Sekhar Chakrabarti

Cell proliferative response to vaccinia virus is mediated by VGF.

VGF, a polypeptide encoded by vaccinia virus, shares amino acid sequence homology and functional properties with cellular growth factors EGF and TGF-alpha. The availability of a VGF minus (VGF-) virus mutant has enabled us to examine the role of VGF in the replication of virus in vitro and in vivo. Studies in vitro with A431 cells (high EGF receptor density) showed that VGF+ wild-type virus induced the rapid formation of a focus of infection (not a plaque) which could be blocked by a monoclonal antibody to the EGF receptor. In vivo experiments with chicken embryos indicated that VGF+ virus stimulated the growth of ectodermal and entodermal cells of the chorioallantoic membrane. At early times, the majority of proliferating cells contained no detectable virus antigen, indicating that cell growth preceded infection and was a consequence of VGF secretion. Relative to VGF- virus, VGF+ virus produced lesions which contained more proliferating cells, more virus antigen, and increased amounts of infectious progeny. Secretion of VGF thus explains the conundrum of a nontransforming, strongly cytopathic virus inducing a hyperplastic cell response.

Synthesis, oligomerization, and biological activity of the human immunodeficiency virus type 2 envelope glycoprotein expressed by a recombinant vaccinia virus

The full-length envelope gene from an infectious human immunodeficiency virus type 2 (HIV-2) molecular clone was expressed in CD4+ and CD4- cells by a recombinant vaccinia virus vector. Pulse-chase experiments indicated that gp160 was processed into gp120 and gp41 subunits. Although large amounts of gp120 were shed into the medium, the recombinant vaccinia virus-infected cells fused with uninfected CD4+ cells. The receptor binding of HIV-2 gp120 was further analyzed using a panel composed of nine soluble CD4 mutants containing insertions of 2 amino acids within the first and second immunoglobulin-like domains. Of three mutations previously shown to interfere with HIV-1 gp120 binding, two also interfered with binding of the HIV-2 glycoprotein indicating use of the same binding site. Chemical crosslinking, sucrose gradient sedimentation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were employed to study the oligomerization of the envelope protein. The data indicated that gp160 assembles posttranslationally into dimers and higher oligomers that are probably tetramers.

Survival and mutagenesis of bacterial plasmids with localized carcinogen adducts

Restriction fragments from nonessential gene regions in bacterial plasmids were covalently modified with benzo[a]pyrene-7,8-dihydrodiol-9,10-oxide (BPDE) and ligated to the remaining unmodified fragment to construct the plasmid with a localized patch of BPDE adducts. These preparations were introduced into appropriate strains of E. coli and the effect of the carcinogen adducts on plasmid survival and mutagenesis determined. Survival as a function of adduct concentration of randomly modified plasmids was the same as that of plasmids with localized adducts in both repair-proficient and -deficient strains, indicating that the simple presence of the carcinogen is the main factor in plasmid mortality. The results of these and other experiments indicate that plasmid survival is a function of the adduct/plasmid molecule ratio not the adduct/nucleotide ratio. Plasmids with mutations produced in the region (tetracycline resistance) containing the localized adducts were selected and the nature of the mutations determined by direct sequence analysis. The mutations included frameshifts, transitions, and transversions.