Cyclin K Inhibits HIV-1 Gene Expression and Replication by Interfering with Cyclin-dependent Kinase 9 (CDK9)-Cyclin T1 Interaction in Nef-dependent Manner

Sohrab Zafar Khan,Debashis Mitra

doi:10.1074/jbc.m110.201194

Abstract

Human immunodeficiency virus-1 (HIV-1) exploits a number of host cellular factors for successful survival and propagation. The viral protein Nef plays an important role in HIV-1 pathogenesis by interacting with various cellular proteins. In the present work, we identified Cyclin K (CycK) as a novel Nef-interacting protein, and for the first time, we showed that CycK inhibits HIV-1 gene expression and replication in a Nef-dependent manner. The positive elongation factor b complex comprising cyclin-dependent kinase 9 (CDK9) and Cyclin T1 is a critical cellular complex required for viral gene expression and replication. Enhanced expression of CycK in the presence of Nef induced CycK-CDK9 binding, which prevented CDK9-Cyclin T1 complex formation and nuclear translocation of CDK9, resulting in inhibition of HIV-1 long terminal repeat-driven gene expression. Furthermore, this effect of CycK was not observed with Nef-deleted virus, indicating the importance of Nef in this phenomenon. Finally, silencing of CycK in HIV-1-infected cells resulted in increased translocation of CDK9 into the nucleus, leading to increased viral gene expression and replication. These data also suggest that endogenous CycK might act as an inhibitory factor for HIV-1 gene expression and replication in T-cells. Thus, our results clearly demonstrate that CycK utilizes HIV-1 Nef protein to displace CycT1 from the positive elongation factor b complex, resulting in inhibition of HIV-1 gene expression and replication.

Highlights

JULY 1, 2011 VOLUME 286 NUMBER 26 any enzymatic activity, but it modulates diverse signaling pathways and the gene expression of host cell proteins [7, 8]
The functions attributed to Nef are believed to be conserved across various Human immunodeficiency virus-1 (HIV-1) subtypes, and to date, the majority of the functional studies have been performed with subtype B Nef protein
The co-immunoprecipitation experiment revealed that in the presence of increasing levels of Cyclin K (CycK) in pNL4-3-transfected cells CycK interaction with cyclin-dependent kinase 9 (CDK9) was increased, whereas CycT1 association with CDK9 was reduced in a dose-dependent manner (Fig. 6A). This result suggests that the increase in CycK levels in the cell reduces the formation of the CDK9-CycT1 complex, which is an active protein complex required for HIV-1 long terminal repeat (LTR)-driven gene expression

Summary

Introduction

JULY 1, 2011 VOLUME 286 NUMBER 26 any enzymatic activity, but it modulates diverse signaling pathways and the gene expression of host cell proteins [7, 8]. To further confirm our finding, a GST-Nef (subtype C) pulldown assay was performed using Jurkat cell lysate as described under “Experimental Procedures.” The result of the pulldown clearly indicates that Cyclin K (CycK) interacts with subtype C Nef but not with GST (Fig. 1C).

Results

Conclusion