Clustered charge-to-alanine mutagenesis of the vaccinia virus H5 gene: isolation of a dominant, temperature-sensitive mutant with a profound defect in morphogenesis.

Vaccinia virus is the prototypical member of the Poxvirus family, members of which include the human pathogens variola virus, the causative agent of smallpox, and molluscum contagiosum virus, which causes benign but long-lived skin lesions. The 192-kb double-stranded DNA genome of vaccinia virus contains approximately 200 genes, enabling the virus to replicate quite autonomously within the cytoplasm of infected cells (17, 31). The virus encodes a complex transcriptional machinery which directs the expression of three classes of temporally regulated genes: early, which encode the proteins required for replication of the viral genome, and intermediate and late, among whose products are those required for virion morphogenesis. Virion formation commences from cytoplasmic sites known as virosomes or viral factories, which contain viral DNA and viral proteins. Membrane crescents form, begin to enclose the virosome contents, and then enlarge and seal to form the visually distinguishable immature (IV) and mature (IMV) forms of the intracellular class of virions. A subset of these virions become wrapped in membranes derived from the trans-Golgi network and exit the cell. The genome also encodes a large number of effector proteins that enable the virus to evade host defenses. Protein phosphorylation is known to play a crucial role in the regulation of many cellular processes such as initiation of DNA replication, signal transduction, and cell cycle control. Similarly, vaccinia virus regulates its life cycle by encoding two protein kinases, B1 and F10, and a protein phosphatase, H1. Their functions have been deduced, primarily, with the powerful genetic tool of conditional lethal mutants. The F10 kinase, which we have recently shown to be a dual specificity kinase that can phosphorylate Ser, Thr, and Tyr residues, is expressed late during infection and is encapsidated into virions (11a, 26, 44, 46). Temperature-sensitive (ts) mutants with lesions in the F10 gene have revealed that the kinase plays an essential role in the initial steps of virion morphogenesis. The H1 phosphatase, a dual-specificity phosphatase (20), is required for the infectivity and transcriptional competence of newly synthesized virions (28). The role of H1 in virion formation has been elucidated and its viral substrates have been identified by the construction of a recombinant virus (vindH1) in which the expression of the H1 protein is under the control of the lac operator/repressor and hence is induced by inclusion of isopropyl-β-d-thiogalactopyranoside (IPTG) in the culture medium. The B1 Ser/Thr kinase (2, 27, 43), which is expressed during the early phase of viral gene expression, plays an essential role in viral DNA replication (35, 36). We have previously described the phenotype of two ts mutants with lesions in the B1 gene. Temperature shift experiments indicate that the B1 kinase is required throughout DNA replication, although its specific role in unknown. The S2 and Sa subunits of the 40S ribosome are cellular substrates of B1, while the only known viral substrate of the kinase is the vaccinia virus H5 protein (1, 4, 5). The H5 gene encodes a 22.3-kDa protein that is synthesized during both the early and late phases of viral gene expression (39). H5 is diffusely localized in the cytoplasm prior to DNA replication and then becomes concentrated within the virosomes (3). H5 undergoes multiple phosphorylation events, and isoforms with isolectric points of 6.8 to 5.5 have been identified (4). H5 appears to be a substrate for both viral and cellular kinases (3, 4; U. Sankar and P. Traktman, unpublished results). The protein has an extremely proline-rich N′ terminus, which has been cited as an explanation for its anomolous electrophoretic migration (35 kDa) during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (18). Several reports detailing possible roles for H5 have appeared in the literature. Kovacs and Moss identified H5 as a viral protein capable of stimulating late transcription two- to sixfold in vitro and designated H5 as a late gene transcription factor (VLTF-4) (25). Black et al. reported that H5 interacted both in vivo and in vitro with the viral proteins G2R and A18R, both of which play a role in the elongation and termination phases of intermediate and late viral transcription (7). Mohandas et al. reported that H5 had some affinity for both membranes and nucleic acids (30). Because H5 is a substrate of the B1 kinase, and in light of our long-standing interest in the role of this kinase in viral replication, we chose to initiate a genetic analysis of the role of H5 in vivo. Unfortunately, no ts mutants with lesions in the H5 gene have been isolated to date, and the expression of H5 at both early and late times precludes the utility of an inducible recombinant. Therefore, we chose to use the method of “clustered charge-to-alanine” mutagenesis as an approach to generating a ts H5 mutant. In this report, we describe our success with this approach and discuss our unexpected finding that the H5 protein appears to play a critical role in viral morphogenesis.