Impact of Nuclear Export Pathway on Cytoplasmic HIV-1 RNA Transport Mechanism and Distribution

Abstract

HIV-1 full-length RNA (referred to as HIV-1 RNA here) serves as the viral genome in virions and as a template for Gag/Gag-Pol translation. We previously showed that HIV-1 RNA, which is exported via the CRM1 pathway, travels in the cytoplasm mainly through diffusion. A recent report suggested that the export pathway used by retroviral RNA could affect its cytoplasmic transport mechanism and localization. HIV-1 RNA export is directed by the viral protein Rev and the cis-acting element, Rev response element (RRE). When Rev/RRE is replaced with the constitutive transport element (CTE) from Mason-Pfizer monkey virus (MPMV), HIV-1 RNA is exported through the NXF1 pathway. To determine the effects of the export pathway on HIV-1 RNA, we tracked individual RNAs and found that the vast majority of cytoplasmic HIV-1 RNAs travel by diffusion regardless of the export pathway. However, CTE-containing HIV-1 RNA diffuses at a rate slower than that of RRE-containing HIV-1 RNA. Using in situ hybridization, we analyzed the subcellular localizations of HIV-1 RNAs in cells expressing a CTE-containing and an RRE-containing provirus. We found that these two types of HIV-1 RNAs have similar subcellular distributions. HIV-1 RNA exported through the NXF1 pathway was suggested to cluster near centrosomes. To investigate this possibility, we measured the distances between individual RNAs to the centrosomes and found that HIV-1 RNAs exported through different pathways do not exhibit significantly different distances to centrosomes. Therefore, HIV-1 RNAs exported through CRM1 and NXF1 pathways use the same RNA transport mechanism and exhibit similar cytoplasmic distributions. IMPORTANCE The unspliced HIV-1 full-length RNA (HIV-1 RNA) is packaged into virions as the genome and is translated to generate viral structural proteins and enzymes. To serve these functions, HIV-1 RNA must be exported from the nucleus to the cytoplasm. It was recently suggested that export pathways used by HIV-1 RNA could affect its cytoplasmic transport mechanisms and distribution. In the current report, we examined the HIV-1 RNA transport mechanism by following the movement of individual RNAs and identifying the distribution of RNA using in situ hybridization. Our results showed that whether exported by the CRM1 or NXF1 pathway, HIV-1 RNAs mainly use diffusion for cytoplasmic travel. Furthermore, HIV-1 RNAs exported using the CRM1 or NXF1 pathway are well mixed in the cytoplasm and do not display export pathway-specific clustering near centrosomes. Thus, the export pathways used by HIV-1 RNAs do not alter the cytoplasmic transport mechanisms or distribution.

5′-Cap sequestration is an essential determinant of HIV-1 genome packaging

“…How does cap exposure prevent packaging? The inability of HIV-1 Gag proteins to package cap-exposed viral RNAs is not likely due to altered intracellular trafficking since live-cell imaging studies have shown that both translating and nontranslating pools of HIV RNAs actively transit sites of virus assembly ( 48 , 49 ). Retroviral transcripts involved in translation and packaging exist in separate, noninterconverting pools ( 22 , 48 – 51 ), and splicing of HIV-1 RNAs transcribed with three 5′ guanosines, which adopt a cap-exposed conformation, occurs at significantly greater levels than those transcribed with a single 5′ guanosine that are known to adopt a cap-sequestered conformation ( 52 ).…”

“…The inability of HIV-1 Gag proteins to package cap-exposed viral RNAs is not likely due to altered intracellular trafficking since live-cell imaging studies have shown that both translating and nontranslating pools of HIV RNAs actively transit sites of virus assembly ( 48 , 49 ). Retroviral transcripts involved in translation and packaging exist in separate, noninterconverting pools ( 22 , 48 – 51 ), and splicing of HIV-1 RNAs transcribed with three 5′ guanosines, which adopt a cap-exposed conformation, occurs at significantly greater levels than those transcribed with a single 5′ guanosine that are known to adopt a cap-sequestered conformation ( 52 ). These findings are collectively consistent with a mechanism in which HIV-1 transcripts containing an exposed 5′ cap are irreversibly sequestered by the cellular processing and translation machinery prior to becoming accessible for Gag-dependent packaging, and that gRNAs avoid this capture by structural sequestration of their 5′ cap ( Fig.…”

Advances in HIV-1 Assembly

“…In support of this idea, one report found that by replacing the RRE in HIV-1 gRNA with multiple copies of the CTE, the subcellular location of the RNA was altered from a diffuse cytoplasmic pattern to one that clustered near the centrosome, suggesting that the mode of nuclear exit indeed determines the subcellular fate of the RNA [ 7 ]. However, Chen and colleagues readdressed this question by comparing the distribution of HIV-1 RNAs exported through the CRM1 or NSF1 pathway using live cell imaging [ 8 ]. They found that CTE-containing HIV-1 RNA and RRE-containing HIV-1 RNA exhibited a similar cytoplasmic distribution and did not observe clustering near the centrosome.…”

Human Retrovirus Genomic RNA Packaging

“…Comparing retroviral gRNA exported by different pathways can provide further insights into potential linkage(s) of these processes. For example, HIV-1 gRNAs exported by the CRM1 or NXF1 pathway were found to have diffusive movement in the cytoplasm, while CTE-containing RNAs diffuse more slowly than RRE-containing RNAs [ 105 ]. While the biology causing observed differences in diffusion rates is unknown, one possible explanation is that RNAs exported by different pathways are associated with different proteins that alter diffusion rates to varying extents.…”

“…For example, components of either system could serve as a signal for a particular cytoplasmic fate. However, one study reported that changes in HIV-1 gRNA localization due to the nuclear export pathway do not change gRNA localization and that the HIV-1 gRNA was not targeted to centrosomes [ 105 ]. The differences in reported observations may involve the use of proviruses to express viral components versus transfected DNAs.…”