Heat Shock Protein 40 Is Necessary for Human Immunodeficiency Virus-1 Nef-mediated Enhancement of Viral Gene Expression and Replication

Abstract

The human immunodeficiency virus-1 (HIV-1) Nef protein, originally identified as a negative factor, has now emerged as one of the most important viral proteins necessary for viral pathogenesis and disease progression. Nef has been also implicated in viral infectivity and replication, however, the molecular mechanism of Nef-induced viral gene expression and replication is not clearly understood. Although involvement of heat shock proteins in viral pathogenesis has been reported earlier, a clear understanding of their role remains to be elucidated. Here we report for the first time that Nef not only interacts with heat shock protein 40 (Hsp40) but it also induces the expression of Hsp40 in HIV-1-infected cells. The interaction between Nef and Hsp40 is important for increased Hsp40 translocation into the nucleus of infected cells, which seems to facilitate viral gene expression by becoming part of the cyclin-dependent kinase 9-associated transcription complex regulating long terminal repeat-mediated gene expression. The finding is consistent with the failure of the nef-deleted virus to induce Hsp40, resulting in reduced virus production. Our data further shows that, whereas, Hsp40 overexpression induces viral gene expression, silencing of Hsp40 reduces the gene expression in a Nef-dependent manner. Thus our results clearly indicate that Hsp40 is crucial for Nef-mediated enhancement of viral gene expression and replication.Viruses are known to modulate cellular proteins for successful replication within the host cells. The sequence of events in the establishment of a productive infection by human immunodeficiency virus type 1 (HIV-1) 3 not only involves interaction between a number of viral and cellular factors but is also accompanied by complex and dynamic changes in the patterns of cellular gene expression. Nef, a 27-30-kDa myristoylated phosphoprotein, encoded by HIV-1 has been shown to play a crucial role in viral pathogenesis by modulating cellular gene expression and signaling pathways (1, 2). nef-deleted viruses fail to replicate efficiently in vivo, and do not develop symptoms of acquired immunodeficiency syndrome (AIDS) (3, 4). Nef is also thought to contribute to viral pathogenesis by down-regulation of CD4 and major histocompatibility complex class I surface molecules preventing viral superinfection and by helping the virus to evade host immune system. Nef has also been implicated in the activation of T cells, making the cells permissible to the virus (5, 6). All these functions of Nef are manifested by a number of important events such as activation of upstream signaling molecules, inhibition of apoptosis in the infected cell, activation and up-regulation of transcription factors, alleviation of repressors of transcription, as well as increase of the infectivity of newly produced virions (1, 7). Even though these functions of Nef have been well studied, controversy exists on its role in viral infectivity and replication as both negative (8, 9) and positive (10, 11) effects are available in the literature...

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

“…As subtype C has become the most prevalent subtype in the world at present, we wanted to identify novel cellular factors, if any, that may interact with subtype C Nef protein. The subtype C Nef from Indian isolate IN301904 (37) used in the yeast two-hybrid screening was shown to have 84% similarity with subtype B NL4-3 Nef (23).…”

“…Plasmids, Cell Lines, and Antibodies-The nef gene from HIV-1 subtype C Indian isolate IN301904 (37) was cloned into pAS2-1 (Clontech) and pcDNA3.1 vector (Invitrogen), respectively, as described earlier (23). The sequence of cloned nef was confirmed by DNA sequencing using an ABI 310 Genetic Analyzer (Applied Biosystems).…”

“…Yeast Two-hybrid Screening-A human leukocyte cDNA library in pACT2 vector (Clontech) was screened for Nef-interacting proteins by co-transformation with the pAS2-1NefC bait plasmid into yeast strain AH109 (Clontech) as described previously (23). Positive clones were selected based on growth in medium lacking adenine, histidine, tryptophan, and leucine and also by expression of ␤-galactosidase.…”

“…HIV-1 Infection and Virus Quantitation-CEM-GFP cells (2 ϫ 10 6 ) were infected with HIV-1 NL4-3 virus at a multiplicity of infection of 0.5 in the presence of Polybrene (1 g/ml) as described earlier (23). 24 h postinfection, cells were transfected with 200 nM siRNA, and then 48 h later, cells were harvested and lysed for preparation of whole cell lysate.…”

“…In another study, Hsp40 was shown to interact and co-localize with Nef in the nucleus and thereby augment LTRdriven gene expression (23). In contrast, HEXIM1 and Hsp70 negatively regulate HIV-1 gene expression and replication (24,25).…”