Binding of Tat to TAR and Recruitment of Positive Transcription Elongation Factor b Occur Independently in Bovine Immunodeficiency Virus

Self Cite

The transcriptional elongation of human immunodeficiency virus type 1 (HIV-1) is mediated by the virally encoded transactivator Tat and its cellular cofactor, positive transcription elongation factor b (P-TEFb). The human cyclin T1 (hCycT1) subunit of P-TEFb forms a stable complex with Tat and the transactivation response element (TAR) RNA located at the 5 end of all viral transcripts. Previous studies have demonstrated that hCycT1 binds Tat in a Zn 2؉ -dependent manner via the cysteine at position 261, which is a tyrosine in murine cyclin T1. In the present study, we mutated all other cysteines and histidines that could be involved in this Zn 2؉ -dependent interaction. Because all of these mutant proteins except hCycT1(C261Y) activated viral transcription in murine cells, no other cysteine or histidine in hCycT1 is responsible for this interaction. Next, we fused the N-terminal 280 residues in hCycT1 with Tat. Not only the full-length chimera but also the mutant hCycT1 with an N-terminal deletion to position 249, which retained the Tat-TAR recognition motif, activated HIV-1 transcription in murine cells. This minimal hybrid mutant hCycT1-Tat protein bound TAR RNA as well as human and murine P-TEFb in vitro. We conclude that this minimal chimera not only reproduces the high-affinity binding among P-TEFb, Tat, and TAR but also will be invaluable for determining the threedimensional structure of this RNA-protein complex.

Section: Discussionsupporting

confidence: 78%

A Minimal Chimera of Human Cyclin T1 and Tat Binds TAR and Activates Human Immunodeficiency Virus Transcription in Murine Cells

Fujinaga¹,

Irwin

Taube

et al. 2002

Self Cite

“…The finding that CycT1 is induced only transiently during macrophage differentiation has implications for lentiviruses in addition to HIV-1, notably bovine immunodeficiency virus and equine infectious viruses (EIAV), both of which also use a viral Tat protein/TAR RNA mechanism, and therefore CycT1, to activate transcription directed by the viral LTR (1,2,39 (38). It is possible that the transient induction of CycT1 in equine macrophages limits the levels of EIAV replication and allows the immune system to contain viral replication to nonpathogenic levels.…”

Section: Discussionmentioning

confidence: 99%

Transient Induction of Cyclin T1 during Human Macrophage Differentiation Regulates Human Immunodeficiency Virus Type 1 Tat Transactivation Function

Liou

Herrmann

Rice

2002

The human immunodeficiency virus type 1 (HIV-1) Tat protein is essential for viral replication and stimulates transcription of the integrated provirus by recruiting the kinase complex TAK/P-TEFb, composed of cyclin T1 (CycT1) and Cdk9, to the viral TAR RNA element. TAK/P-TEFb phosphorylates the RNA polymerase II complex and stimulates transcriptional elongation. In this report, we investigated the regulation of TAK/P-TEFb in primary human macrophages, a major target cell of HIV infection. While Cdk9 levels remained constant, CycT1 protein expression in freshly isolated monocytes was very low, increased early during macrophage differentiation, and, unexpectedly, decreased to very low levels after about 1 week in culture. The kinase activity of TAK/P-TEFb paralleled the changes in CycT1 protein expression. RNA analysis indicated that the transient induction of CycT1 protein expression involves a posttranscriptional mechanism. In transient transfection assays, the ability of Tat to transactivate the HIV long terminal repeat (LTR) in the late differentiated macrophages was greatly diminished relative to its ability to transactivate the HIV LTR in early differentiated cells, strongly suggesting that CycT1 is limiting for Tat function in late differentiated macrophages. Interestingly, lipopolysaccharide, a component of the cell wall of gram-negative bacteria, reinduced CycT1 expression late in macrophage differentiation. These results raise the possibility that regulation of CycT1 expression may be involved in establishing latent infection in macrophages and that opportunistic infection may reactivate the virus by inducing CycT1 expression.The two major cell types targeted by human immunodeficiency virus type 1 (HIV-1) are helper T cells and macrophages, as these cell types express CD4 and a chemokine receptor, either CCR5 or CXCR4, on their surfaces. CD4 is the primary receptor for the virus, while CCR5 or CXCR4 serves as a coreceptor that mediates fusion of the enveloped virion with the cell membrane. In most individuals, HIV-1 infection leads to an inevitable erosion of the immune system and development of AIDS. Analyses of patients treated with drugs that effectively block HIV-1 replication in vivo have revealed that infected CD4 ϩ T cells produce the majority (Ͼ90%) of virus during the prolonged infection period before the onset of AIDS (reviewed in reference 4). These infected CD4 ϩ T cells are short-lived, with a half-life estimated to be Ͻ1 day due to direct cytopathic effects of infection and to clearance of infected cells by immune-mediated mechanisms, especially that of cytotoxic CD8 ϩ T lymphocytes. A second population of productively infected cells has a half-life estimated to be 1 to 4 weeks; this population produces a few percent of virus in infected individuals prior to AIDS. Although the nature of this longer-lived population of infected cells is uncertain, macrophages are a likely candidate cell type, as they are resistant to the cytopathic effects of infection in vitro and the half-life of tissue ma...

“…In these cases, cyclin T1 is not required to form stable, high-affinity RNA complexes and the loops do not contribute to recognition (2,6,11,49). Despite the structural similarity of the TAR sites (50), BIV Tat binds HIV TAR only weakly and cannot use cyclin T1 as a cofactor to bind TAR.…”

mentioning

confidence: 99%

Selection of TAR RNA-Binding Chameleon Peptides by Using a Retroviral Replication System

Xie

Calabro

Wainberg

et al. 2004

The interaction between the arginine-rich motif (ARM) of the human immunodeficiency virus (HIV) Tat protein and TAR RNA is essential for Tat activation and viral replication. Two related lentiviruses, bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV), also require Tat ARM-TAR interactions to mediate activation, but the viruses have evolved different RNA-binding strategies. Interestingly, the JDV ARM can act as a "chameleon," adopting both the HIV and BIV TAR binding modes. To examine how RNA-protein interactions may evolve in a viral context and possibly to identify peptides that recognize HIV TAR in novel ways, we devised a retroviral system based on HIV replication to amplify and select for RNA binders. We constructed a combinatorial peptide library based on the BIV Tat ARM and identified peptides that, like the JDV Tat ARM, also function through HIV TAR, revealing unexpected sequence characteristics of an RNAbinding chameleon. The results suggest that a retroviral screening approach may help identify high-affinity TAR binders and may provide new insights into the evolution of RNA-protein interactions.