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holesterol affinity. We demonstrate that drug-induced cholesterol sequestration in late endosomes not only redirects nsP1 to this compartment but also dramatically decreases genome replication, suggesting the functional importance of nsP1 targeting to cholesterol-rich plasma membrane microdomains. Finally, we evidence that nsP1 from Chikungunya and Sindbis viruses display different sensitivity to cholesterol sequestering agents, that parallel with their difference in the requirement for nsP1 palmitoylation for replication. This research, therefore, gives new insight into the functional role of palmitoylated cysteines in nsP1 for the assembly of functional alphavirus replication complexes in their mammalian host. Copyright © 2020 American Society for Microbiology.The entry/fusion complex (EFC) consists of eleven conserved proteins embedded in the membrane envelope of mature poxvirus particles. Poxviruses also encode proteins that localize in cell membranes and negatively regulate superinfection and syncytium formation. The vaccinia virus (VACV) A56/K2 fusion regulatory complex associates with the G9/A16 EFC subcomplex, but functional support for the importance of this interaction was lacking. Here we describe serially passaging VACV in non-permissive cells expressing A56/K2 as an unbiased approach to isolate and analyze escape mutants. Viruses forming large plaques in A56/K2 cells increased in successive rounds of infection indicating the occurrence and enrichment of adaptive mutations. selleck chemical Sequencing genomes of passaged and cloned viruses revealed mutations near the N-terminus of the G9 open-reading-frame but none in A16 or other genes. The most frequent mutation was His to Tyr at amino acid 44; additional escape mutants had a His to Arg mutation at amino acid 44 or a duer of the poxvirus family, also encodes fusion regulatory proteins A56 and K2 that are displayed on the plasma membrane and may be beneficial by preventing reinfection and cell-cell fusion. Previous studies showed that A56/K2 interacts with the G9/A16 EFC subcomplex in detergent-treated cell extracts. Functional evidence for the importance of this interaction was obtained by serially passaging wild-type VACV in cells that are non-permissive because of A56/K2 expression. VACV mutants with amino acid substitutions or duplications near the N-terminus of G9 were enriched because of their ability to overcome the block to entry imposed by A56/K2. Copyright © 2020 American Society for Microbiology.The TEAD family of transcription factors requires associating cofactors to induce gene expression. TEAD1 is known to activate the early promoter of human papillomavirus (HPV), but the precise mechanisms of TEAD1-mediated transactivation of the HPV promoter, including its relevant cofactors, remain unexplored. Here we reveal that VGLL1, a TEAD-interacting cofactor, contributes to HPV early gene expression. Knockdown of VGLL1 and/or TEAD1 led to a decrease in viral early gene expression in human cervical keratinocytes and cervical cancer cell lines. We identified 11 TEAD-target sites in the HPV16 long control region (LCR) by in vitro DNA-pulldown assays; eight of these sites contributed to transcriptional activation of the early promoter in luciferase reporter assays. VGLL1 bound to the HPV16 LCR via its interaction with TEAD1, both in vitro and in vivo Furthermore, introducing HPV16 and HPV18 whole-genomes into primary human keratinocytes led to increased levels of VGLL1, due in part to upregulation of TEADs. for HPV-associated cancers. Copyright © 2020 American Society for Microbiology.For cell entry, vaccinia virus requires fusion with host membrane via a viral fusion complex of 11 proteins, but the mechanism remains unclear. It was shown previously that viral proteins A56 and K2 are expressed on infected cells to prevent superinfection by extracellular vaccinia virus through binding to two components of the viral fusion complex (G9 and A16), thereby inhibiting membrane fusion. To investigate how the A56/K2 complex inhibits membrane fusion, we performed experimental evolutionary analyses by repeatedly passaging vaccinia virus in HeLa cells overexpressing A56 and K2 proteins to isolate adaptive mutant viruses. Genome sequencing of adaptive mutants revealed that they had accumulated a unique G9R ORF mutation, resulting in a single His44Tyr amino acid change. We engineered recombinant vaccinia virus to express G9H44Y mutant protein and it readily infected HeLa-A56/K2 cells. Moreover, similar to ΔA56 virus, G9H44Y mutant virus on HeLa cells had a cell fusion phenotype, indicating that G9H44Y-me membrane fusion inhibition mediated by the A56/K2 protein complex. We show that H44Y mutation of G9 protein is sufficient to overcome A56/K2-mediated membrane fusion inhibition. Treatment of virus-infected cells with different pH indicated that the H44Y mutation lowers the threshold of fusion inhibition by A56/K2. Our study provides evidence that A56/K2 inhibits the viral fusion complex via the latter's G9 subcomponent. Although G9H44Y mutant protein still binds to A56/K2 at neutral pH, it is less dependent on low pH for fusion activation, implying that it may adopt a subtle conformational change that mimics a structural intermediate induced by low pH. Copyright © 2020 American Society for Microbiology.The nuclear factor kappa B (NF-κB) is a potent transcription factor, activation of which typically results in robust pro-inflammatory signalling and triggering of fast negative feedback modulators to avoid excessive inflammatory responses. Here, we report that infection of epithelial cells, including primary porcine respiratory epithelial cells, with the porcine alphaherpesvirus pseudorabies virus (PRV) results in gradual and persistent activation of NF-κB, illustrated by proteasome-dependent degradation of the inhibitory NF-κB regulator IκB and nuclear translocation and phosphorylation of the NF-κB subunit p65. PRV-induced persistent activation of NF-κB does not result in expression of negative feedback loop genes like IκBα or A20 and does not trigger expression of prototypical pro-inflammatory genes like TNFα or IL-6. In addition, PRV infection inhibits TNFα-induced canonical NF-κB activation. Hence, PRV infection triggers persistent NF-κB activation in an unorthodox way and dramatically modulates the NF-κBNF-κB activation by the inflammatory cytokine TNFα. Aberrant PRV-induced NF-κB activation may therefore paradoxically serve as a viral immune evasion strategy and may represent an important tool to unravel currently unknown mechanisms and consequences of NF-κB activation. Copyright © 2020 American Society for Microbiology.RNA viruses form a dynamic distribution of mutant swarm (termed "quasispecies") due to the accumulation of mutations in the viral genome. The genetic diversity of a viral population is affected by several factors, including a bottleneck effect. Human-to-human transmission exemplifies a bottleneck effect in that only part of a viral population can reach the next susceptible hosts. In the present study, two lineages of the rhesus rotavirus (RRV) strain of Rotavirus A were serially passaged five times at a multiplicity of infection (MOI) of 0.1 or 0.001, and three phenotypes (infectious titer, cell binding ability and specific growth rate) were used to evaluate the impact of a bottleneck effect on the RRV population. The specific growth rate values of lineages passaged under the stronger bottleneck (MOI of 0.001) were higher after five passages. The nucleotide diversity also increased, which indicated that the mutant swarms of the lineages under the stronger bottleneck effect were expanded through the serial pasa sequencing error. The results of this study suggest that the genetic drift caused by a bottleneck in a human-to-human transmission explains the random appearance of new genetic lineages causing viral outbreaks, which can be expected by the molecular epidemiology using next generation sequencing in which the viral genetic diversity within a viral population is investigated. Copyright © 2020 Kadoya et al.A novel lytic bacteriophage ValSw3-3, which efficiently infects pathogenic strains of Vibrio alginolyticus, was isolated from sewage water and characterized by microbiological and in silico genomic analyses. Transmission electron microscopy indicated that ValSw3-3 had the morphology of siphoviruses. This phage can infect four species in the Vibrio genus and has a latent period of 15 min and a burst size of 95 ± 2 PFU/infected bacterium. Genome sequencing results show that ValSw3-3 has a 39,846-bp double stranded DNA genome with a GC content of 43.1%. The similarity between the genome sequences of ValSw3-3 and other phages recorded in GenBank database was below 50% (42%), suggesting that ValSw3-3 significantly differed from previously reported phages at the DNA level. Multiple genome comparisons and phylogenetic analysis based on major capsid protein revealed that phage ValSw3-3 was grouped in a clade with other five phages, including Listonella phage phiHSIC (NC_006953.1), Vibrio phage P23 (MK097141.1), Vibrihost range and infectivity, growth characteristics, stability under various conditions, and genomic features. Our results show that ValSw3-3 could be a potent candidate for phage therapy to treat V. alginolyticus infections due to its strong infectivity and better pH and thermal stability compared with previously reported Vibrio phages. Moreover, genome sequence alignments, phylogenetic analysis, in silico proteomic comparison, and core-gene analysis all support that this novel phage ValSw3-3 and five unclassified phages form a clade distant from other known genera ratified by ICTV. We thus propose a new viral genus within the Siphoviridae family to accommodate this clade with ValSw3-3 as a representative member. Copyright © 2020 Chen et al.Members of the flavivirus genus share a high level of sequence similarity and often circulate in the same geographical regions. However, whether T cells induced by one viral species cross-react with other related flaviviruses has not been globally addressed. Here, we tested pools of epitopes derived from dengue (DENV), zika (ZIKV), Japanese Encephalitis (JEV), West Nile (WNV), and yellow fever (YFV) viruses by Intracellular Cytokine Staining (ICS) using PBMCs of individuals naturally exposed to DENV or immunized with DENV (TV005) or YF17D vaccines. CD8 T cell responses recognized epitopes from multiple flaviviruses, however, the magnitude of cross-reactive responses was consistently several-fold lower than those to the autologous epitope pools, and associated with lower expression of activation markers such as CD40L, CD69, and CD137. Next, we characterized the antigen sensitivity of short-term T cell lines (TCL) representing twenty-nine different individual epitope/donor combinations. TCL derived from DENV moas YF) as a delivery vector. This backbone comprises the non-structural (NS) and capsid (C) antigens which are dominant targets of T cell responses. Here, we demonstrate that cross-reactivity at the level of T cell responses amongst different flaviviruses is very limited, despite high levels of sequence homology. Thus, the use of heterologous flavivirus species as a live attenuated vaccine vector is not likely to generate optimal T cell responses, and might thus impair vaccine performance. Copyright © 2020 American Society for Microbiology.