Human T-cell leukemia virus (HTLV) Tax protein has been implicated in the HTLV oncogenic process, primarily due to its pleiotropic effects on cellular genes involved in growth regulation and cell cycle control. To date, several approaches attempting to correlate Tax activation of the CREB/activating transcription factor (ATF) or NFB/Rel transcriptional activation pathway to cellular transformation have yielded conflicting results. In this study, we use a unique HTLV-2 provirus (HTLV c-enh ) that replicates by a Tax-independent mechanism to directly assess the role of Tax transactivation in HTLV-mediated T-lymphocyte transformation. A panel of well-characterized tax-2 mutations is utilized to correlate the respective roles of the CREB/ATF or NFB/Rel signaling pathway. Our results demonstrate that viruses expressing tax-2 mutations that selectively abrogate NFB/Rel or CREB/ATF activation display distinct phenotypes but ultimately fail to transform primary human T lymphocytes. One conclusion consistent with our results is that the activation of NFB/Rel provides a critical proliferative signal early in the cellular transformation process, whereas CREB/ATF activation is required to promote the fully transformed state. However, complete understanding will require correlation of Tax domains important in cellular transformation to those Tax domains important in the modulation of gene transcription, cell cycle control, induction of DNA damage, and other undefined activities.

Poxviruses that are attenuated for growth in human cells provide a safe means of HIV antigen expression and are capable of eliciting HIV-specific immune responses, including CD8+ cytotoxic T-lymphocyte (CTL) responses. HIV-1 antigen expression in human cells by attenuated poxvirus vectors may be limited by interferon-mediated host defense mechanisms. To enhance HIV antigen expression in human cells, the vaccinia virus E3L and K3L genes were inserted into a canarypox vector that expresses HIV-1 Gag, Env, and a Nef/Pol polyepitope string. E3L and K3L markedly reduced the activation of the double-stranded RNA-dependent protein kinase, PKR, and led to a significant reduction in apoptosis in HeLa cells. Production and release of HIV-1 antigen in the form of pseudovirions was enhanced in both duration and magnitude by this vector modification. The addition of immunomodulatory genes to attenuated poxviruses represents a novel strategy for enhancing antigen production by live vector HIV vaccine candidates.

Canarypox viruses undergo abortive replication in mammalian cells. Despite this restriction on replication in mammalian cells, significant immune responses have been shown in animals and in humans receiving recombinant canarypox vaccine vectors expressing heterologous immunogens. A recombinant canarypox vaccine candidate (vCP205), which expresses human immunodeficiency virus (HIV)-1 Gag, Env, and protease proteins, is presently under investigation in phase I and phase II human trials in the United States and elsewhere. In this study, the ability of vCP205 to elicit HIV Gag-Env pseudovirion formation in avian and mammalian cells was investigated. Gag-Env pseudovirions were produced from both avian and mammalian cell lines infected by this vaccine vector. A subset of mammalian cells was identified in which pseudovirion production and release was very efficient, surpassing the production from infected avian cells. The production of Gag-Env pseudovirions by canarypox HIV vaccine vectors may have important implications for future HIV vaccine design.

A mutational analysis of human T-cell leukemia virus type 2 (HTLV-2) Tax (Tax-2) was performed to identify regions within Tax-2 important for activation of promoters through the CREB/ATF or NF-B/Rel signaling pathway. Tax-2 mutations within the putative zinc-binding region as well as mutations at the carboxy terminus disrupted CREB/ATF transactivation. A single mutation within the central proline-rich region of Tax-2 disrupted the transactivation of the NF-B/Rel pathway. Surprisingly, this mutation, which is thought to be in a separate activation domain, was suppressed by mutations within or around the putative zinc-binding region, suggesting an interaction between these two regions. These analyses indicate that the functional regions or domains important for transactivation through the CREB/ATF or NF-B/Rel signaling pathway are similar, but not identical, in Tax-1 and Tax-2. Identification of these distinct Tax-2 mutants should facilitate comparative biological studies of HTLV-1 and HTLV-2 and ultimately lead to the determination of the functional importance of Tax transacting capacities in T-lymphocyte transformation by HTLV.