Cell, Vol. 62, 621-626,

August

24, 1990, Copyright

0 1990 by Cell Press

Poxviruses: An Emerging Portrait of Biological Strategy Paula Traktman Departments of Cell Biology and Microbiology Cornell University Medical College New York, New York 10021

During the history of medicine and microbiology, poxviruses have emerged periodically to play leading roles. Early attempts to confer immunity to smallpox by variolation were practiced for centuries before Jenner perceived in 1798 that scarification with the related cowpox could prevent smallpox infection. Vaccinia emerged as the vaccine of choice and, as the agent that rid the world of smallpox, played its first scientific role. Vaccinia’s second prominent role owed much to its large size and the ease of virion isolation. It was the first virus to be visualized by light microscopy and among the first to be seen in greater detail with the newly developed electron microscope. It was also the first animal virus to be grown in tissue culture. As well as providing the first estimates of the chemical composition of animal viruses, studies on vaccinia were the first to reveal that virions were not merely inert particles of nucleic acid and structural proteins. The discovery of RNA polymerase within vaccinia virions was the first observation of an encapsidated enzyme. Detection of the mRNA capping enzyme and poly(A) polymerase were seminal discoveries in dissecting mRNA biogenesis. In the mid-1970s these early morphologic and biochemical advantages were overshadowed by the difficulties inherent in subjecting such a complex virus to genetic study. This temporary eclipse in the study of poxviruses, however, was surpassed with the advent of modern molecular biology. In the 10 years since the first genomic clones were generated, poxviruses have emerged as an excellent system in which to dissect transcription and replication; as well, the intricacies of the virus’s domination over cellular processes and the host’s immune system are compelling. At the 8th International Symposium on Poxviruses and Iridoviruses, held on May 15-20 at the Wintergreen Conference Center, the maturation of the field was highlighted by the presentation of the complete sequence of the Copenhagen strain of vaccinia virus. It seems a fitting time to review our understanding of poxvirus biology (primarily vaccinia virus) in the context of the advances presented at this meeting. The Genome

Takes Center

Stage

The first talk of the meeting catapulted the field onto a new plane of knowledge and potential with the presentation of the entire sequence of the Copenhagen strain of vaccinia virus (E. Paoletti, Virogenetics). For the first time, the genomic organization could be seen in toto and a complete search for open reading frame (ORF) homologies was offered. The Copenhagen sequence predicts a genome of 191,636 bp that is 67% AT and has 198 ORFs of

Meeting Review

at least 60 amino acids. (ORFs are defined by position [ascending from left to right with respect to the genome] within their genomic Hindlll fragment [A-P]). The value of this data is enhanced by the ability to perform comparisons with the sequence data generated in recent years for the WR strain of vacciniavirus. The published and unpublished WR sequences will be compiled within the year by G. Smith (University of Oxford) and B. Moss (NIH). The genome of vaccinia (Figure 1) and most other poxviruses contains inverted terminal repeats; in vaccinia, approximately 10 kb of genomic sequence is found in this conformation, making on the order of ten genes diploid. At the extreme termini are hairpin loops of approximately 100 nucleotides; these are found in two isoforms that are inverted and complementary with respect to one another. Curiously, only one isoform of the hairpin was found in the compilation of the Copenhagen sequence. Just internal to the hairpin is a sequence essential for resolution of replicated concatemers; adjacent to this element are numerous tandem repeats of short sequence elements. Homologous recombination, vigorous during poxvirus infection, leads to variation in the number of these repeated motifs. Several key generalizations regarding the genetic structure of vaccinia were confirmed by the new sequence information. First, the genome is densely packed with genes that are virtually contiguous. Second, early and late genes are interspersed throughout the genome. Third, the polarity of the genes displays local organization. Genes in the leftmost 35 kb are transcribed almost exclusively in the leftward direction, whereas those in the rightmost 35 kb are transcribed almost exclusively in the rightward direction. The central portion of the genome contains transcriptional units of both polarities. The transcriptional units have no intervening sequences, consistent with the restriction of the poxvirus infectious cycle to the cytoplasm. In general, 5’ and 3’ untranslated leaders are very short. Comparison of the deduced protein sequences derived from poxvirus genes reveals an extraordinary number of strong homologies with previously characterized proteins. Conserved motifs such as nucleotide binding sites, zinc fingers, and leucine zippers have been detected; enzymes involved in nucleic acid metabolism, such as type I topoisomerase, RNA polymerase, DNA ligase, DNA polymerase, thymidine kinase, thymidylate kinase, and ribonucleotide reductase show significant similarity to their mammalian and yeast counterparts. Vaccinia also encodes proteins with homology to growth fac-

Figure

1. The Vaccinia

Genome

Arrows indicate the general direction of transcription. ITR, inverted terminal repeat; T, tandem repeats; A, resolution signal; Ii. hairpin.

Cell 622

tars, complement regulatory proteins, and serine protease inhibitors. Homologies to profilin, superoxide dismutase, and tumor necrosis factor receptor were among the most provocative findings provided by sequence analysis of the WR and Copenhagen strains of vaccinia as well as the leporipoxvirus Shope fibroma virus. The Cascade of Gene Expression-A in Three Acts

Play

Within the core of the vaccinia virion, the genome is assembled with all of the enzymatic machinery required for early gene expression (for review see Moss, 1990). Upon entry into a host cell, an uncoating process releases the intact core and transcription commences (Figure 2). The viral RNA polymerase consists of at least seven subunits; the genes for five of these have been identified (A24, D7, E4, J4, J6). The two largest subunits display sequence homology with eukaryotic polymerases; one of the small subunits shows homology to the eukaryotic S-II transcription elongation factor (E. Paoletti; B. Moss). The polymerase itself cannot initiate or terminate with specificity, and can only transcribe a single-stranded DNA template. Another component of the transcriptional machinery has 96 and 33 kd subunits (Dl, D12) and functions both as the capping enzyme and termination protein. The genes encoding both subunits have been cloned and expressed in E. coli (E. Niles, SUNY Buffalo; B. Moss; S. Shuman, Sloan-Kettering): the large subunit possesses triphosphatase and guanylyl transferase activities: the small subunit is required for methyl transferase activity (S. Shuman). In

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Poxviruses: an emerging portrait of biological strategy.

The meeting offered ample evidence that the field of poxvirology has truly come of age. Many investigators were attracted to the field by its experime...
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