Double-stranded RNA is a potent inducer of interferon, a modulator of the expression of a number of other genes involved in the response of cells to virus infection, an activator of the interferon-induced antiviral state, and may be involved in differentiation, induction of apoptosis, and control of oncogenic transformation. This review will attempt to summarize what is known about the cellular proteins that act to mediate the response of cells to double-stranded RNA and the viral and cellular macromolecules that may be able to modulate these responses.

A vaccinia virus-encoded double-stranded RNA-binding protein, p25, has been previously implicated in inhibition of the interferon-induced, double-stranded RNAactivated protein kinase. In this study, we have identified the vaccinia viral gene (WR strain) that encodes p25. Amino acid sequence analysis of a chymotryptic fragment of p25 revealed a close match to the vaccinia virus (Copenhagen strain) E3L gene. The WR strain E3L gene was cloned and expressed either in COS-1 cells or in rabbit reticulocyte lysates in vitro. A M, 25,000 polypeptide that could bind to poly(rI) poly(rC)-agarose and that reacted with p25-specific antiserum was produced in each case. In addition, COS cells expressing E3L gene products inhibited activation of the double-stranded RNA-activated protein kinase in extracts from interferon-treated cells. Removal of E3L-encoded products by adsorption with anti-p25 antiserum resulted in loss of kinase inhibitory activity. These results demonstrate that the vaccinia virus E3L gene encodes p25 and that the products of the E3L gene have kinase inhibitory activity. Comparison of the deduced amino acid sequence of the E3L gene products with the protein sequence data base revealed a region closely related to the human interferon-induced, double-stranded RNA-activated protein kinase.

The N-terminal domain of the E3L protein of vaccinia virus has sequence similarity to a family of Z-DNA binding proteins of defined three-dimensional structure and it is necessary for pathogenicity in mice. When other Z-DNA-binding domains are substituted for the similar E3L domain, the virus retains its lethality after intracranial inoculation. Mutations decreasing Z-DNA binding in the chimera correlate with decreases in viral pathogenicity, as do analogous mutations in wild-type E3L. A chimeric virus incorporating a related protein that does not bind Z-DNA is not pathogenic, but a mutation that creates Z-DNA binding makes a lethal virus. The ability to bind the Z conformation is thus essential to E3L activity. This finding may allow the design of a class of antiviral agents, including agents against variola (smallpox), which has an almost identical E3L.

Vaccinia virus (VACV) has been used more extensively for human immunization than any other vaccine. For almost two centuries, VACV was employed to provide cross-protection against variola virus, the causative agent of smallpox, until the disease was eradicated in the late 1970s. Since that time, continued research on VACV has produced a number of modified vaccines with improved safety profiles. Attenuation has been achieved through several strategies, including sequential passage in an alternative host, deletion of specific genes or genetic engineering of viral genes encoding immunomodulatory proteins. Some highly attenuated third-and fourth-generation VACV vaccines are now being considered for stockpiling against a possible re-introduction of smallpox through bioterrorism. Researchers have also taken advantage of the ability of the VACV genome to accommodate additional genetic material to produce novel vaccines against a wide variety of infectious agents, including a recombinant VACV encoding the rabies virus glycoprotein that is administered orally to wild animals. This review provides an in-depth examination of these successive generations of VACV vaccines, focusing on how the understanding of poxviral replication and viral gene function permits the deliberate modification of VACV immunogenicity and virulence.

The vaccinia virus (VV) E3L gene is responsible for providing interferon (IFN) resistance and a broad host range to VV in cell culture. The E3L gene product contains two distinct domains. A conserved carboxy-terminal domain, which is required for the IFN resistance and broad host range of the virus, has been shown to bind double-stranded RNA (dsRNA) and inhibit the antiviral dsRNA-dependent protein kinase, PKR. The aminoterminal domain, while conserved among orthopoxviruses, is dispensable in cell culture. To study the role of E3L in whole-animal infections, WR strain VV recombinants either lacking E3L (VV⌬E3L) or expressing an amino-terminal (VVE3L⌬83N) or carboxy-terminal (VVE3L⌬26C) truncation of E3L were constructed. Whereas wild-type VV had a 50% lethal dose of approximately 10 4 PFU after intranasal infection, and elicited severe weight loss and morbidity, VV⌬E3L was apathogenic, leading to no death, weight loss, or morbidity. VV⌬E3L was also apathogenic after intracranial injection. Although the amino-terminal domain of E3L is dispensable for infection of cells in culture, both the amino-and carboxy-terminal domains of E3L were required for full pathogenesis in intranasal infections. These results demonstrate that the entire E3L gene is required for pathogenesis in the mouse model.

Although hepatitis C virus (HCV) infection is an emerging global epidemic causing severe liver disorders, the molecular mechanisms of HCV pathogenesis remain elusive. The NS5A nonstructural protein of HCV contains several proline-rich sequences consistent with Src homology (SH) 3-binding sites found in cellular signaling molecules. Here, we demonstrate that NS5A specifically bound to growth factor receptor-bound protein 2 (Grb2) adaptor protein. Immunoblot analysis of anti-Grb2 immune complexes derived from HeLa S3 cells infected with a recombinant vaccinia virus (VV) expressing NS5A revealed an interaction between NS5A and Grb2 in vivo. An inactivating point mutation in the N-terminal SH3 domain, but not in the C-terminal SH3 domain, of Grb2 displayed significant diminished binding to NS5A. However, the same mutation in both SH3 regions completely abrogated Grb2 binding to NS5A, implying that the two SH3 domains bind in cooperative fashion to NS5A. Further, mutational analysis of NS5A assigned the SH3-binding region to a proline-rich motif that is highly conserved among HCV genotypes. Importantly, phosphorylation of extracellular signalregulated kinases 1 and 2 (ERK1͞2) was inhibited in HeLa S3 cells infected with NS5A-expressing recombinant VV but not recombinant VV control. Additionally, HeLa cells stably expressing NS5A were refractory to ERK1͞2 phosphorylation induced by exogenous epidermal growth factor. Moreover, the coupling of NS5A to Grb2 in these cells was induced by epidermal growth factor stimulation. Therefore, NS5A may function to perturb Grb2-mediated signaling pathways by selectively targeting the adaptor. These findings highlight a viral interceptor of cellular signaling with potential implications for HCV pathogenesis.

Vaccinia virus encodes two regulators of the cellular antiviral response. The E3L gene is thought to act primarily by sequestering double-stranded RNA, whereas the K3L gene is thought to act as a competitive inhibitor of the double-stranded RNA-dependent protein kinase, PKR. The broad host range associated with vaccinia virus replication appears to be related to the presence of these genes. The E3L gene is required for replication in HeLa cells, but is not required for replication in BHK cells. On the contrary, the K3L gene is required for replication in BHK cells, but is dispensable for replication in HeLa cells. Our results suggest that these cell lines varied in the expression of endogenous activatable PKR and that replication of vaccinia virus in different cell lines led to altered levels of double-stranded RNA synthesis from the virus. Vaccinia virus was able to overcome these cellular variations by regulating PKR activity through the synthesis of either E3L or K3L. The results suggest that vaccinia virus has evolved a broad host range by maintaining both the E3L and the K3L genes.

The Sonora tlger salamander Ambystoma tignnuni stebb~nsi Lowe is a genetically d~stinct race restricted to about 30 small ponds in the San Rafael Valley 111 southern Arizona, USA, which was added recently to the USA Federal List of Endangered Species Populations of these salamanders periodically expenence decimating epizootics. Virus was ~solated from diseased salamanders using fish cell cultures, injected into healthy laboratory-reared salamanders, and then reisolated in cell culture. Electron microscopy of thin sections from dying salamanders revealed abundant enveloped and nonenveloped icosahedral virus particles approximately 160 to 180 nm in diameter in the cytoplasm of skin and liver cells and free in the intercellular spaces. This virus, believed to be an iridovirus based on viral morphology and host pathology, was demonstrated to be the primary pathogen in these epizootics, and is the first lethal epizootic virus reported from salamanders. We have named the virus Ambystoma tigrinum Virus (ATV). Hemolytic bacteria were isolated from sick individuals, but we were unable to induce the disease by exposing salamanders to isolated bacteria at concentrations up to 10' ml-' KEY WORDS: Salamander. Ambystoma tigrinum stebbinsi. Virus. Indovirus. Amphibian decline