Notes

Framework and hybridization qualities of phosphoryl guanidine oligonucleotides beneath crowding together situations.
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. learn more 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 passages.
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