Transient dominant host-range selection using Chinese hamster ovary cells to generate marker-free recombinant viral vectors from vaccinia virus.

Recombinant vaccinia viruses (rVACVs) are promising antigen-delivery systems for vaccine development that are also useful as research tools. Two common methods for selection during construction of rVACV clones are (i) co-insertion of drug resistance or reporter protein genes, which requires the use of additional selection drugs or detection methods, and (ii) dominant host-range selection. The latter uses VACV variants rendered replication-incompetent in host cell lines by the deletion of host-range genes. Replicative ability is restored by co-insertion of the host-range genes, providing for dominant selection of the recombinant viruses. Here, we describe a new method for the construction of rVACVs using the cowpox CP77 protein and unmodified VACV as the starting material. Our selection system will expand the range of tools available for positive selection of rVACV during vector construction, and it is substantially more high-fidelity than approaches based on selection for drug resistance.

An interaction domain in human SAMD9 is essential for myxoma virus host-range determinant M062 antagonism of host anti-viral function.

In humans, deleterious mutations in the sterile α motif domain protein 9 (SAMD9) gene are associated with cancer, inflammation, weakening of the immune response, and developmental arrest. However, the biological function of SAMD9 and its sequence-structure relationships remain to be characterized. Previously, we found that an essential host range factor, M062 protein from myxoma virus (MYXV), antagonized the function of human SAMD9. In this study, we examine the interaction between M062 and human SAMD9 to identify regions that are critical to SAMD9 function. We also characterize the in vitro kinetics of the interaction. In an infection assay, exogenous expression of SAMD9 N-terminus leads to a potent inhibition of wild-type MYXV infection. We reason that this effect is due to the sequestration of viral M062 by the exogenously expressed N-terminal SAMD9 region. Our studies reveal the first molecular insight into viral M062-dependent mechanisms that suppress human SAMD9-associated antiviral function.