Nuclear RNA Export and Packaging Functions of HIV-1 Rev Revisited

Abstract

Although the viral Rev protein is necessary for HIV replication, its main function in the viral replication cycle has been controversial. Reinvestigating the effect of Rev on the HIV-1 RNA distribution in various cell lines and primary cells revealed that Rev enhanced cytoplasmic levels of the unspliced HIV-1 RNA, mostly 3-to 12-fold, while encapsidation of the RNA and viral infectivity could be stimulated >1,000-fold. Although this clearly questions the general notion that the nuclear export of viral RNAs is the major function of Rev, mechanistically encapsidation seems to be linked to nuclear export, since the tethering of the nuclear export factor TAP to the HIV-1 RNA also enhanced encapsidation. Interference with the formation of an inhibitory ribonucleoprotein complex in the nucleus could lead to enhanced accessibility of the cytoplasmic HIV-1 RNA for translation and encapsidation. This might explain why Rev and tethered TAP exert the same pattern of pleiotropic effects.In contrast to simple retroviruses, HIV-1, a lentivirus, utilizes several trans-acting regulatory proteins which fulfill important functions throughout the different phases of the lentiviral replication cycle (recently reviewed in references 16, 30, and 34). One of those regulators is the 16-kDa HIV-1 Rev protein, which is required for the expression of the structural proteins during the late phase of the HIV-1 replication cycle. This effect of Rev has been attributed to its nuclear export activity. After initial transcription of the integrated proviral genome, "early" transcripts are subjected to the cellular splicing machinery. Alternative splicing events caused by the complex genome organization of HIV-1 lead to expression of Rev from a multiply spliced mRNA. Once Rev is translated in the cytoplasm, it is imported into the nucleus, where it binds to a viral RNA element, the Rev response element (RRE), which is present on singly spliced and unspliced transcripts. Rev seems to circumvent further splicing and leads to an increase of the cytoplasmic levels of unspliced and singly spliced viral transcripts by tethering these transcripts to the Crm1 export pathway (13, 28; reviewed in reference 36). The unspliced and singly spliced transcripts serve as templates for translation, and the unspliced RNA is also encapsidated into assembling virus particles.The magnitude by which Rev enhances lentiviral RNA levels in the cytoplasm has been controversial. Under some experimental conditions, unspliced viral RNA levels in the cytoplasm were detectable only in the presence of Rev, while others observed just a 4-fold enhancement of these cytoplasmic RNA levels by Rev (2,8,9,12,26,36,43,45). Since Rev was found to stimulate protein levels encoded by the Rev-dependent RNAs to a much larger extent than the cytoplasmic levels of these RNAs, Rev also seems to stimulate translation (6, 25, 35; recently reviewed in reference 17). Consistently, Rev was furthermore shown to enhance the association of the Rev-dependent viral RNA with polysomes (9).In trying to dev...

Cytoplasmic Utilization of Human Immunodeficiency Virus Type 1 Genomic RNA Is Not Dependent on a Nuclear Interaction with Gag

“…In Hgp syn ⌬MA, the MA domain of Hgp syn was replaced with the myristoylation signal of the protooncogene c-src (nt 450 to 491 of GenBank entry NM_005417.3). Proviral and vector constructs HXB2⌬env⌬nefCAT (formerly pHXB⌬envCAT [64]), NL4.3Re (formerly HIV RevϪ [6]), and V H (7) were described previously. Plasmids NL4.3Reg**, NL4.3Re-mutCA, and NL4.3Re⌬MA are derivatives of NL4.3Re.…”

“…Nuclear and cytoplasmic fractions were isolated essentially as described previously (6,7,36). Two days after transfection, HEK 293T cells were detached and washed in cold PBS (Invitrogen).…”

The human immunodeficiency virus type 1 Rev protein: ménage à trois during the early phase of the lentiviral replication cycle

“…Consistent with an enhanced polysomal association of the RRE-containing RNAs, Rev was also found to stimulate protein expression levels to a greater extent than the cytoplasmic mRNA levels, suggesting an independent stimulatory effect of Rev on translation (recently reviewed by Groom et al, 2009). More recently, we also observed that Rev enhances encapsidation of the genomic RNA into virions to a much larger extent than it affects cytoplasmic RNA levels (Blissenbach et al, 2010;Brandt et al, 2007). Nuclear RNA export, the corresponding modulation of the ratio of differentially spliced transcripts in nucleus and cytoplasm, the increase in translational efficiency, and stimulation of encapsidation are all exerted during the late phase of the virus replication cycle and, until now, no Rev mutants have been reported to be differentially impaired for one or several of these functions.…”

Rev-ing up post-transcriptional HIV-1 RNA expression

“…36,37 Additionally, evidence suggests that the presence of Rev than 1,000 fold. [60][61][62][63] Interestingly, this latter activity of Rev was in part linked to the nuclear export of RNA suggesting that this apparent activity of Rev may lie in its ability to modulate nuclear RNP (ribonuclear protein) assembly on RRE containing RNAs. [60][61][62] It was observed that in cells expressing similar amounts of different forms of HIV-1 RNA, Rev/ RRE dependent viral RNA encapsidation was 100 fold higher than similar viral Figure 1.…”

“…[60][61][62] It remains to be determined if a TMG-PIMT/Rev interaction may also influence the encapsidation of viral genomic RNA. Preliminary analyses of HIV-1 virion RNAs suggest the presence of TMG-capped moieties (Yedavalli VS, unpublished results).…”