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We have previously reported that a murine anti-Tat sFv intrabody, termed sFvtat1Ck, directed against the proline-rich N-terminal activation domain of HIV-1, is a potent inhibitor of HIV-1 replication [Mhashilkar, A. M., et al. (1995). EMBO J. 14, 1542-1551]. In this study, the protective effect of sFvtat1Ck expression on HIV-1 replication in both acutely infected and persistently infected CD4+ cells was examined. Stably transfected CD4+ SupT1 cells were resistant to HIV-1 infection at high MOI with both the laboratory isolate HxB2 and six syncytium-inducing (SI) primary isolates. Persistently infected U1 cells, which can be induced to increase HIV-1 mRNA synthesis on addition of PMA or TNF-alpha, showed decreased production of HIV-1 in the presence of sFvtat1Ck. In transduced CD4+-selected, CD8+-depleted, and total PMBCs, the sFvtat1Ck-expressing cells showed marked inhibition of HIV-1 replication. The anti-Tat sFv was subsequently humanized by substituting compatible human framework regions that were chosen from a large database of human V(H) and V(L) sequences on the basis of high overall framework matching, similar CDR length, and minimal mismatching of canonical and V(H)/V(L) contact residues. One humanized anti-Tat sFv intrabody, termed sFvhutat2, demonstrated a level of anti-HIV-1 activity that was comparable to the parental murine sFv when transduced PBMCs expressing the murine or humanized sFv intrabodies were challenged with HxB2 and two SI primary isolates. Because Tat is likely to have both direct and indirect effects in the pathogenesis of AIDS through its multiple roles in the HIV-1 life cycle and through its effects on the immune system, the strategy of genetically blocking Tat protein function with a humanized anti-Tat sFv intrabody may prove useful for the treatment of HIV-1 infection and AIDS, particularly when used as an adjuvant gene therapy together with highly active antiretroviral therapies that are currently available.

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The variceUa-zoster virus (VZV) glycoprotein B (gB) is a major viral antigen which elicits immunity and neutralizing antibodies. In this study, the genomic map position and DNA sequence of a simian varicella virus (SVV) homologue of the VZV gB gene was identified and the transcript analysed. A 32P-labelled VZV gB DNA probe hybridized to a subclone of the SVV BamHI B restriction endonuclease fragment indicating the fine map position of SVV DNA sequences homologous to the VZV gB gene. The SVV gB DNA sequence was determined and analysis revealed a 2751 base pair open reading frame (ORF) with 71.1% identity to the VZV gB gene and 53-8 % identity to the herpes simplex type 1 gB gene. The SVV gB ORF encodes a 916 amino acid polypeptide with a predicted molecular mass of 104K. The deduced SVV and VZV gB polypeptides share 78.9 % amino acid identity and predicted N-linked glycosylation sites, cleavage sites and transmembrane regions. 3~P-labelled SVV gB DNA and RNA probes hybridized to a 3"5 kilobase SVV polyadenylated transcript. Primer extension experiments identified transcript start sites for the SVV and VZV gB genes and permitted a comparison of the sequences upstream of the SVV and VZV gB ORFs. The SW and VZV gB promoter elements are similar in content and align closely. The VZV gB transcript start site suggests a gB polypeptide initiation site which is inconsistent with the previously reported ATG start codon.