Mechanisms of Human Immunodeficiency Virus Type 2 RNA Packaging: Efficient trans Packaging and Selection of RNA Copackaging Partners

Abstract

Human immunodeficiency virus type 2 (HIV-2) has been reported to have a distinct RNA packaging mechanism, referred to as cis packaging, in which Gag proteins package the RNA from which they were translated. We examined the progeny generated from dually infected cell lines that contain two HIV-2 proviruses, one with a wild-type gag/gag-pol and the other with a mutant gag that cannot express functional Gag/Gag-Pol. Viral titers and RNA analyses revealed that mutant viral RNAs can be packaged at efficiencies comparable to that of viral RNA from which wild-type Gag/Gag-Pol is translated. These results do not support the cis-packaging hypothesis but instead indicate that trans packaging is the major mechanism of HIV-2 RNA packaging. To further characterize the mechanisms of HIV-2 RNA packaging, we visualized HIV-2 RNA in individual particles by using fluorescent protein-tagged RNA-binding proteins that specifically recognize stem-loop motifs in the viral genomes, an assay termed single virion analysis. These studies revealed that >90% of the HIV-2 particles contained viral RNAs and that RNAs derived from different viruses were copackaged frequently. Furthermore, the frequencies of heterozygous particles in the viral population could be altered by changing a 6-nucleotide palindromic sequence at the 5-untranslated region of the HIV-2 genome. This finding indicates that selection of copackaging RNA partners occurs prior to encapsidation and that HIV-2 Gag proteins primarily package one dimeric RNA rather than two monomeric RNAs. Additionally, single virion analyses demonstrated a similar RNA distribution in viral particles regardless of whether both viruses had a functional gag or one of the viruses had a nonfunctional gag, providing further support for the trans-packaging hypothesis. Together, these results revealed mechanisms of HIV-2 RNA packaging that are, contrary to previous studies, in many respects surprisingly similar to those of HIV-1.

Determining the Frequency and Mechanisms of HIV-1 and HIV-2 RNA Copackaging by Single-Virion Analysis

“…The HIV-2 constructs 2-GagCeFP-BglSL, 2-Gag-BglSL, and 2-6G-GagCeFP-BSL have been previously described (27); for clarity, these constructs are referred to as HIV-2-GagCeFP-BglSL, HIV-2-Gag-BglSL, and HIV-2-DIS6G-GagCeFP-BglSL in this report. These two constructs were derived from the ROD12 molecular clone and contained all cis-acting elements important for viral replication and inactivating mutations in pol, vpr, and env.…”

“…Additionally, both constructs contained 18 copies of stem-loops (BglSL) in pol recognized by the E. coli BglG protein. Construct HIV-2-GagCeFP-BglSL expresses Gag tagged with CeFP, whereas HIV-2-Gag-BglSL expresses untagged Gag (27). As with HIV-1, only the GagCeFP construct is mentioned, although both CeFP-tagged HIV-2 Gag and wild-type HIV-2 Gag were coexpressed in all experiments.…”

“…To prepare particles for microscopy, clarified culture supernatant was mixed with Polybrene and the mixture was plated on a 35-mmdiameter glass-bottom plate (MatTek Corp.) and incubated at 37°C for 2 h. An inverted Nikon Eclipse Ti microscope with a 100ϫ oil objective and a numerical aperture of 1.40 and an X-Cite 120 system (EXFO Photonic Solutions Inc.) were used for image capture as previously described (27). The filter settings were 427/10 nm and 480/40 nm (CeFP), 504/12 nm and 542/27 nm (YFP), and 577/25 nm and 632/60 nm (mCherry) for excitation and emission, respectively.…”

“…The HIV-2-DIS6G-GagCeFP-BglSL-noTatRev construct was generated using the same procedure by starting with HIV-2-DIS6G-GagCeFP-BglSL plasmid. The HIV-2-noGag-BglSL was derived from a previously described 2*T-17 mutant that contained two stop codons in gag, one at codon 17 of Gag in MA and one at codon 171 of CA (27). DIS mutants in the HIV-2-noGag-BglSL context were constructed using 2-step PCR.…”

“…It is thought that the DIS sequences of the two RNAs form intermolecular base pairings to initiate the dimerization or the RNA partner selection process. Our recent studies showed that the RNA packaging mechanisms of HIV-2 have many features similar to those of HIV-1, including RNA partner selection prior to encapsidation and the involvement of a palindromic sequence in this process (27).…”