Poxviruses are large DNA viruses that include the causal agent of human smallpox and vaccinia virus. Poxviruses replicate in cytoplasmic foci known as DNA factories. Here we show that a virus-encoded transcription factor, viral mRNA, cellular RNA-binding protein heterodimer G3BP/Caprin-1 (p137), translation initiation factors eIF4E and eIF4G, and ribosomal proteins are concentrated in the same subdomains of cytoplasmic DNA factories. Furthermore, a cell coinfected with two recombinant vaccinia viruses expressing a virus core protein fused to cyan or yellow fluorescent protein displayed separate cyan and yellow factories, indicating that each factory formed from a single genome and was the site of transcription and translation as well as DNA replication. Hijacking of the host translation apparatus within the factory likely enhances the efficiency of virus replication and contributes to the suppression of host protein synthesis, thereby facilitating poxvirus subjugation of the cell.

A new human herpesvirus has been isolated from CD4' T cells purified from peripheral blood mononuclear cells of a healthy individual (RK), following incubation of the cells under conditions promoting T-cell activation. The virus could not be recovered from nonactivated cells. Cultures of lymphocytes infected with the RK virus exhibited a cytopathic effect, and electron microscopic analyses revealed a characteristic herpesvirus structure. RK virus DNA did not hybridize with large probes derived from herpes simplex virus, EpsteinBarr virus, varicella-zoster virus, and human cytomegalovirus. The genetic relatedness of the RK virus to the recently identified T-lymphotropic human herpesvirus 6 (HHV-6) was investigated by restriction enzyme analyses using 21 different enzymes and by blot hybridization analyses using 11 probes derived from two strains of HHV-6 (Z29 and U1102 MATERIALS AND METHODSPurification of CD4+ T Cells and T-Cell Activation. CD4+ T cells were isolated from peripheral blood lymphocytes (PBLs) by negative selection using immunoadsorption with goat anti-mouse immunoglobulin-coated magnetic particles, as previously described (10, 11). The cells were >99% CD2+ and >96% CD4+, as determined by flow cytometry. Monocytes were <0.1% as determined by staining with nonspecific esterase. For T-cell activation the cultures were incubated for 2 days with plastic-immobilized CD3 monoclonal antibody (mAb) G19-4 (11). To maintain cell proliferation, the cells were further cultured with interleukin 2 (IL-2; Calbiochem) at 30 units/ml or with CD28 mAb 9.3 (12). Cultures were restimulated at weekly intervals with plastic-immobilized CD3 mAb. The cells were cryopreserved in 7.5% dimethyl sulfoxide.Virus Propagation. The Z29 strain of HHV-6 (6) was obtained from C. Lopez (Centers for Disease Control, Atlanta). The U1102 strain (5) was obtained from R. W. Honess (National Institute of Medical Research, London). HHV-6 (Z29) was propagated in PBLs as described (13). Briefly, PBLs were precultured for 3 days in RPMI-10% medium [RPMI 1640 medium plus gentamicin (50 jig/ml) with 10% heat-inactivated fetal bovine serum] containing phytohemagglutinin (PHA; Difco) at 10 ,ug/ml. Infection was done in RPMI-10% medium with PHA at 5 ,ug/ml. HHV-6 (U1102) and HHV-7 (RK) were similarly propagated in PHApretreated PBLs. However, the infection was done in RPMI-10% medium.Electron tTo whom reprint requests should be addressed. 748The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

SUMMARY Poxvirus replication involves synthesis of double stranded RNA (dsRNA), which can trigger antiviral responses by inducing phosphorylation-mediated activation of protein kinase R (PKR) and stimulating 2’5’-oligoadenylate synthetase (OAS). PKR inactivates the translation initiation factor eIF2α via phosphorylation, while OAS induces the endonuclease RNase L to degrade RNA. We show that poxvirus decapping enzymes D9 and D10, which remove caps from mRNAs, inhibit these antiviral responses by preventing dsRNA accumulation. Catalytic site mutations of D9 and D10, but not of either enzyme alone, halt vaccinia virus late protein synthesis and inhibit virus replication. Infection with the D9-D10 mutant was accompanied by massive mRNA reduction, cleavage of ribosomal RNA and phosphorylation of PKR and eIF2α that correlated with a ~15-fold increase in dsRNA compared to wild-type virus. Additionally, mouse studies show extreme attenuation of the mutant virus. Thus, vaccinia virus decapping, in addition to targeting mRNAs for degradation, prevents dsRNA accumulation and anti-viral responses.