Human Immunodeficiency Virus Type 1 Reverse Transcription Is Stimulated by Tat from Other Lentiviruses

Abstract

The tat gene is required by HIV-1 for efficient reverse transcription and this function of Tat can be distinguished from its role in transcription by RNA polymerase II using tat point mutations that abrogate each function independently. The mechanism of Tat's role in reverse transcription, however, is not known, nor is it known whether this role is conserved among trans-activating factors in other retroviruses. Here we examine the abilities of heterologous viral trans-activating proteins from jembrana disease virus (jTat), HIV-2 (Tat2), and equine infectious anemia virus (eTat) to substitute for HIV-1 Tat (Tat1) and restore reverse transcription in HIV-1 carrying an inactivated tat gene. Natural endogenous reverse transcription assays showed that trans-activators from some retroviruses (Tat2 and jTat, but not eTat) could substitute for Tat1 in complementation of HIV-1 reverse transcription. Finally, we show that Y47 is critical for Tat1 to function in reverse transcription, but not HIV-1 gene expression. We mutated the homologous position in jTat to H62Y and found it did not improve its ability to stimulate reverse transcription, but an H62A mutation did inhibit jTat complementation. These data highlight the finding that the role of Tat in reverse transcription is not related to trans-activation and demonstrate that other tat genes conserve this function.

Human Immunodeficiency Virus Type 1 Protease Regulation of Tat Activity Is Essential for Efficient Reverse Transcription and Replication

“…Finally, this study confirms previous reports that Tat Y47 was essential for virus replication in H9 cells and further showed that a Y47D mutation in Tat imparted a significant reverse transcription defect that, in conjunction with a small gene expression defect, impeded virus replication (12,26). Our data indicate that the reduced viral fitness observed by Verhoef et al (26) was likely due to combined inefficiencies in transcription and reverse transcription.…”

“…We previously reported that HIV-1 Tat improved the efficiency of minus-strand SS DNA synthesis using a transcomplementation system (9,12,22). Briefly, we made a 293 cell line stably transfected with HIV-1 with a functional deletion of the tat gene, called 293HIV⌬tat.…”

“…twofold) and reverse transcription (ca. fourfold) (12,26). Therefore, we asked whether other mutations of the proviral tat gene at Y47 had similar effects on replication and/or reverse transcription and whether these mutations also affected cleavage of Tat by PR.…”

“…The protein content of cell lysates was measured using a modified Bradford assay (Bio-Rad). ␤-Galactosidase activity was determined using a chlorophenol red galactopyranoside assay (12). CAT activity was measured using the Roche Diagnostics CAT ELISA kit.…”

Construction of a Doxycycline-Dependent Simian Immunodeficiency Virus Reveals a Nontranscriptional Function of Tat in Viral Replication

“…It seems unlikely that this second Tat function involves binding to TAR RNA, since the Tat-TAR binding study demonstrated that the mutations in the SIV-rtTA TAR element prevent Tat binding. Multiple additional roles for Tat have been suggested in HIV-1 biology; for example, Tat has been reported to affect mRNA capping, splicing, and translation (14,18,20,21,23,67,69,78), to stimulate reverse transcription (1,36,37,42,43,71), and to suppress RNA interference (7,35). Moreover, Huang et al demonstrated that HIV-1 variants regulated by the Gal4-VP16 transactivator protein require Tat for infectivity (38), and we recently observed that the introduction of a frameshift mutation in the HIV-1 Tat gene, which prevents Tat production, abolished HIV-rtTA replication.…”