The Human Rev Interacting Protein (hRIP) is Required for Rev Function and HIV-1 Replication: a Dissertation
Graduate School of Biomedical Sciences, Department of Molecular Genetics and Microbiology
HIV-1; RNA-Binding Proteins; Nuclear Pore Complex Proteins; Virus Replication; RNA, Messenger; Academic Dissertations
Life Sciences | Medicine and Health Sciences
Retroviruses have evolved sophisticated mechanisms to ensure timely export of incompletely spliced viral messenger ribonucleic acids (mRNAs) for gene expression and for viral packaging. For example, the Human Immunodeficiency Virus type 1 (HIV-1) encodes the Rev regulatory protein, a sequence-specific RNA-binding protein that is responsible for the cytoplasmic accumulation of intron-containing viral mRNAs.
The HIV-1 Rev protein contains an amino terminal (N-terminal) Arginine-Rich Motif (ARM) RNA-binding domain (RBD) and a carboxy terminal (C-terminal) leucine-rich activation domain which functions as a Nuclear Export Signal (NES). The Rev ARM interacts in a sequence-specific manner with a cis-acting viral RNA stem-loop structure, the Rev Responsive Element (RRE), located in all incompletely spliced viral mRNAs. This initial interaction is followed by the recruitment of additional Rev molecules to form a RiboNucleoProtein (RNP) complex involving the RRE and Rev molecules.
The cytoplasmic accumulation of the Rev:RRE RNP complex is dependent on the interaction of Rev with key cellular cofactors. Rev activation domain mutants exhibit a trans-dominant negative phenotype, suggesting that this domain of Rev interacts with cellular proteins required for Rev function. Biochemical and genetic studies have identified several cellular proteins that bind to the activation domain of Rev and are therefore candidate cofactors for Rev function. Amongst these is the human Rev Interacting Protein [hRIP, 79], which is also known as the Rev/Rex activation domain-binding protein [Rab, 18].
hRIP was identified in a yeast two-hybrid assay with the HIV-1 Rev and its functionally equivalent Human T-cell Leukemia Virus type-1 (HTLV-1) Rex protein as baits. The interaction between hRIP and HIV-1 Rev is dependent on a functional Rev NES, as predicted for a bona fide Rev cellular cofactor, and the Nucleoporin-like (Nup-like) repeats in the C-terminus of hRIP (18, 79]. Additional genetic studies indicated that the interaction between hRIP and Rev is indirect and is most likely mediated by the cellular export receptor CRM1 (Chromosomal Region Maintenance 1) [1, 153].
A role for hRIP in Rev function or HIV-1 replication has remained elusive. The goal of this dissertation was to determine whether hRIP is required for Rev function and HIV-1 replication. We used two approaches, a dominant-negative mutant and RNA interference (RNAi), to ablate hRIP activity and analyzed Rev function and HIV-1 replication using standard assays.
The results of this dissertation demonstrate that hRIP is required for Rev function and HIV-1 replication. We show that Rev function is inhibited upon ablation of hRIP activity by either a trans-dominant negative mutant or RNAL Furthermore, we find that depletion of endogenous hRIP by RNAi results in the loss of viral replication in human cell lines and primary human macrophages. Unexpectedly, in the absence of functional hRIP, RRE-containing viral RNAs accumulate in the nuclear periphery where hRIP is localized. Comparable ablation of hRIP activity did not affect the intracellular localization or trafficking of a variety of proteins or cellular poly (A+ mRNA, suggesting that the inhibition of Rev-directed RNA export is specific.
In conclusion, the results of this dissertation demonstrate that hRIP is involved in the movement of Rev-directed RNAs from the nuclear periphery to the cytoplasm. Therefore, hRIP is required for Rev function and HIV-1 replication. The hRIP protein is not essential for the maintenance of cell viability and thus might represent a novel target for the development of antiviral agents for HIV-1.
Sánchez-Velar, Nuria, "The Human Rev Interacting Protein (hRIP) is Required for Rev Function and HIV-1 Replication: a Dissertation" (2005). University of Massachusetts Medical School. GSBS Dissertations and Theses. Paper 312.
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