Genetically complex retroviruses, including HIV-1, encode the Rev posttranscriptional regulatory protein, which is absolutely necessary for nuclear export of the intron-containing RNA (15
). The nucleocytoplasmic shuttling ability of Rev is disrupted by actD (23
). Therefore, actD plays a redundant role in curtailing cytoplasmic accumulation of genomic RNA in HIV-1-infected cells: inhibition of RNA synthesis and prevention of nuclear export.
We treated HIV-1-infected human T cells with actD and analyzed the cytoplasmic fate of HIV-1 genomic RNA by RPA (7
). First, conditions were determined under which RNA synthesis was inhibited to 15% of that of the mock-treated control with no overt effect on the viability of the infected CEM(A) T cells. Second, we evaluated the effect of 4 h of actD treatment on viral particle assembly and established that virion assembly was sustained. Radioimmunoprecipitation assays indicated that HIV-1 Gag protein synthesis was sustained at 93% during the 4-h experiment. Pulse-chase experiments showed that newly synthesized Gag protein begins to be assembled into virions by 2 h postsynthesis. Assembly and release of the virions, and processing of the Gag precursor protein within the progeny virions, were also sustained despite the reduction in de novo RNA synthesis. Another critical finding was that Gag protein synthesized prior to actD treatment remains competent for assembly into HIV-1 virions for at least 4 h postsynthesis.
Quantitative RPAs on RNAs collected 4 h post-actD treatment detected an expected decrease in cytoplasmic HIV-1 genome-length RNA (to 30% of the level of the mock-treated control). Virion-associated RNA from equivalent amounts of virions that were standardized by Gag p24 enzyme-linked immunosorbent assay was also decreased (to 60%). The results show that in contrast to MLV, de novo RNA synthesis is not necessary for packaging of HIV-1 genomic RNA. The HIV-1 results are similar to the ASV findings and are consistent with the existence of a single multifunctional RNA population (Fig. , model 2).
Dorman and Lever analyzed RNAs from HIV-1- or HIV-2-infected Jurkat T cells after treatment with actD or leptomycin B (LMB) (11
). Similar to actD, LMB inhibits Rev function and disrupts nuclear export of intron-containing HIV-1 RNA (42
). Therefore, LMB provided a second approach to curtailing cytoplasmic accumulation of genome-length RNA in HIV-1-infected cells. The effect of actD or LMB on the activity of HIV-2 Rev has not been characterized.
RPAs detected coordinate decreases in HIV-1 and HIV-2 cytoplasmic and virion-associated RNA levels following actD treatment (11
). For HIV-1, cytoplasmic RNA and virion RNA levels declined to 40% of those of the controls after 9 h of actD treatment. The decline in virion RNA was calculated after standardization of virion abundance by exogenous RT assay. Unexpectedly, HIV-2 cytoplasmic and virion RNA levels were sustained at 80 to 100% of control levels at 12 h post-actD treatment. The relative persistence of the HIV-2 RNA implies that cytoplasmic HIV-2 proviral transcripts are inherently more stable in the actD assay than their HIV-1 counterparts. Surprisingly, particle abundance, as measured by exogenous RT activity, declined to 65% despite the maintenance of the steady-state cytoplasmic RNA. A possible explanation is that actD treatment produces a defect in RT function or virion assembly.
LMB treatment also produced proportionate responses in HIV-1 and HIV-2 cytoplasmic and virion-associated RNA levels. HIV-1 cytoplasmic RNA was reduced to 25% and virion-associated RNA levels declined to 45% after a 12-h incubation with LMB. Again, HIV-2 genome-length cytoplasmic RNA and virion-associated RNA levels were sustained at 100 to 140% of control levels at 12 h post-LMB treatment. Similar to the actD result for HIV-2, virion abundance was reduced (to 40%) without reduction of the cytoplasmic RNA level. The possibility that HIV-2 virion assembly is disrupted by actD or LMB treatment was not addressed, but the data imply a requirement for an unknown labile cofactor that is important for virion production. The results of the actD and LMB experiments are in agreement regarding the theory that HIV-1 and HIV-2 genome-length RNAs do not segregate into functionally independent populations of mRNA and genomic RNA. The cytoplasmic fate of HIV-1 and HIV-2 genomic RNA is similar to that of ASV RNA, and the data are consistent with a single RNA population functioning as both mRNA and genomic RNA (Fig. , model 2).
In summary, RNA analysis of genomic RNA in infected cells or progeny virus after actD treatment has been an important tool to examine two hypothetical models of genomic RNA trafficking in simple and complex retroviruses. Results with MLV genome-length RNA indicate that the unspliced transcript segregates into two functionally distinct populations in the cytoplasm (Fig. , model 1). In contrast, results with ASV, HIV-1, and HIV-2 genome-length RNAs support the existence of a single RNA population that functions interchangeably as both mRNA and genomic RNA (Fig. , model 2). These experiments did not address whether genome-length RNA is obligated to function as mRNA prior to being packaged as genomic RNA (model 2A versus model 2B). A modification of the experimental approach was implemented in the HIV-1 system to address this important open question.