Vaccinia Virus Virulence Research Articles

Deletion of the vaccinia virus growth factor gene reduces virus virulence.

The vaccinia virus growth factor (VGF) gene encodes a polypeptide with amino acid sequence homology to epidermal growth factor (EGF) and transforming growth factor alpha and is present twice, once at each end of the virus genome within the inverted terminal repetition. Recombination procedures were used to replace more than half of both VGF genes with a beta-galactosidase cassette which served as a color indicator for isolating an unconditionally viable VGF- mutant. The VGF- mutant genotype and phenotype were confirmed by Southern blot analysis and assays for functional growth factor. The plaque-forming efficiencies of VGF- and wild-type (WT) viruses were similar in a variety of cell types containing low or high densities of EGF receptors, suggesting a lack of a specific requirement for either VGF or the EGF receptor in the initiation of virus infection. The yield of VGF- virus was similar to that of WT virus in growing BS-C-1 and Swiss 3T3 cells, but lower in resting Swiss 3T3 cells. The greatest differences between VGF- and WT virus occurred in vivo: higher doses of VGF- virus than WT virus were required for intracranial lethality in mice and for production of skin lesions in rabbits. Thus, expression of the VGF gene is important to the virulence of vaccinia virus.

Association of non-viral proteins with recombinant vaccinia virus virions.

Evidence is presented which suggests that recombinant vaccinia virus particles (VV:CAT), containing the bacterial chloramphenicol acetyl transferase gene, are capable of encapsidating both the foreign protein which they encode (CAT) as well as cellular enzymes such as thymidine kinase. These results are discussed with respect to using VV to passively introduce biologically-active proteins into cells or organisms.

Isolation and characterization of attenuated mutants of vaccinia virus

Attenuated variants of vaccinia virus with two specific and stable genetic markers were obtained after long-term passage of persistently infected Friend erythroleukemia (FEL) cells. The genetic markers were an 8-MDa deletion on the left HindIII-C terminus of the viral genome and sequence alterations localized in the middle of the HindIII-A DNA fragment. This latter genetic marker led to small plaque size phenotype of these variants. The mode of replication of these variants in tissue culture cells and their virulence in mice were analyzed. In cultured cells, these variants have greatly reduced virus yields in cell lines of different origins. These variants penetrate into cells, synthesize early and late viral proteins, and replicate their DNA with about the same efficiency as wild-type virus. The defect of these variants appears at some step during virus maturation. When groups of BALB/c mice were inoculated intraperitoneally (ip) with these variants, 50% of the mice survived with ⩾ 1 × 10 9 plaque-forming units (PFU) as opposed to about 50% survival for mice inoculated with 1 × 10 6 PFU of wild-type virus. Mice inoculated with these variants were fully protected when challenged ip with lethal doses of wild-type virus. The reduced virulence of these variants correlated with the 8-MDa deletion; in addition, the plaque size phenotype marker contributes to a further decrease of the virulence of vaccinia virus. Due to their limited virus production and protective immune response, these variants may be potentially useful as vaccines.

A 14K envelope protein of vaccinia virus with an important role in virus-host cell interactions is altered during virus persistence and determines the plaque size phenotype of the virus

The phenomenon of genetic variability and attenuation or virulence of poxviruses is poorly understood. We have identified mutants of vaccinia virus from untreated and interferon (IFN)-treated persistently infected Friend erythro-leukemia (FEL) cells that have major alterations in the size of a virus structural protein. This protein is part of the virus envelope as documented with specific monoclonal antibody (mAbC3). This protein, under reducing conditions, has a molecular weight of about 14,000 (14K) Da in wild-type virus but gained 1.5 K in mutants from untreated, persistently infected cells and about 0.5K in mutants from IFN-treated persistently infected cells. Under nonreducing conditions, this protein forms covalently linked oligomers which also differ in size between wild-type and mutant viruses. The 14K protein elicits humoral immune response as assessed by immunoblots of two-dimensional SDS-PAGE analysis using rabbit anti-vaccinia serum. Two molecular forms of the 14K protein with different isolectric points were found only in mutants from untreated, persistently infected cells. Protein modifications were the result of DNA sequence alterations in the virus population since the 15.5K protein could be reverted to 14K after marker-rescue with the cloned 14K encoding gene. We provide direct evidence that changes in size of the 14K envelope protein are responsible for the small plaque size phenotype of these variants. From our previous studies ( J. F. Rodriguez, R. Janeczko, and M. Esteban, 1985. J. Virol. 56, 352–356; J. F. Rodriguez, E. Paez, and M. Esteban, 1987, J. Virol. 61, 395–404) and those described here, we established that the 14K protein plays an important role in virus-host cell interactions. This is because this protein is part of the virus envelope, is highly antigenic, elicits neutralizing antibodies, has a role in virus penetration acting in cell fusion, is involved in the virus plaque size phenotype and is highly conserved among Orthopoxviruses. In addition, because mutants with altered 14K protein that have gained 1.5K in size are attenuated ( S. Dallo and M. Esteban, 1987, Virology 159, 408–422), it suggests that a modified form of this protein contributes to the decrease of the virulence of vaccinia virus.