Bacterial artificial chromosome (BAC) clones are rendered electrocompetent and transformed with the recombinant shuttle vector, pLD53SCAB/AB-box. Cointegrates are selected by growth on chloramphenicol and ampicillin to ensure recombination of the shuttle vector into the BAC. © 2020 Cold Spring Harbor Laboratory Press.Plasmid DNA is prepared from the recombinant shuttle vector pLD53.SCAB/A-B created by cloning of the A and B homology arms for two-step bacterial artificial chromosome (BAC) engineering. To confirm that the A-box and B-box arms have been successfully incorporated into pLD53.SCAB, the pattern of enzyme digestion of the modified plasmid is compared with that of the unmodified pLD53.SCAB. Once the shuttle vector is shown to carry the proper sequences, it is ready for transfer into the BAC host. © 2020 Cold Spring Harbor Laboratory Press.This protocol describes the preparation of the shuttle vector before its introduction into bacterial artificial chromosome (BAC) host cells for BAC two-step engineering. The homology arm sequences, prepared previously, are introduced by ligation into the digested shuttle vector DNA to provide sites for recombination within the BAC clone. Crude lysates of individual bacterial transformants serve as templates in polymerase chain reaction (PCR) analysis to confirm the presence of the homology arms in the recombinant shuttle vector. © 2020 Cold Spring Harbor Laboratory Press.The 700-bp A homology arm (A-box) and the 700-bp B homology arm (B-box) are amplified by polymerase chain reaction (PCR) using purified bacterial artificial chromosome (BAC) DNA as template for two-step BAC engineering. The resulting A-box PCR product contains an AscI site at its 5' end (the 5' primer incorporates an AscI site, and the 3' primer does not incorporate any restriction sites). The B-box PCR product contains an XmaI site at its 3' end (the 5' primer does not incorporate any restriction sites, and the 3' primer incorporates an XmaI site). The amplification products are then digested with the appropriate restriction endonucleases to render them suitable for cloning into the shuttle vector. © 2020 Cold Spring Harbor Laboratory Press.In two-step bacterial artificial chromosome (BAC) engineering, a single plasmid is introduced into the BAC-carrying cell lines. The shuttle vector pLD53.SCAB (or pLD53.SCAEB) carries the recA gene and the R6Kγ origin, which requires the π protein to replicate. PIR2 cells, expressing π, are typically used for the amplification of the vector and maintain about 15 copies/cell of the donor vector, which is relatively stable in this host. © 2020 Cold Spring Harbor Laboratory Press.Tetherin/BST-2 is an antiviral protein that blocks the release of enveloped viral particles by linking them to the membrane of producing cells. At first, BST-2 genes were described only in humans and other mammals. Recent work identified BST-2 orthologs in non-mammalian vertebrates, including birds. Here, we identify the BST-2 sequence in domestic chicken (Gallus gallus) for the first time and demonstrate its activity against Avian sarcoma and leukosis virus (ASLV). We generated BST-2 knockout in chicken cells and showed that BST-2 is a major determinant of an interferon-induced block of ASLV release. Ectopic expression of chicken BST-2 blocks the release of ASLV in chicken cells and of Human immunodeficiency virus type 1 (HIV-1) in human cells. Using metabolic labeling and pulse-chase analysis of HIV-1 Gag proteins, we verified that chicken BST-2 blocks the virus at the release stage. Further, we describe BST-2 orthologs in multiple avian species from 12 avian orders. Previously, some of these species were rrmed virus particles from infected cells. Recent work identified BST-2 in non-mammalian vertebrate species, including birds. Here, we report the BST-2 sequence in domestic chicken and describe its antiviral activity against a prototypical avian retrovirus, ASLV. We also identify BST-2 genes in multiple avian species and show that they evolve rapidly in birds, which is an important indication of their relevance for antiviral defense. Analysis of avian BST-2 genes will shed light on defense mechanisms against avian viral pathogens. Copyright © 2020 American Society for Microbiology.Cyclic GMP-AMP synthase (cGAS) senses double-stranded DNA and synthesizes the second messenger cyclic GMP-AMP (cGAMP) which binds to mediator of IRF3 activation (MITA) and initiates MITA-mediated signaling, leading to induction of type I interferons (IFNs) and other antiviral effectors. Human cytomegalovirus (HCMV), a widespread and opportunistic pathogen, antagonizes host antiviral immune response to establish latent infection. Here we identified HCMV tegument protein UL94 as an inhibitor of cGAS-MITA-mediated antiviral response. Ectopic expression of UL94 impaired cytosolic dsDNA- and DNA virus-triggered induction of type I IFNs and enhanced viral replication. Conversely, UL94-deficiency potentiated HCMV-induced transcription of type I IFNs and downstream antiviral effectors and impaired viral replication. UL94 interacted with MITA, disrupted the dimerization and translocation of MITA, and impaired the recruitment of TBK1 to the MITA signalosome. These results suggest that UL94 plays an important role in the immune evasion of HCMV.Importance Human cytomegalovirus (HCMV), a large dsDNA virus, encodes more than 200 viral proteins. HCMV infection causes irreversible abnormalities of central nervous system in newborns and severe syndromes in organ transplantation patients or AIDS patients. It has been demonstrated that HCMV has evolved multiple immune evasion strategies to establish latent infection. Previous studies pay more attention to the mechanism by which HCMV evades immune response in the early phase of infection. In this study, we identified UL94 as a negative regulator of innate immune response, which functions in the late phase of HCMV infection. Copyright © 2020 American Society for Microbiology.Beta genus human papillomaviruses (β-HPVs) cause cutaneous squamous cell carcinomas (cSCCs) in a subset of immunocompromised patients. However, β-HPVs are not necessary for tumor maintenance in the general population. Instead, they may destabilize the genome in the early stages of cancer development. Supporting this idea, β-HPV's 8E6 protein attenuates p53 accumulation after failed cytokinesis. This paper offers mechanistic insight into how β-HPV E6 causes this change in cell signaling. An in silico screen and characterization of HCT 116 cells lacking p300 suggested that the histone acetyltransferase is a negative regulator of Hippo pathway (HP) gene expression. HP activation restricts growth in response to stimuli, including failed cytokinesis. Loss of p300 resulted in increased HP gene expression, including pro-proliferative genes associated with HP inactivation.  https://www.selleckchem.com/products/mivebresib-abbv-075.html  β-HPV 8E6 expression recapitulates some of these phenotypes. We used a chemical inhibitor of cytokinesis (dihydrocytochalasin B or H2CB) to induce failed cytokinesis.