As a member of the retrovirus family, HIV-1 packages its RNA genome into particles and replicates through a DNA intermediate that integrates into the host cellular genome. The multiple genes encoded by HIV-1 are expressed from the same promoter and their expression is regulated by splicing and ribosomal frameshift. The full-length HIV-1 RNA plays a central role in viral replication as it serves as the genome in the progeny virus and is used as the template for Gag and GagPol translation. In this review, we summarize findings that contribute to our current understanding of how full-length RNA is expressed and transported, cis- and trans-acting elements important for RNA packaging, the locations and timing of RNA:RNA and RNA:Gag interactions, and the processes required for this RNA to be packaged into viral particles.

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Type-III interferons (IFNs) are important mediators of antiviral immunity. IFN-λ4 is a unique type-III IFN because it is produced only in individuals who carry a dG allele of a genetic variant rs368234815-dG/TT. Counterintuitively, those individuals who can produce IFN-λ4, an antiviral cytokine, are also less likely to clear HCV infection. Here, we searched for unique functional properties of IFN-λ4 that might explain its negative effect on HCV clearance. We used fresh primary human hepatocytes (PHH) treated with recombinant type-III IFNs or infected with Sendai virus (SeV) to model acute viral infection, and subsequently validated our findings in HepG2 cell line models. Endogenous IFN-λ4 protein was detectable only in SeV-infected PHH from individuals with the dG allele, where it was poorly secreted but highly functional even at concentrations below 50 pg/ml. IFN-λ4 acted faster than other type-III IFNs in inducing antiviral genes as well as negative regulators of IFN response, such as USP18 and SOCS1. Transient treatment of PHH with IFN-λ4 but not IFN-λ3 caused a strong and sustained induction of SOCS1, and refractoriness to further stimulation with IFN-λ3. Our results suggest unique functional properties of IFN-λ4 that can be important in viral clearance and other clinical conditions.

How retroviruses regulate the amount of RNA genome packaged into each virion has remained a long-standing question. Our previous study showed that most HIV-1 particles contain two copies of viral RNA, indicating that the number of genomes packaged is tightly regulated. In this report, we examine the mechanism that controls the number of RNA genomes encapsidated into HIV-1 particles. We hypothesize that HIV-1 regulates genome packaging by either the mass or copy number of the viral RNA. These two distinct mechanisms predict different outcomes when the genome size deviates significantly from that of wild type. Regulation by RNA mass would result in multiple copies of a small genome or one copy of a large genome being packaged, whereas regulation by copy number would result in two copies of a genome being packaged independent of size. To distinguish between these two hypotheses, we examined the packaging of viral RNA that was larger (≈17 kb) or smaller (≈3 kb) than that of wild-type HIV-1 (≈9 kb) and found that most particles packaged two copies of the viral genome regardless of whether they were 17 kb or 3 kb. Therefore, HIV-1 regulates RNA genome encapsidation not by the mass of RNA but by packaging two copies of RNA. To further explore the mechanism that governs this regulation, we examined the packaging of viral RNAs containing two packaging signals that can form intermolecular dimers or intramolecular dimers (self-dimers) and found that one self-dimer is packaged. Therefore, HIV-1 recognizes one dimeric RNA instead of two copies of RNA. Our findings reveal that dimeric RNA recognition is the key mechanism that regulates HIV-1 genome encapsidation and provide insights into a critical step in the generation of infectious viruses.