Abstract
Overexpression of Sam68 functionally substitutes for, as well as synergizes with, human immunodeficiency virus type 1 (HIV-1) Rev in RRE (Rev response element)-mediated gene expression and virus replication. In addition, COOH-terminal deletion and/or point mutants of Sam68 exhibit a transdominant negative phenotype for HIV replication. Sam68 is a member of KH domain family that includes SLM-1, SLM-2 (Sam68 like mammalian); and QKI-5, QKI-6, and QKI-7 (mouse quaking) proteins. The objective of this study was to examine the effects of these KH family proteins on RRE- and CTE (constitutive transport element of type-D retrovirus)-mediated transactivation. We now report that SLM-1 and SLM-2 proteins, which are the closest relatives of Sam68, marginally enhanced RRE-mediated transactivation, while QK isoforms that are distant relatives of Sam68 had no effect. Interestingly, these proteins still enhanced the effect of Rev in RRE-mediated gene expression. The increase in chloramphenicol acetyltransferase activity was also reflected at the levels of cytoplasmic RRE-chloramphenicol acetyltransferase mRNAs, indicating that Sam68 and KH proteins may have been involved in the stability or export of unspliced RNA. The increase in Rev activity was sensitive to leptomycin B, but not to olomoucine, indicating that the effect of SLM-1, SLM-2, QKI-5, QKI-6, and QKI-7 is exerted through a CRM-1-dependent mRNA export pathway. Thus, KH family proteins play an important role in the post-transcriptional regulation of HIV.
Highlights
The Rev protein of human immunodeficiency virus (HIV)1 facilitates the nuclear export of unspliced or singly spliced viral mRNA [1]
Because of the sequence homology among these proteins, we have investigated the role of KH proteins in the post-transcriptional regulation of HIV replication
To explore a potential role of KH family proteins in RRE- and CTE-mediated transactivation, we examined the effect of exogenously expressed SLM-1, SLM-2, QKI-5, QKI-6, and QKI-7 under the control of the CMV promoter on a RRE-regulated reporter gene expression in transient co-transfection assays
Summary
The Rev protein of human immunodeficiency virus (HIV) facilitates the nuclear export of unspliced or singly spliced viral mRNA [1]. Substitutions of leucine residues within nuclear export sequence yielded a mutant Rev protein (RevM10) with a dominant negative phenotype [3]. We report that KH proteins (SLM-1, SLM-2, QKI-5, QKI-6, and QKI-7), that have partial homology with Sam failed to transactivate RRE-directed reporter gene expression independent of Rev, but are able to enhance Rev transactivation on RRE. Our results provide the first direct evidence that other members of KH proteins are involved in post-transcriptional regulation of HIV gene expression
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