Poxvirus Gene Expression and DNA Replication

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

Abstract

Poxviruses encode enzymes and factors needed for transcription and replication of their genomes within the cytoplasm of infected cells. Vaccinia virus, the prototypic member of the poxvirus family, provides a unique system for combining biochemical and genetic approaches for investigating mechanisms of gene regulation, mRNA biosynthesis and DNA synthesis. Studies with vaccinia virus indicated that the genes are divided into three temporal classes - early, intermediate and late. Each gene class has a consensus DNA promoter sequence and corresponding transcription factors that interact with the virus-encoded multisubunit RNA polymerase. The transcription system for early genes is packaged within the infectious virus particle during its assembly, whereas the factors for intermediate and late gene transcription are synthesized successively after infection and localize within cytoplasmic factory areas. Poxviruses also encode enzymes that modify their mRNA by adding a cap structure to the 5' end and a poly(A) tail to the 3' end, which are necessary for efficient translation and stability. The shut down of cellular protein synthesis and the tight regulation of viral protein synthesis are regulated by poxvirus enzymes that cleave the cap structure. Using new generation DNA sequencing, we have made a complete transcription and translation map of the vaccinia virus genome and defined the RNA start sites and the sequences adjacent to the poly(A) tail. These studies have revealed numerous previously unannotated transcripts. In addition, the effects of vaccinia virus infection on host mRNAs have been defined. In 2020 we discovered that mutations in the decapping enzyme enhance the replication of modified vaccinia virus Ankara (MVA), an attenuated virus that is approved as a smallpox vaccine and is in clinical trials as a vector for other pathogens. The safety of MVA is due in large part to its inability to replicate in mammalian cells. Although, host-range restriction is considered a stable feature of the virus, we described the occurrence of spontaneous mutations in MVA that increase replication considerably in monkey BS-C-1 cells but only slightly in human cells. The mutants contain single nucleotide changes that lead to amino acid substitutions in one of the two decapping enzymes. Although the spontaneous mutations are distant from the decapping enzyme active site, engineered active site-mutations also increased virus replication in BS-C-1 cells. The D10 mutations occurred at N- or C-terminal locations distal from the active site, suggesting an indirect effect on decapping or on another previously unknown role of D10. Although increased amounts of viral mRNA and proteins were found in BS-C-1 cells infected with the mutants compared to parental MVA, the increase was much less than the one to two logs higher virus yields. Nevertheless, a contributing role for diminished decapping in overcoming the host range defect was consistent with increased replication and viral protein synthesis in BS-C-1 cells infected with an MVA engineered to have active site mutations that abrogate decapping activity entirely. Optimal decapping may vary depending on the biological context. In 2020, we initiated a new project to determine the mode of action of the intermediate transcription factor that is comprised of subunits of 34- and 45-kDa. The subunits, containing a 6-histidine tag were expressed in E. coli and purified by chromatography. Efforts are now being made to determine DNA binding and to crystallize the protein for determination of the structure. An understanding of the regulation of poxvirus gene expression and DNA replication will help to design vaccines and identify targets for antiviral therapy and will contribute to our understanding of these processes in other viruses and cells.