Retroviruses package two copies of full-length RNA in one virus particle (virion). One of the consequences of packaging two RNAs is frequent recombination during DNA synthesis when reverse transcriptase uses parts of both RNAs as templates. Although frequent recombination can occur during DNA synthesis of all virions, a genotypically different recombinant can only be generated from virions that package two different RNAs (heterozygous virions). High genetic diversity of HIV-1 presents a difficult barrier for drug treatment and vaccine development.
Using a recombination assay, researchers have shown that RNA molecules derived from two similar HIV-1 proviruses can randomly assort and be efficiently copackaged into virions. However, heterozygous virions are formed less efficiently when the two proviruses contain variations in their dimerization initiation signal (DIS). Located at the loop of stem-loop 1 of the 5′ untranslated region, the DIS is a 6-nt palindromic sequence that forms the initial interaction between the two HIV-1 RNAs. The Gag polyproteins of HIV-1 interact with, and specifically package, the viral RNA to generate infectious viruses. We have previously examined whether RNA dimerization occurs prior to virus assembly using HIV-1 variants with DIS mutations that abolish their palindromic nature (for example, from GCGCGC to GGGGGG) but can form perfect base pairs with the DIS of a partner virus (such as a virus with CCCCCC at the DIS). In the coinfected cells if dimeric RNAs are packaged, then the GGGGGG viral RNA would preferentially pair with CCCCCC viral RNA, and an increase in the formation of heterozygous virions would be observed. In contrast, if two monomeric RNAs are packaged, then there would not be an increase in heterozygous viruses. Results reveal that most of the virions from coinfected cells were heterozygous, indicating that copackaged RNA partner selection, i.e. dimerization, occurs prior to the packaging of virion RNA.
HIV-1 full-length RNAs serve at least two functions: as a template for Gag/Gag-Pol translation, and as genetic material packaged in the virion. Many cellular factors ensure the correct macromolecular trafficking between nucleus and cytoplasm; specifically, mechanisms exist to prevent the export of intron-containing transcripts, such as the full-length HIV-1 RNA. Most cellular mRNAs are fully spliced before export and many are believed to exit the nucleus via the NXF1-dependent pathway. However, many proteins and some RNAs use an alternative, CRM-1-dependent pathway to migrate out of the nucleus. The extent to which these two pathways are linked or overlap is currently unknown, and the reason for their differential use is subject to speculation.
Once transcribed, the nascent full-length RNA of HIV-1 must travel to the appropriate host cell sites to be translated or to find a partner RNA for copackaging to form newly generated viruses. In this report, scientists sought to identify the location where HIV-1 RNA initiates dimerization and the influence of the RNA transport pathway used by the virus on downstream events essential to viral replication. Using a cell-fusion-dependent recombination assay, they were able to demonstrate that the two RNAs destined for copackaging into the same virion select each other mostly within the cytoplasm. Moreover, by manipulating the RNA export element in the viral genome, they showed that the export pathway taken is important for the ability of RNA molecules derived from two viruses to interact and be copackaged. These results further illustrate that at the point of dimerization the two main cellular export pathways are partially distinct. Lastly, by providing Gag in trans, they demonstrated that Gag is able to package RNA from either export pathway, irrespective of the transport pathway used by the gag mRNA. These findings provide unique insights into the process of RNA export in general, and more specifically, of HIV-1 genomic RNA trafficking.
Probing the HIV-1 Genomic RNA Trafficking Pathway and Dimerization by Genetic Recombination and Single Virion Analyses. 2009 PLoS Pathog 5(10): e1000627 doi:10.1371/journal.ppat.1000627
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Influenza A virus is the prototype of the Orthomyxoviridae, and like all members of this family, the negative-sense RNA that comprises its genome is divided into separate segments. These vRNA segments share a common organisation; a long central coding region (in antisense), sometimes encoding more than one polypeptide, flanked by relatively short untranslated regions (UTRs) and at the termini, sequences conserved between segments that show partial complementarity. The vRNA segments are separately encapsidated into ribonucleoprotein (RNP) structures by viral polypeptides. These RNPs act as independent units for the purposes of viral RNA synthesis, which occurs in the nuclei of infected cells. Replicated vRNAs are exported (as RNPs) from the nucleus via the cellular CRM1 pathway, and at the final stage of viral assembly, are incorporated into the virion as it buds from the apical plasma membrane of the cell. The process of virion assembly is not well understood but is thought to involve a series of protein-protein interactions between the cytoplasmic tails of the viral integral membrane proteins, the matrix protein and the RNPs.
