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

Abstract

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.

A single point mutation in the nuclear localization domain of Sam68 blocks the Rev/RRE-mediated transactivation

“…There was a good correlation between the localization of Sam68 P439?R mutant and its e ect on the inhibition of HIV-1 replication. An interesting parallel with our previous studies is that the C' deletion mutant, which is localized in the cytoplasm, and does not bind to RRE, also inhibited HIV-1 replication (Reddy et al, 1999). In light of these ®ndings, we conclude that the integrity of the C-terminal tyrosine rich domain is required for the nuclear localization of wild type Sam68.…”

“…Since endogenous levels of Sam68 are low, it is easy to distinguish the Sam68 proteins (wild type and mutants) expressed by the transgene from endogenous Sam68 (Reddy et al, 1999 and Figure 2). Figure 2a presents results that demonstrate that Sam68 (wild type) was localized in the nucleus, which is in agreement with the previous observation that Sam68 is a nuclear protein (Ishidate et al, 1997;Reddy et al, 1999). Similarly, Sam68 RP420?NR mutant was also localized in the nucleus (Figure 2b), indicating that downstream amino acid residues may be important for the nuclear localization of Sam68.…”

“…We and others have demonstrated that Sam68 proteins lacking C-terminal domain are localized in the cytoplasm (Reddy et al, 1999;Ishidate et al, 1997). In addition, Ishidate et al (1997) demonstrated that mutation of Arginine-429?Alanine in the PPXXR motif of the NLS will induce the translocation of Sam68 protein from nucleus to cytoplasm.…”

“…Recently, we have demonstrated that Sam68 binds to RRE in vitro and in vivo, and functionally replaces as well as synergizes with HIV-1 Rev in RRE-mediated gene expression and virus replication (Reddy et al, 1999). Furthermore, Cterminally deleted mutants of Sam68 show a dominant negative phenotype in HIV replication (Reddy et al, 1999).…”

“…Recently, we have demonstrated that Sam68 binds to RRE in vitro and in vivo, and functionally replaces as well as synergizes with HIV-1 Rev in RRE-mediated gene expression and virus replication (Reddy et al, 1999). Furthermore, Cterminally deleted mutants of Sam68 show a dominant negative phenotype in HIV replication (Reddy et al, 1999). Mutation of Arginine 429 to an Alanine residue in this C-terminal domain (NLS) of Sam68 aa 420 ± 423 fused to GFP has been shown to disrupt the nuclear localization of Sam68 (Ishidate et al, 1997).…”