Olga A. Nikolaitchik

High efficiency of HIV-1 genomic RNA packaging and heterozygote formation revealed by single virion analysis

A long-standing question in retrovirus biology is how RNA genomes are distributed among virions. In the studies presented in this report, we addressed this issue by directly examining HIV-1 RNAs in virions using a modified HIV-1 genome that contained recognition sites for BglG, an antitermination protein in the Escherichia coli bgl operon, which was coexpressed with a fragment of BglG RNA binding protein fused to a fluorescent protein. Our results demonstrate that the majority of virions (>90%) contain viral RNAs. We also coexpressed HIV-1 genomes containing binding sites for BglG or the bacteriophage MS2 coat protein along with 2 fluorescent protein-tagged RNA binding proteins. This method allows simultaneously labeling and discrimination of 2 different RNAs at single-RNA-detection sensitivity. Using this strategy, we obtained physical evidence that virions contain RNAs derived from different parental viruses (heterozygous virion) at ratios expected from a random distribution, and we found that this ratio can be altered by changing the dimerization sequences. Our studies of heterozygous virions also support a generally accepted but unproven assumption that most particles contain 1 dimer. This study provides answers to long-standing questions in HIV-1 biology and illustrates the power and sensitivity of the 2-RNA labeling method, which can also be adapted to analyze various issues of RNA biogenesis including the detection of different RNAs in live cell imaging.

Genetic Recombination of Human Immunodeficiency Virus Type 1 in One Round of Viral Replication: Effects of Genetic Distance, Target Cells, Accessory Genes, and Lack of High Negative Interference in Crossover Events

ABSTRACT Recombination is a major mechanism that generates variation in populations of human immunodeficiency virus type 1 (HIV-1). Mutations that confer replication advantages, such as drug resistance, often cluster within regions of the HIV-1 genome. To explore how efficiently HIV-1 can assort markers separated by short distances, we developed a flow cytometry-based system to study recombination. Two HIV-1-based vectors were generated, one encoding the mouse heat-stable antigen gene and green fluorescent protein gene (GFP), and the other encoding the mouse Thy-1 gene and GFP. We generated derivatives of both vectors that contained nonfunctional GFP inactivated by different mutations. Recombination in the region between the two inactivating mutations during reverse transcription could yield a functional GFP. With this system, we determined that the recombination rates of markers separated by 588, 300, 288, and 103 bp in one round of viral replication are 56, 38, 31, and 12%, respectively, of the theoretical maximum measurable recombination rate. Statistical analyses revealed that at these intervals, recombination rates and marker distances have a near-linear relationship that is part of an overall quadratic fit. Additionally, we examined the segregation of three markers within 600 bp and concluded that HIV-1 crossover events do not exhibit high negative interference. We also examined the effects of target cells and viral accessory proteins on recombination rate. Similar recombination rates were observed when human primary CD4+ T cells and a human T-cell line were used as target cells. We also found equivalent recombination rates in the presence and absence of accessory genes vif, vpr, vpu, and nef. These results illustrate the power of recombination in generating viral population variation and predict the rapid assortment of mutations in the HIV-1 genome in infected individuals.

Probing the HIV-1 Genomic RNA Trafficking Pathway and Dimerization by Genetic Recombination and Single Virion Analyses

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, we sought to delineate 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, we 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, we show 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, we have 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.

Dimer Initiation Signal of Human Immunodeficiency Virus Type 1: Its Role in Partner Selection during RNA Copackaging and Its Effects on Recombination

ABSTRACT Frequent human immunodeficiency virus type 1 (HIV-1) recombination occurs during DNA synthesis when portions of the two copackaged RNAs are used as templates to generate a hybrid DNA copy. Therefore, the frequency of copackaging of genomic RNAs from two different viruses (heterozygous virion formation) affects the generation of genotypically different recombinants. We hypothesized that the selection of copackaged RNA partners is largely determined by Watson-Crick pairing at the dimer initiation signal (DIS), a 6-nucleotide palindromic sequence at the terminal loop of stem-loop 1 (SL1). To test our hypothesis, we examined whether heterozygous virion formation could be encouraged by manipulation of the DIS. Three pairs of viruses were generated with compensatory DIS mutations, designed so that perfect DIS base pairing could only occur between RNAs derived from different viruses, not between RNAs from the same virus. We observed that vector pairs with compensatory DIS mutations had an almost twofold increase in recombination rates compared with wild-type viruses. These data suggest that heterozygous virion formation was enhanced in viruses with compensatory DIS mutations (from 50% to more than 90% in some viral pairings). The role of the SL1 stem in heterozygous virion formation was also tested; our results indicated that the intermolecular base pairing of the stem sequences does not affect RNA partner selection. In summary, our results demonstrate that the Watson-Crick pairing of the DIS is a major determinant in the selection of the copackaged RNA partner, and altering the base pairing of the DIS can change the proportion of heterozygous viruses in a viral population. These results also strongly support the hypothesis that HIV-1 RNA dimers are formed prior to encapsidation.

Dimeric RNA Recognition Regulates HIV-1 Genome Packaging

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.

Mechanisms and factors that influence high frequency retroviral recombination.

With constantly changing environmental selection pressures, retroviruses rely upon recombination to reassort polymorphisms in their genomes and increase genetic diversity, which improves the chances for the survival of their population. Recombination occurs during DNA synthesis, whereby reverse transcriptase undergoes template switching events between the two copackaged RNAs, resulting in a viral recombinant with portions of the genetic information from each parental RNA. This review summarizes our current understanding of the factors and mechanisms influencing retroviral recombination, fidelity of the recombination process, and evaluates the subsequent viral diversity and fitness of the progeny recombinant. Specifically, the high mutation rates and high recombination frequencies of HIV-1 will be analyzed for their roles in influencing HIV-1 global diversity, as well as HIV-1 diagnosis, drug treatment, and vaccine development.

Effects of mutations in the human immunodeficiency virus type 1 Gag gene on RNA packaging and recombination.

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) recombination occurs during reverse transcription when parts of the two copackaged RNAs are used as templates for DNA synthesis. It was previously hypothesized that HIV-1 Gag polyproteins preferentially encapsidate the RNA from which they were translated (cis-packaging hypothesis). This hypothesis implies that mutants encoding Gag that cannot efficiently package viral RNA are selected against at two levels: these mutants do not generate infectious virus, and these mutants are not efficiently rescued by the wild-type virus because the mutant RNAs are packaged at much lower levels than are those of the wild-type genome. Therefore, genetic information encoded by gag mutants can be rapidly lost in the viral population. To test this prediction of the cis-packaging hypothesis, we examined several gag mutants by measuring the efficiencies of the mutant RNAs in being packaged in trans in the presence of wild-type virus and determining the rates of recombination between gag mutants and wild-type viruses. We observed that the viral RNAs from the nucleocapsid zinc finger or the capsid truncation mutant were packaged efficiently in trans, and these mutant viruses also frequently recombined with the wild-type viruses. In contrast, viral RNAs from mutants containing a 6-nucleotide substitution encompassing the gag AUG were not efficiently encapsidated, resulting in a low rate of recombination between the mutants and wild-type viruses. Further analyses revealed that other, more subtle mutations changing the gag AUG and abolishing Gag translation did not interfere with efficient encapsidation of the mutant RNA. Our results indicated that neither the gag AUG sequence nor Gag translation is essential for viral RNA encapsidation, and Gag can package both wild-type and gag mutant RNAs with similar efficiencies. Therefore, we propose that HIV-1 RNA encapsidation occurs mainly in trans, and most gag mutants can be rescued by wild-type virus; therefore, they are unlikely to face the aforementioned double-negative selection.

Cytoplasmic HIV-1 RNA is mainly transported by diffusion in the presence or absence of Gag protein

Significance HIV-1 full-length RNA must go to specific subcellular compartments to carry out its functions as a template for translation of structural and enzymatic proteins and as the genetic material for new virions. RNA mislocalization can affect the functions of the RNA and its encoded proteins, causing defects in viral replication. Currently, little is known about how HIV-1 RNA is transported in the cytoplasm. Here, we demonstrate that HIV-1 full-length RNAs use diffusion as the major mechanism for cytoplasmic transport in the absence of viral group-specific antigen (Gag) proteins and even in the presence of sufficient Gag proteins for virus assembly, indicating that Gag does not alter the RNA transport mechanism. These studies provide insights into mechanisms essential to viral replication. Full-length HIV-1 RNA plays a central role in viral replication by serving as the mRNA for essential viral proteins and as the genome packaged into infectious virions. Proper RNA trafficking is required for the functions of RNA and its encoded proteins; however, the mechanism by which HIV-1 RNA is transported within the cytoplasm remains undefined. Full-length HIV-1 RNA transport is further complicated when group-specific antigen (Gag) protein is expressed, because a significant portion of HIV-1 RNA may be transported as Gag–RNA complexes, whose properties could differ greatly from Gag-free RNA. In this report, we visualized HIV-1 RNA and monitored its movement in the cytoplasm by using single-molecule tracking. We observed that most of the HIV-1 RNA molecules move in a nondirectional, random-walk manner, which does not require an intact cytoskeletal structure, and that the mean-squared distance traveled by the RNA increases linearly with time, indicative of diffusive movement. We also observed that a single HIV-1 RNA molecule can move at various speeds when traveling through the cytoplasm, indicating that its movement is strongly affected by the immediate environment. To examine the effect of Gag protein on HIV-1 RNA transport, we analyzed the cytoplasmic HIV-1 RNA movement in the presence of sufficient Gag for virion assembly and found that HIV-1 RNA is still transported by diffusion with mobility similar to the mobility of RNAs unable to express functional Gag. These studies define a major mechanism of HIV-1 gene expression and resolve the long-standing question of how the RNA genome is transported to the assembly site.

Patterns of Human Immunodeficiency Virus Type 1 Recombination Ex Vivo Provide Evidence for Coadaptation of Distant Sites, Resulting in Purifying Selection for Intersubtype Recombinants during Replication

ABSTRACT High-frequency recombination is a hallmark of HIV-1 replication. Recombination can occur between two members of the same subtype or between viruses from two different subtypes, generating intra- or intersubtype recombinants, respectively. Many intersubtype recombinants have been shown to circulate in human populations. We hypothesize that sequence diversity affects the emergence of viable recombinants by decreasing recombination events and reducing the ability of the recombinants to replicate. To test our hypothesis, we compared recombination between two viruses containing subtype B pol genes (B/B) and between viruses with pol genes from subtype B or F (B/F). Recombination events generated during a single cycle of infection without selection pressure on pol gene function were analyzed by single-genome sequencing. We found that recombination occurred slightly (∼30%) less frequently in B/F than in B/B viruses, and the overall distribution of crossover junctions in pol was similar for the two classes of recombinants. We then examined the emergence of recombinants in a multiple cycle assay, so that functional pol gene products were selected. We found that the emerging B/B recombinants had complex patterns, and the crossover junctions were distributed throughout the pol gene. In contrast, selected B/F recombinants had limited recombination patterns and restricted crossover junction distribution. These results provide evidence for the evolved coadapted sites in variants from different subtypes; these sites may be segregated by recombination events, causing the newly generated intersubtype recombinants to undergo purifying selection. Therefore, the ability of the recombinants to replicate is the major barrier for many of these viruses.

HIV-1 RNA genome dimerizes on the plasma membrane in the presence of Gag protein

Significance Dimerization of the RNA genome is a key event in HIV-1 virion assembly and has a strong impact in viral replication and evolution. Packaging the dimeric genome allows frequent recombination to rescue genetic information in damaged RNAs and to generate variants that can evade the host immune response or resist antiviral treatments. Furthermore, genome packaging is regulated by recognition of dimeric RNA. Our studies demonstrate that HIV-1 RNAs dimerize not in the cytoplasm but on the plasma membrane, often early during the assembly process, and that Gag protein is required for maintenance of the RNA dimer. These studies address the timing, location, and partners involved in RNA dimerization, an important process for HIV-1 replication. Retroviruses package a dimeric genome comprising two copies of the viral RNA. Each RNA contains all of the genetic information for viral replication. Packaging a dimeric genome allows the recovery of genetic information from damaged RNA genomes during DNA synthesis and promotes frequent recombination to increase diversity in the viral population. Therefore, the strategy of packaging dimeric RNA affects viral replication and viral evolution. Although its biological importance is appreciated, very little is known about the genome dimerization process. HIV-1 RNA genomes dimerize before packaging into virions, and RNA interacts with the viral structural protein Gag in the cytoplasm. Thus, it is often hypothesized that RNAs dimerize in the cytoplasm and the RNA–Gag complex is transported to the plasma membrane for virus assembly. In this report, we tagged HIV-1 RNAs with fluorescent proteins, via interactions of RNA-binding proteins and motifs in the RNA genomes, and studied their behavior at the plasma membrane by using total internal reflection fluorescence microscopy. We showed that HIV-1 RNAs dimerize not in the cytoplasm but on the plasma membrane. Dynamic interactions occur among HIV-1 RNAs, and stabilization of the RNA dimer requires Gag protein. Dimerization often occurs at an early stage of the virus assembly process. Furthermore, the dimerization process is probably mediated by the interactions of two RNA–Gag complexes, rather than two RNAs. These findings advance the current understanding of HIV-1 assembly and reveal important insights into viral replication mechanisms.