Vaccinia virus DNA binding proteins and their role in virion morphogenesis

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Poxviruses remain a fascinating biological puzzle, 35 years after variola was eradicated and smallpox ceased to be a medical problem. The prototypic poxvirus, vaccinia virus (VAC), encodes >200 genes within a ~200 kbp duplex DNA genome, and virion synthesis requires many carefully orchestrated biochemical steps. During this process the virus must replicate its genome and package it into mature virus particles, whilst trying to avoid triggering, or actively inhibiting, the innate immune system that would otherwise block infection. VAC encodes numerous DNA binding proteins, of which many are enzymes required to catalyze transcription and DNA replication. Interestingly, the literature and bioinformatics identify at least seven more virus genes, encoding DNA binding proteins, many of which are essential and where the function(s) of some are still unclear. We will use the RNA interference methods developed with our current NSERC award, in conjunction with advanced microscopy, virus-reverse genetics, and other technologies to further explore the functions served by these poorly characterized proteins. We predict that they support viral replication, genome packaging, and/or viral attenuation of a dsDNA-triggered host response. As a first step we have established a panel of siRNA’s that selectively inhibit VAC gene expression as judged by reverse transcriptase PCR. Plaque assays have been used to measure effects on virus growth. To date we have established siRNA’s targeting I3L, I6L, or E8R that clearly decrease both gene expression and virus replication in human cells. Similar, but not so strongly penetrant effects have been seen with siRNAs against E5R, and J1R and we are screening additional siRNAs to improve the methods. Using super resolution fluorescence microscopy, we have also observed a novel viral DNA structure that may represent an early packaging intermediate. From electron microscopy it has been suggested that mature viral particles enclose tubular structures composed of DNA wrapped around a protein core. Our observation of tubular viral DNA structures, prior to virion assembly and packaging, is the first discovery of such structures outside of the mature particle. Having devised a way to visualize these putative packaging intermediates, we will determine the composition of these structures and test which VAC DNA binding proteins play a role in their assembly or processing. Lastly, we have been intrigued by the fact that over a 24 hr replication cycle, an infected cell makes almost as much viral DNA as is found in the nucleus and does so despite the presence of a growing list of cellular DNA sensors (e.g. cGAS, DAI, Pol-III, STING, and TLR9). VAC is known to block detection of certain pathogen-associated molecular patterns (e.g. dsRNA and 5’-triphosphate capped RNA) and also inhibits the downstream signaling that would otherwise induce inflammation, apoptosis, and/or interferons. We hypothesize that one or more of the VAC-encoded DNA-binding proteins may also serve a still unidentified role as inhibitors of cytoplasmic DNA sensors. Of particular interest is the cell’s DNA damage sensing system, since virus replication produces DNA intermediates that should trigger a damage response. This can block the cell cycle at G2-M, which is poorly supportive of virus replication. In fact, we have observed that VAC infection does not trigger a DNA damage response despite the presence of fragmented dsDNA, as indicated by TUNNEL assay. These preliminary data provide a basis for a program of studies designed to better define the biology of these VAC DNA-binding proteins. These studies will also provide high-quality early-career training in advanced virology, molecular biology, and state-of-the-art microscopic imaging.