Description: Myxoma virus (MYXV) and vaccinia virus (VACV), two distinct members of the family Poxviridae , are both currently being developed as oncolytic virotherapeutic agents. Recent studies have demonstrated that ex vivo treatment with MYXV can selectively recognize and kill contaminating cancerous cells from autologous bone marrow transplants without perturbing the engraftment of normal CD34 + hematopoietic stem and progenitor cells. However, the mechanism(s) by which MYXV specifically recognizes and eliminates the cancer cells in the autografts is not understood. While little is known about the cellular attachment factor(s) exploited by MYXV for entry into any target cells, VACV has been shown to utilize cell surface glycosaminoglycans such as heparan sulfate (HS), the extracellular matrix protein laminin, and/or integrin β1. We have constructed MYXV and VACV virions tagged with the Venus fluorescent protein and compared their characteristics of binding to various human cancer cell lines as well as to primary human leukocytes. We report that the binding of MYXV or VACV to some adherent cell lines could be partially inhibited by heparin, but laminin blocked only VACV binding. In contrast to cultured fibroblasts, the binding of MYXV and VACV to a wide spectrum of primary human leukocytes could not be competed by either HS or laminin. Additionally, MYXV and VACV exhibited very different binding characteristics against certain select human leukocytes, suggesting that the two poxviruses utilize different cell surface determinants for the attachment to these cells. These results indicate that VACV and MYXV can exhibit very different oncolytic tropisms against some cancerous human leukocytes.

Description: Antigen receptor signaling to NF-B, essential for normal lymphocyte activation, is dysregulated in several types of lymphoma. During normal signaling, the multidomain adapter CARD11 transitions from a closed, inactive state to an open, active scaffold that assembles a multiprotein complex, leading to NF-B activation. The regulation of CARD11 scaffold function is bypassed by lymphoma-associated oncogenic CARD11 mutations that induce spontaneous signaling. We report an unbiased high-throughput quantitative signaling screen that identifies new CARD11 hyperactive variants and defines a LATCH domain that functions with the CARD to promote CARD11 autoinhibition. Gain-of-function mutations in the LATCH or CARD disrupt inhibitory domain binding, promote Bcl10 association, and induce Bcl10 ubiquitination, NF-B activation, and human lymphoma cell survival. Our results identify CARD11 mutations with oncogenic potential, provide a mechanistic explanation for their signaling potency, and offer a straightforward method for the discovery of variants that promote the tumorigenesis of NF-B-dependent lymphomas.

Description: We report the complete genome sequences of two novel isolates of norovirus isolated from the fecal swab specimens of dogs in Hong Kong. The complete viral genome is approximately 7.6 kb in length and consists of 3 overlapping open reading frames encoding the ORF1 polyprotein, VP1, and VP2, respectively. Analysis of the VP1 sequence suggested that these noroviruses are divergent from known noroviruses and may represent a novel phylogenetic clade within the genus.

Description: We report two genome sequences of novel noroviruses isolated from fecal swab specimens of brown rats in Hong Kong. The complete genome is approximately 7.5 kb in length and consists of 3 overlapping open reading frames encoding ORF1 polyprotein, VP1, and VP2, respectively. Sequence analysis suggested that these noroviruses should be classified in genogroup V, but they are distinct from other known rodent noroviruses and represent a novel cluster within the genogroup.

Description: There are two mechanisms for the incorporation of B5 into the envelope of extracellular virions produced by orthopoxviruses, one that requires A33 and one that does not. We have hypothesized that the A33-dependent mechanism requires a direct interaction between A33 and B5. In this study, chimeric constructs of A33 and B5/B5-green fluorescent protein (GFP) were used to show that the two proteins interact through their lumenal domains and that the coiled-coil domain of B5 is sufficient for an interaction with A33. Furthermore, our experiments reveal that a transmembrane domain, not necessarily its own, is requisite for the lumenal domain of B5 to interact with A33. In contrast, the lumenal domain of A33 is sufficient for interaction with B5. Furthermore, the lumenal domain of A33 is sufficient to restore the proper localization of B5-GFP in infected cells. Taken together, our results demonstrate that the lumenal domains of A33 and B5 interact and that the interaction is required for the incorporation of B5-GFP into extracellular virions, whereas the incorporation of A33 is independent of B5. These results suggest that viral protein incorporation into extracellular virions is an active process requiring specific protein-protein interactions.

Description: Two mechanisms exist for the incorporation of B5 into extracellular virions, one of which is dependent on A33. In the companion to this paper ( W. M. Chan and B. M. Ward, J. Virol. 86:8210–8220, 2012 ), we show that the lumenal domain of A33 is sufficient for interaction with the coiled-coil domain of B5 and capable of directing B5-green fluorescent protein (GFP) into extracellular virions. Here, we have created a panel of charge-to-alanine mutations in the lumenal domain of A33 to map the B5 interaction site. While none of these mutations abolished the interaction with B5, a subset displayed an increased interaction with both B5 and B5-GFP. Both B5 and B5-GFP recombinant viruses expressing these mutant proteins in place of normal A33 had a small-plaque phenotype. The increased interaction of the mutant proteins was detected during infection, suggesting that normally the interaction is either weak or transient. In addition, the increased A33-B5 interaction was detected on virions produced by recombinant viruses and correlated with reduced target cell binding. Taken together, these results show that both B5 and B5-GFP interact with A33 during infection and that the duration of this interaction needs to be regulated for the production of fully infectious extracellular virions.

Description: Dehalococcoides mccartyi strains are obligate organohalide-respiring bacteria harboring multiple distinct reductive dehalogenase (RDase) genes within their genomes. A major challenge is to identify substrates for the enzymes encoded by these RDase genes. We demonstrate an approach that involves blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by enzyme activity assays with gel slices and subsequent identification of proteins in gel slices using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). RDase expression was investigated in cultures of Dehalococcoides mccartyi strain BAV1 and in the KB-1 consortium growing on chlorinated ethenes and 1,2-dichloroethane. In cultures of strain BAV1, BvcA was the only RDase detected, revealing that this enzyme catalyzes the dechlorination not only of vinyl chloride, but also of all dichloroethene isomers and 1,2-dichloroethane. In cultures of consortium KB-1, five distinct Dehalococcoides RDases and one Geobacter RDase were expressed under the conditions tested. Three of the five RDases included orthologs to the previously identified chlorinated ethene-dechlorinating enzymes VcrA, BvcA, and TceA. This study revealed substrate promiscuity for these three enzymes and provides a path forward to further explore the largely unknown RDase protein family.

Description: NF-B activation downstream of antigen receptor engagement is a highly regulated event required for lymphocyte activation during the adaptive immune response. The pathway is often dysregulated in lymphoma, leading to constitutive NF-B activity that supports the aberrant proliferation of transformed lymphocytes. To identify novel regulators of antigen receptor signaling to NF-B, we developed bioluminescence resonance energy transfer-based interaction cloning (BRIC), a screening strategy that can detect protein-protein interactions in live mammalian cells in a high-throughput manner. Using this strategy, we identified the RING finger protein RNF181 as an interactor of CARD11, a key signaling scaffold in the antigen receptor pathway. We present evidence that RNF181 functions as an E3 ubiquitin ligase to inhibit antigen receptor signaling to NF-B downstream of CARD11. The levels of the obligate signaling protein Bcl10 are reduced by RNF181 even prior to signaling, and Bcl10 can serve as a substrate for RNF181 E3 ligase activity in vitro . Furthermore, RNF181 limits the proliferation of human diffuse large B cell lymphoma cells that depend upon aberrant CARD11 signaling to NF-B for growth and survival in culture. Our results define a new regulatory checkpoint that can modulate the output of CARD11 signaling to NF-B in both normal and transformed lymphocytes.

Description: Interferon-inducible transmembrane proteins (IFITMs) can restrict the entry of a wide range of viruses. IFITM3 localizes to endosomes and can potently restrict the replication of influenza A viruses (IAV) and several other viruses that also enter host cells through the endocytic pathway. Here, we investigate whether IFITMs are involved in protection in ducks, the natural host of influenza virus. We identify and sequence duck IFITM1 , IFITM2 , IFITM3 , and IFITM5 . Using quantitative PCR (qPCR), we demonstrate the upregulation of these genes in lung tissue in response to highly pathogenic IAV infection by 400-fold, 30-fold, 30-fold, and 5-fold, respectively. We express each IFITM in chicken DF-1 cells and show duck IFITM1 localizes to the cell surface, while IFITM3 localizes to LAMP1-containing compartments. DF-1 cells stably expressing duck IFITM3 (but not IFITM1 or IFITM2) show increased restriction of replication of H1N1, H6N2, and H11N9 IAV strains but not vesicular stomatitis virus. Although duck and human IFITM3 share only 38% identity, critical residues for viral restriction are conserved. We generate chimeric and mutant IFITM3 proteins and show duck IFITM3 does not require its N-terminal domain for endosomal localization or antiviral function; however, this N-terminal end confers endosomal localization and antiviral function on IFITM1. In contrast to mammalian IFITM3, the conserved YXX endocytosis signal sequence in the N-terminal domain of duck IFITM3 is not essential for correct endosomal localization. Despite significant structural and amino acid divergence, presumably due to host-virus coevolution, duck IFITM3 is functional against IAV. IMPORTANCE Immune IFITM genes are poorly conserved across species, suggesting that selective pressure from host-specific viruses has driven this divergence. We wondered whether coevolution between viruses and their natural host would result in the evasion of IFITM restriction. Ducks are the natural host of avian influenza A viruses and display few or no disease symptoms upon infection with most strains, including highly pathogenic avian influenza. We have characterized the duck IFITM locus and identified IFITM3 as an important restrictor of several influenza A viruses, including avian strains. With only 38% amino acid identity to human IFITM3, duck IFITM3 possesses antiviral function against influenza virus. Thus, despite long coevolution of virus and host effectors in the natural host, influenza virus evasion of IFITM3 restriction in ducks is not apparent.