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Principal Investigator: Bernard Moss
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2020
Award: $574,999
Funding agency: National Institute of Allergy and Infectious Diseases
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 2019, we demonstrated the mRNA of an abundant orthopoxvirus gene is translated at the site of function. On-site translation of mRNAs provides an efficient means of subcellular protein localization. In eukaryotic cells, the transport of cellular mRNAs to membrane-less sites usually occurs prior to translation and involves specific sequences known as zip codes that interact with RNA-binding and motor proteins. Poxviruses replicate in specialized cytoplasmic factory regions where DNA synthesis, transcription, translation and virion assembly occur. Some poxviruses embed infectious virus particles outside of factories in membrane-less protein bodies with liquid gel-like properties known as A-type inclusions (ATIs) that are comprised of numerous copies of the viral 150-kDa ATI protein. Here, we demonstrate by fluorescent in situ hybridization that these inclusions are decorated with ATI mRNA. On-site translation is supported by the localization of a translation initiation factor eIF4E and by ribosome-bound nascent chain ribopuromycylation. Nascent peptide-mediated anchoring of ribosome-mRNA translation complexes to the inclusions is suggested by release of the mRNA by puromycin, a peptide chain terminator. Following puromycin washout, re-localization of ATI mRNA at inclusions depends on RNA and protein synthesis but requires neither microtubules nor actin polymerization. Further studies show that the ATI mRNAs remain near the sites of transcription in the factory regions when stop codons are introduced near the N-terminus of the ATI or large truncations are made at the N- or C-termini. Instead of using a zipcode, we propose that ATI mRNA localization is mediated by ribosome-bound nascent ATI polypeptides that interact with ATI protein in inclusions and thereby anchor the complex for multiple rounds of mRNA translation. Association of ATI mRNA with inclusion bodies allows multiple rounds of local translation and prevents premature ATI protein aggregation and trapping of virions within the factory.
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.
Terms: <Actins><Anabolism><Antiviral Agents><Antiviral Drugs><Antiviral Therapy><Antivirals><Area><Basal Transcription Factor><Basal transcription factor genes><Biochemical Genetics><Cell Body><Cells><Cellular Inclusions><Cleaved cell><Code><Coding System><Complex><Consensus><Cytoplasm><DNA><DNA Replication><DNA Synthesis><DNA biosynthesis><DNA seq><DNA sequencing><DNA-Dependent RNA Polymerases><DNA-Directed RNA Polymerase><DNAseq><Deoxyribonucleic Acid><Enzyme Gene><Enzymes><Eukaryotic Cell><FISH Technic><FISH Technique><FISH analysis><FISH assay><Family><Fluorescence In Situ Hybridization><Fluorescent in Situ Hybridization><Focal Infection><Gel><Gene Action Regulation><Gene Expression><Gene Expression Regulation><Gene Regulation><Gene Regulation Process><Gene Transcription><General Transcription Factor Gene><General Transcription Factors><Generations><Genes><Genetic Transcription><Genetic Translation><Genome><Goals><Inclusion Bodies><Initiation Factors><Late Gene Transcriptions><Liquid substance><Maps><Mediating><Membrane><Messenger RNA><Micro-tubule><Microtubules><Motor><Non-Polyadenylated RNA><Nucleic Acids><Orthopox virus><Orthopoxvirus><Peptide Initiation Factors><Peptides><Poly(A) Tail><Poxviridae><Poxvirus officinale><Poxviruses><Process><Promoter Regions><Promotor Regions><Property><Protein Analysis><Protein Biosynthesis><Proteins><Puromicina><Puromycin><Puromycine><Puromycinum><RNA><RNA Binding><RNA Expression><RNA Gene Products><RNA Polymerases><RNA bound><RNA chemical synthesis><RNA synthesis><Reagent><Regulation><Ribonucleic Acid><Ribosomal Peptide Biosynthesis><Ribosomal Protein Biosynthesis><Ribosomal Protein Synthesis><Ribosomes><Site><Stop Codon><Structure><System><Termination Codon><Terminator Codon><Transcript><Transcription><Transcription Factor Proto-Oncogene><Transcription factor genes><Translating><Translation Initiation Factor><Translation Stop Signal><Translational Initiation Factor><Translations><Vaccine Design><Vaccinia virus><Viral><Viral Diseases><Viral Gene Products><Viral Gene Proteins><Viral Genome><Viral Proteins><Virion><Virus><Virus Diseases><Virus Particle><anti-viral agents><anti-viral drugs><anti-viral therapy><anti-virals><biosynthesis><cleaved><fluid><genetic approach><genetic promoter element><genetic promoter sequence><genetic strategy><improved><infection localized><insoluble aggregate><liquid><local infection><mRNA><mRNA Translation><member><membrane structure><plasmid vaccine><polymerization><polypeptide><pox virus><poxvirus vectors><premature><prematurity><prevent><preventing><promoter sequence><protein aggregate><protein aggregation><protein protein interaction><protein synthesis><prototype><recombinant vaccinia virus><tissue culture><transcription factor><vector vaccine><viral infection><viral infectious disease treatment><virus genome><virus infection><virus protein><virus-induced disease>