Thipparthi R. Reddy

Inhibition of HIV replication by dominant negative mutants of Sam68, a functional homolog of HIV-1 Rev

The HIV-1 Rev protein facilitates the nuclear export of mRNA containing the Rev response element (RRE) through binding to the export receptor CRM-1. Here we show that a cellular nuclear protein, Sam68 (Src-associated protein in mitosis), specifically interacts with RRE and can partially substitute for as well as synergize with Rev in RRE-mediated gene expression and virus replication. Differential sensitivity to leptomycin B, an inhibitor of CRM-1, indicates that the export pathways mediated by Rev and Sam68 are distinct. C-terminally deleted mutants of Sam68 inhibited the transactivation of RRE-mediated expression by both wild-type Sam68 and Rev. They were retained in the cytoplasm and impeded the nuclear localization of Rev in co-expressed cells. These mutants also inhibited wild-type HIV-1 replication to the same extent as the RevM10 mutant, and may be useful as anti-viral agents in the treatment of AIDS.

Sam68, RNA helicase A and Tap cooperate in the post-transcriptional regulation of human immunodeficiency virus and type D retroviral mRNA

Unlike cellular mRNA, retroviral mRNA bypasses the tight coupling of the splicing and nuclear export steps to allow the export of intron-containing viral RNA transcripts to the cytoplasm. Two distinct nuclear export pathways for retroviral mRNA have been described: a CRM-1 dependent pathway mediated by the HIV-1 Rev protein and the Rev Response Element (RRE), and a CRM-1 independent pathway mediated by the Constitutive Transport Element (CTE) of type D retroviruses. Two CTE-binding proteins, RNA helicase A (RHA) and Tap, have been implicated in the nuclear export of CTE-containing RNA. Recently, we reported that expression of RRE-containing RNA could also be mediated by a cellular protein, Sam68, independently of Rev. Here we report evidence that Sam68, RHA and Tap cooperate in the nuclear export of both CTE- and RRE-containing RNA. RHA binds to Sam68 and to Tap both in vivo and in vitro. Over-expression of Sam68 activates both RRE- and CTE-regulated reporter gene expression in human cells and in quail cells in the presence of human Tap. This activation was competitively inhibited by the nuclear transport domain (NTD) of RHA and a transdominant negative mutant of Tap. Conversely, the activation of CTE by Tap in quail cells was inhibited by a transdominant mutant of Sam68 and NTD. We propose that both HIV and type D retroviruses may access the same constitutive RNA nuclear export pathway involving RHA, Tap and Sam68, even though HIV also utilizes the Rev protein for more efficient nuclear export. it is likely that this constitutive export pathway is also used by cellular mRNA, but at a different interface with the splicing process.

Functional interaction of the HTLV-1 transactivator Tax with activating transcription factor-4 (ATF4)

The Tax protein of the Human T-cell Leukemia Virus (HTLV) activates the expression of viral mRNA through a three 21 bp repeat enhancer located within the HTLV-1 LTR. Since Tax does not bind to the 21 bp DNA repeats directly, it has been speculated that Tax interacts with cellular protein(s) which mediate binding to the enhancer. We employed the yeast two hybrid system to identify host proteins that are potentially relevant to Tax transactivation. We identified a Tax binding protein encoded from a cDNA expression library derived from peripheral blood lymphocytes. The corresponding cDNA has sequence identity with a known transcription factor, activating factor-4 (ATF-4). ATF-4 also binds to GST-Tax fusion protein in vitro. Tax mutants that did not transactivate the HTLV-1 LTR also failed to bind ATF-4. The critical domain for Tax binding resides in a 85 amino acid stretch in the C-terminus of ATF-4, which contains the basic domain and leucine zipper. We further demonstrated that both full length and N-terminal truncated ATF-4 were able to enhance Tax transactivation. Thus, ATF-4 may act as an adapter between Tax and the TRE (Tax responsive element), and play an important role in Tax-mediated transactivation.

Cellular Proteins and HIV-1 Rev Function

The human immunodeficiency virus (HIV-1) differentially controls viral protein expression at the level of splicing as well as nuclear export of incompletely spliced viral RNA. This process, mediated by the Rev protein, interfaces with cellular components involved in post-transcriptional gene regulation. While a number of reviews have focused on the host proteins (i.e., Crm1, importin-beta and nucleoporins) that specifically regulate shuttling of Rev between the nucleus and cytoplasm, we could find no systematic review of other cellular proteins implicated in Rev function. Therefore, we will here focus on other Rev cofactors (eIF5a, hRIP, Sam68, RNA helicases, etc) and the role they play in Rev/RRE function and HIV-1 replication.

General effect of Sam68 on Rev/Rex regulated expression of complex retroviruses

We have previously demonstrated that overexpression of Sam68 functionally substitutes for, as well as synergizes with, HIV-1 Rev in RRE-mediated gene expression and virus replication. In addition, C-terminal deletion mutants of Sam68 exhibit a transdominant negative phenotype in HIV replication. We now report that Sam68 also enhances the activities of Rev-like proteins of other complex retroviruses (e.g. HTLV-1 and EIAV) on their respective RNA targets. Furthermore, we demonstrate that Sam68 can function alone as well as synergize with Rev-MS2 and/or Rex-MS2 chimeric proteins on expression mediated by the corresponding RRE-MS2 fusion RNA element. Additionally, dominant negative mutants of Sam68 also repressed the synergistic activation of Sam68 with Rex, E-Rev, and/or Rev-MS2/Rex-MS2 on their corresponding RNA targets. Thus, Sam68 may play an important role in the post-transcriptional regulation of all complex retroviruses.

Functional interaction of Sam68 and heterogeneous nuclear ribonucleoprotein K.

Sam68 is a target of the c-Src tyrosine kinase. We previously showed that overexpression of Sam68 functionally substitutes for, as well as synergies with, HIV-1 Rev in Rev-response element (RRE)-mediated gene expression and virus replication. Here we describe the identification of heterogeneous nuclear ribonucleoprotein K (hnRNP K) as a protein that specifically interacts with Sam68 in vitro and in vivo. HnRNP K did not bind to RRE-RNA directly, but formed a super complex with Sam68 and RRE in vitro. RNase treatment did not change the strength of binding of hnRNP K to Sam68. We demonstrated that hnRNP K significantly inhibited Sam68-mediated, but not Rev-mediated, RRE-dependent gene expression. We further showed that Sam68, but not a non-functional mutant Sam68p21, inhibited transcriptional activation of CT element by hnRNP K. Interestingly, the Sam68p21 with a single amino acid substitution in the nuclear localization domain exhibited less affinity for hnRNP K in vitro. We propose that the direct interaction of Sam68 and hnRNP K adversely affect the activities of both proteins in signal transduction pathways of both transcriptional and post-transcriptional events.

A Novel hnRNP Specifically Interacts with HIV-1 RRE RNA.

We have identified and obtained the full-length clone of RREBP49, a human nuclear factor which specifically interacts with the Rev-responsive element (RRE) sequence of human immunodeficiency virus type 1. Sequence analysis revealed that RREBP49 is highly homologous to hnRNP F protein and contains three repeated RNA-binding domains. Binding assays demonstrated that Rev and RREBP49 bind to different subregions on the RRE sequence and that binding is mutually nonexclusive. Blocking of endogenous RREBP49 expression by an antisense construct increases Rev activity in CV-1 cells, indicating that RREBP49 and Rev may play antagonistic roles in HIV-1 replication. RREBP49 may function as a splicing factor or a nuclear retention factor for unspliced mRNAs. However, only a slight decrease of Rev activity was observed when exogenous RREBP49 was introduced into CV-1 cells by pSVL-RREBP49 expression vector. This may be explained by a high endogenous level of RREBP49 which is above optimal. Alternatively, additional cellular factors may be required for RREBP49-mediated inhibition of Rev. Copyright 1996 S. Karger AG, Basel