Microbial superantigens (SAGs) have been implicated in the pathogenesis of human autoimmune diseases. Preferential expansion of the Vveta7 T cell receptor positive T cell subset in patients suffering from acute-onset type I diabetes has indicated the presence of a surface membrane-bound SAG. Here, we have isolated a novel mouse mammary tumor virus-related human endogenous retrovirus. We further show that the N-terminal moiety of the envelope gene encodes an MHC class II-dependent SAG. We propose that expression of this SAG, induced in extrapancreatic and professional antigen-presenting cells, leads to beta-cell destruction via the systemic activation of autoreactive T cells. The SAG encoded by this novel retrovirus thus constitutes a candidate autoimmune gene in type I diabetes.

A cDNA clone encoding phenol hydroxylase from the soil yeast Trichosporon cutaneum was isolated and characterized. The clone was identified by hybridization screening of a bacteriophage lambda ZAP-based cDNA library with an oligonucleotide probe which corresponded to the N-terminal amino acid sequence of the purified enzyme. The cDNA encodes a protein consisting of 664 amino acids. Amino acid sequences of a number of peptides obtained by Edman degradation of various cleavage products of the purified enzyme were identified in the cDNA-derived sequence. The phenol hydroxylase cDNA was expressed in Escherichia coli to yield high levels of active enzyme. The E. coli-derived phenol hydroxylase is very similar to the T. cutaneum enzyme with respect to the range of substrates acted upon, inhibition by excess phenol, and the order of magnitude of kinetic parameters in the overall reaction. Southern blot analysis revealed the presence of phenol hydroxylase gene-related sequences in a number of T. cutaneum and Trichosporon beigelii strains and in Cryptococcus elinovii but not in Trichosporon pullulans, Trichosporon penicillatum, or Candida tropicalis.

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.

We have recently shown that live attenuated virus vaccines produced on chicken-derived cells contain low levels of particle-associated reverse transcriptase (RT). In both virus and corresponding control harvests produced on chicken embryo fibroblasts, these activities were present at significantly higher concentrations than in the vaccines. In order to identify the putative retrovirus sequence responsible for this activity, a novel method for the selective PCR amplification of particle-associated retrovirus RNA that uses DNA primers complementary to the primer binding sites of the known exogenous retroviruses in combination with an anchor primer was applied. A product of the endogenous avian retrovirus family EAV-0, termed EAV-0 B1 , was reproducibly generated with a tRNA Trp -derived primer from the RT peak fraction of a sucrose density gradient run with a harvest of a live attenuated measles vaccine. In contrast, no products were detected with primers derived from tRNA Pro , tRNA 1,2 Lys or tRNA 3 Lys . In the same fraction, genomic RNA of EAV-0 B1 was demonstrated by long PCR. Analysis of several sucrose density gradients from different harvests of various manufacturers demonstrated accumulation of, and colocalization with, RT activity for the EAV-0 B1 RNA but not for a chicken cellular mRNA. Synthesis of cDNA from EAV-0 B1 RNA was shown by endogenous RT reaction. Furthermore, complexes of naturally primed EAV-0 B1 RNA with RT were demonstrated. Taken together, these data strongly suggest that EAV-0 is able to produce virus-like particles with an active RT.