliable quantitation of proteins in complex samples from limited material. As proteomics provides essential information on the biologically active entity namely the protein, including its posttranslational modifications and its interactions with other proteins, it is an indispensable method in the virologist's toolbox. Here we review protein interactions during virus entry and compare classical biochemical methods to study entry with novel technically advanced quantitative proteomics techniques. We highlight the value of quantitative proteomics in mapping functional virus entry networks, discuss the benefits and limitations and illustrate how the methodology will help resolve unsettled questions in virus entry research in the future. [ABSTRACT FROM AUTHOR]

The human herpesvirus-6 (HHV-6) infection induces cell-cycle arrest. In this study, we found that the HHV-6-encoded U14 protein induced cell-cycle arrest at G2/M phase via an association with the cellular protein EDD, a mediator of DNA-damage signal transduction. In the early phase of HHV-6 infection, U14 colocalized with EDD dots in the nucleus, and similar colocalization was also observed in cells transfected with a U14 expression vector. When the carboxyl-terminal region of U14 was deleted, no association of U14 and EDD was observed, and the percentage of cells in G2/M decreased relative to that in cells expressing wild-type U14, indicating that the C-terminal region of U14 and the U14–EDD association are critical for the cell-cycle arrest induced by U14. These results indicate that U14 is a G2/M checkpoint regulator encoded by HHV-6. [ABSTRACT FROM AUTHOR]

Helper-dependent adenovirus vectors (HDVs) are safe and efficient tools for gene transfer with high cloning capacity. However, the multiple amplification steps needed to produce HDVs hamper a robust production process and in turn the availability of high-quality vectors. To understand the factors behind the low productivity, we analyzed the progression of HDV life cycle. Canine adenovirus (Ad) type 2 vectors, holding attractive features to overcome immunogenic concerns and treat neurobiological disorders, were the focus of this work. When compared with E1-deleted (ΔE1) vectors, we found a faster helper genome replication during HDV production. This was consistent with an upregulation of the Ad polymerase and pre-terminal protein and led to higher and earlier expression of structural proteins. Although genome packaging occurred similarly to ΔE1 vectors, more immature capsids were obtained during HDV production, which led to a ~4-fold increase in physical-to-infectious particles ratio. The higher viral protein content in HDV-producing cells was also consistent with an increased activation of autophagy and cell death, in which earlier cell death compromised volumetric productivity. The increased empty capsids and earlier cell death found in HDV production may partially contribute to the lower vector infectivity. However, an HDV-specific factor responsible for a defective maturation process should be also involved to fully explain the low infectious titers. This study showed how a deregulated Ad cycle progression affected cell line homeostasis and HDV propagation, highlighting the impact of vector genome design on virus-cell interaction. [ABSTRACT FROM AUTHOR]

Viruses commonly modify host endomembranes to facilitate biological processes in the viral life cycle. Infection by viruses belonging to the genus Bymovirus (family Potyviridae) has long been known to induce the formation of large membranous inclusion bodies in host cells, but their assembly and biological roles are still unclear. Immunoelectron microscopy of cells infected with the bymovirus Wheat yellow mosaic virus ( WYMV) showed that P1, P2 and P3 are the major viral protein constituents of the membranous inclusions, whereas NIa-Pro (nuclear inclusion-a protease) and VPg (viral protein genome-linked) are probable minor components. P1, P2 and P3 associated with the endoplasmic reticulum ( ER), but only P2 was able to rearrange ER and form large aggregate structures. Bioinformatic analyses and chemical experiments showed that P2 is an integral membrane protein and depends on the active secretory pathway to form aggregates of ER membranes. In planta and in vitro assays demonstrated that P2 interacts with P1, P3, NIa-Pro or VPg and recruits these proteins into the aggregates. In vivo RNA labelling using WYMV-infected wheat protoplasts showed that the synthesis of viral RNAs occurs in the P2-associated inclusions. Our results suggest that P2 plays a major role in the formation of membranous compartments that house the genomic replication of WYMV. [ABSTRACT FROM AUTHOR]

Abstract: Viral channel forming proteins are known for their capability to make the lipid membrane of the host cell and its subcellular compartments permeable to ions and small compounds. There is increasing evidence that some of the representatives of this class of proteins are also strongly interacting with host proteins and the effectiveness of this interaction seems to be high. Interaction of viral channel proteins with host factors has been proposed by bioinformatics approaches and has also been identified experimentally. An overview of the interactions with host proteins is given for Vpu from HIV-1, E5 from HPV-16 and p7 from HCV. This article is part of a Special Issue entitled: Viral Membrane Proteins — Channels for Cellular Networking. [Copyright &y& Elsevier]

Cell cultures used routinely in proteomic experiments may contain proteins from other species because of infection, transfection or just contamination. Since infection or contamination may affect the results of a biological experiment, it is important to test the samples for the presence of “alien” proteins. Usually cells are tested only for the most common infections, and most of the existing tests are targeting specific contaminations. Here we describe a three-step procedure for reliable untargeted detection of viral proteins using proteomics data, and recommend this or similar procedure to be applied to every proteomics dataset submitted for publication. [ABSTRACT FROM AUTHOR]

Marek's disease (MD) is an economically significant disease in chickens that is caused by the highly oncogenic Marek's disease virus (MDV). A major unanswered question is the mechanism of MDV-induced tumor formation. Meq, a bZIP transcription factor discovered in the 1990s, is critically involved in viral oncogenicity, but only a few of its host target genes have been described, impeding our understanding of MDV-induced tumorigenesis. Using chromatin immunoprecipitation-sequencing (ChIP-seq) and microarray analysis, a high-confidence list of Meq binding sites in the chicken genome and a global transcriptome of Meq-responsive genes were generated. Meq binding sites were found to be enriched in the promoter regions of upregulated genes but not in those of downregulated genes. ChIP-seq was also performed for c-Jun, a known heterodimeric partner of Meq. The close location of binding sites of Meq and c-Jun was noted, suggesting cooperativity between these two factors in modulating transcription. Pathway analysis indicated that Meq transcriptionally regulates many genes that are part of several signaling pathways including the extracellular signal-regulated kinase /mitogen-activated protein kinase (ERK/MAPK), Jak-STAT, and ErbB pathways, which are critical for oncogenesis and/or include signaling mediators involved in apoptosis. Meq activates oncogenic signaling cascades by transcriptionally activating major kinases in the ERK/MAPK pathway and simultaneously repressing phosphatases, as verified using inhibitors of MEK and ERK1/2 in a cell proliferation assay. This study provides significant insights into the mechanistic basis of Meq-dependent cell transformation. [ABSTRACT FROM AUTHOR]

Positive-strand RNA viruses are known to rearrange the endomembrane network to make it more conducive for replication, maturation, or egress. Our previous transmission electron microscopic (TEM) analysis showed that ectopic expression of wildtype (wt) capsid protein (CP) of Brome mosaic virus (BMV) has an intrinsic property of modifying the endoplasmic reticulum (ER) to induce vesicles similar to those present in wt BMV infection. In this study, we evaluated the functional significance of CP-mediated vesicle induction to the BMV infection cycle in planta. Consequently, the cytopathologic changes induced by wt CP or its mutants defective in virion assembly due to mutations engineered in either N- or C-proximal domains were comparatively analyzed by TEM in two susceptible (Nicotiana benthamiana and Chenopodium quinoa) and one nonhost (N. clevelandii) plant species. The results showed that in susceptible hosts, CP-mediated ER-derived vesicle induction is contingent on the expression of encapsidation-competent CP. In contrast, unlike in N. benthamiana and C. quinoa, transient expression of wt CP in nonhost N. clevelandii plants eliminated vesicle induction. Additionally, comparative source-to-sink analysis of virus spread in leaves of N. benthamiana and N. clevelandii coexpressing wt BMV and Cucumber mosaic virus (CMV) showed that despite trans-encapsidation, CMV failed to complement the defective cell-to-cell movement of BMV. The significance and relation of CP-mediated vesicle induction to virus cell-to-cell movement are discussed. [ABSTRACT FROM AUTHOR]

Viruses commonly manipulate cell cycle progression to create cellular conditions that are most beneficial to their replication. To accomplish this feat, viruses often target critical cell cycle regulators in order to have maximal effect with minimal input. One such master regulator is the large, multisubunit E3 ubiquitin ligase anaphase-promoting complex (APC) that targets effector proteins for ubiquitination and proteasome degradation. The APC is essential for cells to progress through anaphase, exit from mitosis, and prevent a premature entry into S phase. These far-reaching effects of the APC on the cell cycle are through its ability to target a number of substrates, including securin, cyclin A, cyclin B, thymidine kinase, geminin, and many others. Recent studies have identified several proteins from a number of viruses that can modulate APC activity by different mechanisms, highlighting the potential of the APC in driving viral replication or pathogenesis. Most notably, human cytomegalovirus (HCMV) protein pUL21a was recently identified to disable the APC via a novel mechanism by targeting APC subunits for degradation, both during virus infection and in isolation. Importantly, HCMV lacking both viral APC regulators is significantly attenuated, demonstrating the impact of the APC on a virus infection. Work in this field will likely lead to novel insights into viral replication and pathogenesis and APC function and identify novel antiviral and anticancer targets. Here we review viral mechanisms to regulate the APC, speculate on their roles during infection, and identify questions to be addressed in future studies. [ABSTRACT FROM AUTHOR]

Abstract: Viral assembly is a key step in the virus life cycle. In this review, we focus mainly on the ability of retroviruses, especially HIV-1, to assemble at the plasma membrane of their host cells. The assembly process of RNA enveloped viruses necessitates a fine orchestration between the different viral components and specific interactions between viral proteins and lipids of the host cell membrane. Searching for a comparison with another RNA enveloped virus, we refer to influenza virus to show how it could share (or not) some common features with HIV-1 assembly since both viruses are believed to assemble mainly in raft microdomains. We also discuss the role of RNA and the cellular actin cytoskeleton in enhancing these viral assembly processes. Finally, based on the literature and on new results we have obtained by molecular docking, we propose another mechanism for HIV-1 assembly in membrane domains. This mechanism involves the trapping of acidic lipids by the viral Gag protein by means of ionic protein–lipid interactions, inducing thereby formation of acidic lipid-enriched microdomains (ALEM). [Copyright &y& Elsevier]

Abstract: Zinc has been shown to mediate antiviral effects against certain viruses. However, the underlying mechanisms are still largely unknown. We investigated the effects of the two zinc salts, zinc chloride (ZnCl2) and zinc sulfate (ZnSO4), on infection of swine testicle (ST) cells with transmissible gastroenteritis virus (TGEV) and compared it to the effects of a control salt, magnesium sulfate (MgSO4). Virus yield reduction experiments showed that ZnCl2 and ZnSO4 did not exhibit direct virucidal effects and did not affect adsorption of TGEV to ST cells. However, ZnCl2 and ZnSO4 markedly reduced viral titers as well as TGEV RNA and viral protein synthesis when applied during virus penetration and at different time points after viral cell entry. The results of the study suggest that zinc salts do not interfere with TGEV-cell binding but that they mediate antiviral effects through inhibition of viral penetration or egress or the intracellular phase of the viral life-cycle. [Copyright &y& Elsevier]

The papillomavirus E2 proteins are indispensable for the viral life cycle, and their functions are subject to tight regulation. The E2 proteins undergo posttranslational modifications that regulate their properties and roles in viral transcription, replication, and genome maintenance. During persistent infection, the E2 proteins from many papillomaviruses act as molecular bridges that tether the viral genomes to host chromosomes to retain them within the host nucleus and to partition them to daughter cells. The betapapillomavirus E2 proteins bind to pericentromeric regions of host mitotic chromosomes, including the ribosomal DNA loci. We recently reported that two residues (arginine 250 and serine 253) within the chromosome binding region of the human papillomavirus type 8 (HPV8) E2 protein are required for this binding. In this study, we show that serine 253 is phosphorylated, most likely by protein kinase A, and this modulates the interaction of the E2 protein with cellular chromatin. Furthermore, we show that this phosphorylation occurs in S phase, increases the half-life of the E2 protein, and promotes chromatin binding from S phase through mitosis. [ABSTRACT FROM AUTHOR]

The cellular receptor of foamy viruses (FVs) is unknown. The broad spectrum of permissive cells suggests that the cellular receptor is a molecular structure with almost ubiquitous prevalence. Here, we investigated the ability of heparan sulfate (HS), a glycosaminoglycan (GAG) present on the extracellular matrix of many cells, to bind FV particles and to permit prototype FV (PFV) and feline FV (FFV) entry. Permissivity of different cell lines for FV entry correlated with the amount of heparan sulfate present on the cell surface. The resulting 50% cell culture infectious doses (CCID50s) were distributed over a range of 4 logs, which means that the most susceptible cell line tested (HT1080) was more than 10,000 times more susceptible for PFV infection than the least susceptible cell line (CRL-2242). HS surface expression varied over a range of 2 logs. HS expression and FV susceptibility were positively correlated (P<0.001). Enzymatic digestion of heparan sulfate on HT1080 cells diminished permissivity for PFV entry by a factor of at least 500. Using fast protein liquid chromatography (FPLC), we demonstrated binding of FV vector particles to a gel filtration column packed with heparin, a molecule structurally related to heparan sulfate, allowing for the purification of infectious particles. Both PFV and FFV infection were inhibited by soluble heparin. Our results show that FVs bind to HS and that this interaction is a pivotal step for viral entry, suggesting that HS is a cellular attachment factor for FVs. [ABSTRACT FROM AUTHOR]

During human adenovirus type 3 (Ad3) infection, an excess of penton base and fiber proteins are produced which form dodecahedral particles composed of 12 pentamers of penton base and 12 trimers of fiber protein. No biological functions have yet been ascribed to Ad3 dodecahedra. Here, we show that dodecahedra compete with Ad3 virions for binding to the cell surface and trigger cell remodeling, giving new insights into possible biological functions of dodecahedra in the Ad3 infectious cycle. [ABSTRACT FROM AUTHOR]