Date Published: March 21, 2013
Publisher: Public Library of Science
Author(s): Olga A. Nikolaitchik, Kari A. Dilley, William Fu, Robert J. Gorelick, S.-H. Sheldon Tai, Ferri Soheilian, Roger G. Ptak, Kunio Nagashima, Vinay K. Pathak, Wei-Shau Hu, Hans-Georg Krausslich.
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.
Retroviruses are RNA viruses that replicate through a DNA phase, in which viral DNA is integrated into the host genome to form a provirus . Retroviral genomes in virions are dimers, consisting of two copies of full-length, unspliced RNA, each of which encodes all of the genetic information needed for virus replication , , , , . Packaging of the retroviral genome is mediated by interactions between the viral structural protein Gag and the cis-acting element(s), collectively called the packaging signal, in the viral RNA , , , , , , , . In some retroviruses, such as HIV-1, RNA partner selection and the initiation of the dimerization process occur in the cytoplasm ; therefore, RNA dimerizes before encapsidation , . A 6-nt sequence located at the 5′ untranslated region of the HIV-1 genome, termed the dimerization initiation signal (DIS), forms intermolecular base-pairs between two HIV-1 RNAs to initiate the dimerization process , , , , . There are multiple DIS sequences in the circulating HIV-1 strains; two of the most common sequences are GCGCGC and GUGCAC, . As base-pairing is involved in RNA partner selection for the initial dimerization process, the identity of the DIS sequence affects the ability of two HIV-1 RNAs derived from different proviruses to be copackaged together to form heterozygous particles .
One of the essential steps in generating an infectious retroviral virion is the packaging of the RNA genome. The mechanism that regulates RNA packaging has many implications for retroviral evolution. For example, if a virion cannot accommodate two copies of full-length genomes exceeding a certain size, then this limitation would be a major factor in shaping the economical organization of the viral genome. Additionally, the mechanism that regulates RNA packaging and heterozygosity can affect genome diversity by altering the potential for recombination. Recombination occurs when reverse transcriptase copies genetic information from different portions of the two copackaged viral RNAs. If a certain mass of RNA is packaged, then multiple copies of smaller RNAs, or one large viral RNA, would be packaged into one virion; therefore, the size of the viral genome would directly affect the recombination potential of the virus. In this report, we address the long-standing question of how a retrovirus regulates the number of viral genomes packaged into each virion and show that HIV-1 regulates its RNA encapsidation not by the mass of the viral genome, but by packaging two copies of RNAs based on the recognition of an RNA dimer structure.