Knockdown of Cellular RNA Helicase DDX3 by Short Hairpin RNAs Suppresses HIV-1 Viral Replication Without Inducing Apoptosis

The targeting of a cellular co-factor, rather than the HIV-1-specific RNAs, by small interfering RNAs holds promise as the rapid mutational ability of the HIV-1 genome may obviate the potential clinical use of RNAi against this virus. The DEAD-box RNA helicase DDX3 is an essential Rev co-factor in the CRM1-Rev-RRE complex that promotes the export of unspliced and single-spliced HIV-1 RNAs from the nucleus to cytoplasm. In this report, human DDX3 was targeted by specific short hairpin RNAs, and the down-regulation of cell's endogenous DDX3 suppressed the nuclear export of unspliced HIV-1 RNAs but did not affect the cell viability. We further showed that the knockdown of cellular DDX3 could effectively inhibit the replication of HIV-1. Therefore, the current results suggest that the RNA helicase DDX3 may become a potential target by RNAi for future genetic therapy of HIV/AIDS.     

The carboxy-terminal region of the human immunodeficiency virus type 1 protein Rev has multiple roles in mediating CRM1-related Rev functions

The human immunodeficiency virus type 1 (HIV-1) regulatory protein, Rev, mediates the nuclear export of unspliced and singly spliced viral mRNAs by bridging viral RNA and export receptor human CRM1 (hCRM1). Ribonucleoprotein complex formation, including the oligomerization of Rev proteins on viral RNA, must occur to allow export. We show here that Rev-Rev interactions, which are a basis of complex formation, can be initiated without cellular factors and are subsequently enhanced by hCRM1-Ran-GTP. Furthermore, we reveal functions for the Rev carboxy-terminal (C-terminal) region, which is well conserved among many HIV-1 strains, and for which no function has been reported. This region is required for the efficient binding of Rev to hCRM1 and consequently for nuclear export, Rev-Rev dimerization, and full Rev transactivator activity. Consistent with these results, a HIV-1 proviral plasmid that expresses a C-terminally truncated Rev mutant protein produces smaller amounts of the p24 antigen than does a plasmid that possesses an intact rev gene. These results indicate the functional importance of the C-terminal region for full Rev activity, which leads to efficient HIV-1 replication.     

HIV Rev-isited

The human immunodeficiency virus type 1 (HIV-1) proteome is expressed from alternatively spliced and unspliced genomic RNAs. However, HIV-1 RNAs that are not fully spliced are perceived by the host machinery as defective and are retained in the nucleus. During late infection, HIV-1 bypasses this regulatory mechanism by expression of the Rev protein from a fully spliced mRNA. Once imported into the nucleus, Rev mediates the export of unprocessed HIV-1 RNAs to the cytoplasm, leading to the production of the viral progeny. While regarded as a canonical RNA export factor, Rev has also been linked to HIV-1 RNA translation, stabilization, splicing and packaging. However, Rev''s functions beyond RNA export have remained poorly understood. Here, we revisit this paradigmatic protein, reviewing recent data investigating its structure and function. We conclude by asking: what remains unknown about this enigmatic viral protein?     

A structured retroviral RNA element that mediates nucleocytoplasmic export of intron-containing RNA.

A common feature of gene expression in all retroviruses is that unspliced, intron-containing RNA is exported to the cytoplasm despite the fact that cellular RNAs which contain introns are usually restricted to the nucleus. In complex retroviruses, the export of intron-containing RNA is mediated by specific viral regulatory proteins (e.g., human immunodeficiency virus type 1 [HIV-1] Rev) that bind to elements in the viral RNA. However, simpler retroviruses do not encode such regulatory proteins. Here we show that the genome of the simpler retrovirus Mason-Pfizer monkey virus (MPMV) contains an element that serves as an autonomous nuclear export signal for intron-containing RNA. This element is essential for MPMV replication; however, its function can be complemented by HIV-1 Rev and the Rev-responsive element. The element can also facilitate the export of cellular intron-containing RNA. These results suggest that the MPMV element mimics cellular RNA transport signals and mediates RNA export through interaction with endogenous cellular factors.     

Modulation of HIV-1 Rev protein abundance and activity by polyubiquitination with unconventional Lys-33 branching

The HIV-1 Rev protein plays a key role in virus replication by allowing export to the cytoplasm of unspliced or singly-spliced RNAs. In this report, we investigated whether Rev is modified by ubiquitination or sumoylation. Whereas no evidence of sumoylation was obtained, transient expression experiments showed that ubiquitin conjugates to Rev as high molecular weight polyubiquitin chains. Mutation of the three lysine residues of Rev showed that the site of ubiquitin conjugation is Lys-115. Experiments with ubiquitin mutants including a single lysine at every seven possible position indicated that branching of the polyubiquitin chains mainly involves Lys-33. Mutation of Rev Lys-115 to arginine reduces markedly the steady state amount of the protein, but does not impair its ability to export RNA via the Rev response element. These observations support the notion that polyubiquitination of Rev stabilizes the viral protein but hinders its activity.     

Cofactor requirements for nuclear export of Rev response element (RRE)- and constitutive transport element (CTE)-containing retroviral RNAs. An unexpected role for actin

Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the export receptor CRM1/exportin1. However, additional protein factors interacting with leucine-rich NESs have been described. Here, we investigate human immunodeficiency virus type 1 (HIV-1) Rev-mediated nuclear export and Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE)-mediated nuclear export in microinjected Xenopus laevis oocytes. We show that eukaryotic initiation factor 5A (eIF-5A) is essential for Rev and Rev-mediated viral RNA export, but not for nuclear export of CTE RNA. In vitro binding studies demonstrate that eIF-5A is required for efficient interaction of Rev-NES with CRM1/exportin1 and that eIF-5A interacts with the nucleoporins CAN/nup214, nup153, nup98, and nup62. Quite unexpectedly, nuclear actin was also identified as an eIF-5A binding protein. We show that actin is associated with the nucleoplasmic filaments of nuclear pore complexes and is critically involved in export processes. Finally, actin- and energy-dependent nuclear export of HIV-1 Rev is reconstituted by using a novel in vitro egg extract system. In summary, our data provide evidence that actin plays an important functional role in nuclear export not only of retroviral RNAs but also of host proteins such as protein kinase inhibitor (PKI).     

Rev inhibition strongly affects intracellular distribution of human immunodeficiency virus type 1 RNAs

To define the human immunodeficiency virus type 1 (HIV-1) RNA maturation pathways, we analyzed the intracellular distribution of HIV-1 RNA and the viral regulatory proteins Rev and Tat in transfected COS cells and HIV-1-infected lymphoid C8166 cells by means of ultrastructural in situ hybridization using antisense RNA probes and immunoelectron microscopy. The intranuclear viral RNA occurs in ribonucleoprotein fibrils in the perichromatin and interchromatin regions. The simultaneous demonstration of Rev, Tat, Br-labeled RNA, and cellular proteins SC35 and CRM1 in such fibrils reveals the potential of Rev to associate with nascent HIV pre-mRNA and its splicing complex and transport machinery. In a rev-minus system, the env intron-containing, incompletely spliced viral RNAs are revealed only in the nucleus, indicating that Rev is required to initiate the transport to the cytoplasm. Moreover, env intron sequences frequently occur in the periphery of interchromatin granule clusters, while the probe containing the rev exon sequence does not associate with this nucleoplasmic domain. When cells were treated with the CRM1 inhibitor leptomycin B in the presence of Rev protein, the env intron containing HIV RNAs formed clusters throughout the nucleoplasm and accumulated at the nuclear pores. This suggests that Rev is necessary and probably also sufficient for the accumulation of incompletely spliced HIV RNAs at the nuclear pores while CRM1 is needed for translocation across the nuclear pore complex.     

Characterization of novel inhibitors of HIV-1 replication that function via alteration of viral RNA processing and rev function

Expression of the complete HIV-1 genome depends on the appropriate processing of viral RNA. Altering the balance of viral RNA processing impairs replication of the virus. In this report, we characterize two small molecule modulators of HIV-1 RNA processing, 8-azaguanine and 2-(2-(5-nitro-2-thienyl)vinyl)quinoline (5350150), which function by distinct mechanisms to suppress viral gene expression. Although only 8-Azaguanine dramatically decreased accumulation of HIV-1 unspliced and singly spliced RNAs and altered splice site usage, both compounds blocked Gag and Env expression without affecting production of Tat (p16) and Rev regulatory proteins. Subsequent analyses suggest that these compounds affect Rev-mediated RNA transport by different mechanisms. Both compounds induced cytoplasmic accumulation of Rev, suggesting that they function, in part, by impairing Rev function. This conclusion is supported by the determination that both drugs block the nuclear export of genomic HIV-1 RNA to the cytoplasm. Testing confirmed that these compounds suppress HIV-1 expression in T cells at doses below those previously used in humans for tumour chemotherapy. Together, our observations demonstrate that small molecules can be used to inhibit HIV-1 replication by altering another avenue of viral RNA processing, offering the potential for the development of novel therapeutics for controlling this disease.     

Analysis of rev gene function on human immunodeficiency virus type 1 replication in lymphoid cells by using a quantitative polymerase chain reaction method.

Most detailed analyses of the human immunodeficiency virus type 1 (HIV-1) rev gene product have relied on transfection of subgenomic env constructs into cells in which amplification of the transfected DNA occurs. This was necessitated by difficulties in quantitating low-abundance HIV-1 mRNA species and in distinguishing different RNAs of similar sizes. We have modified the conventional polymerase chain reaction method for general use as an extremely sensitive procedure for quantitative analysis of RNA species. Using this method, we assessed the role of the HIV-1 rev gene in viral replication following mutagenesis of an infectious molecular clone, HIV-1JR-CSF. Following transfection of wild-type and mutant proviral constructs, we can specifically detect unspliced RNA and distinguish between the spliced tat-rev and nef mRNAs, which are not resolved by standard RNA analyses. Our results show that the rev protein of HIV-1JR-CSF simultaneously down regulates the expression of tat-rev and nef RNAs and up regulates the level of unspliced full-length HIV-1 RNA. A cis-acting element(s), located exclusively within the env sequences, is essential to exhibit this regulation. Fractionation of cells shows that the ultimate effect of Rev is to direct the appearance of unspliced or singly spliced RNAs in the cytoplasm. Models are discussed for possible mechanisms of Rev action.