Alice Telesnitsky

NMR Detection of Structures in the HIV-1 5′-Leader RNA That Regulate Genome Packaging

An RNA structural switch regulates whether the HIV genome is translated or dimerized and packaged. The 5′-leader of the HIV-1 genome regulates multiple functions during viral replication via mechanisms that have yet to be established. We developed a nuclear magnetic resonance approach that enabled direct detection of structural elements within the intact leader (712-nucleotide dimer) that are critical for genome packaging. Residues spanning the gag start codon (AUG) form a hairpin in the monomeric leader and base pair with residues of the unique-5′ region (U5) in the dimer. U5:AUG formation promotes dimerization by displacing and exposing a dimer-promoting hairpin and enhances binding by the nucleocapsid (NC) protein, which is the cognate domain of the viral Gag polyprotein that directs packaging. Our findings support a packaging mechanism in which translation, dimerization, NC binding, and packaging are regulated by a common RNA structural switch.

The Remarkable Frequency of Human Immunodeficiency Virus Type 1 Genetic Recombination

SUMMARY The genetic diversity of human immunodeficiency virus type 1 (HIV-1) results from a combination of point mutations and genetic recombination, and rates of both processes are unusually high. This review focuses on the mechanisms and outcomes of HIV-1 genetic recombination and on the parameters that make recombination so remarkably frequent. Experimental work has demonstrated that the process that leads to recombination—a copy choice mechanism involving the migration of reverse transcriptase between viral RNA templates—occurs several times on average during every round of HIV-1 DNA synthesis. Key biological factors that lead to high recombination rates for all retroviruses are the recombination-prone nature of their reverse transcription machinery and their pseudodiploid RNA genomes. However, HIV-1 genes recombine even more frequently than do those of many other retroviruses. This reflects the way in which HIV-1 selects genomic RNAs for coencapsidation as well as cell-to-cell transmission properties that lead to unusually frequent associations between distinct viral genotypes. HIV-1 faces strong and changeable selective conditions during replication within patients. The mode of HIV-1 persistence as integrated proviruses and strong selection for defective proviruses in vivo provide conditions for archiving alleles, which can be resuscitated years after initial provirus establishment. Recombination can facilitate drug resistance and may allow superinfecting HIV-1 strains to evade preexisting immune responses, thus adding to challenges in vaccine development. These properties converge to provide HIV-1 with the means, motive, and opportunity to recombine its genetic material at an unprecedented high rate and to allow genetic recombination to serve as one of the highest barriers to HIV-1 eradication.

Determination of the site of first strand transfer during Moloney murine leukemia virus reverse transcription and identification of strand transfer‐associated reverse transcriptase errors

Reverse transcriptase must perform two specialized template switches during retroviral DNA synthesis. Here, we used Moloney murine leukemia virus‐based vectors to examine the site of one of these switches during intracellular reverse transcription. Consistent with original models for reverse transcription, but in contrast to previous experimental data, we observed that this first strand transfer nearly always occurred precisely at the 5′ end of genomic RNA. This finding allowed us to use first strand transfer to study the classes of errors that reverse transcriptase can and/or does make when it switches templates at a defined position during viral DNA synthesis. We found that errors occurred at the site of first strand transfer ∼1000‐fold more frequently than reported average reverse transcriptase error rates for template‐internal positions. We then analyzed replication products of specialized vectors that were designed to test possible origins for the switch‐associated errors. Our results suggest that at least some errors arose via non‐templated nucleotide addition followed by mismatch extension at the point of strand transfer. We discuss the significance of our findings as they relate to the possible contribution that template switch‐associated errors may make to retroviral mutation rates.

Structure of the HIV-1 RNA packaging signal

Structural signals that direct HIV packaging During the viral replication cycle of HIV, unspliced dimeric RNA genomes are efficiently packaged into new virions at the host cell membrane. Packaging is directed by a region at the start of the genome, the 5′ leader. The architecture of the 5′ leader remains controversial. Keane et al. developed nuclear magnetic resonance methods to determine the structure of a 155-nucleotide-long region of the 5′ leader that can direct viral packaging. The structure shows how the 5′ leader binds to the HIV protein that directs packaging, how unspliced dimeric genomes are selected for packaging, and how translation is suppressed when the genome dimerizes. Science, this issue p. 917 A nuclear magnetic resonance structure of a region of the HIV-1 RNA 5′ leader gives insight into how the viral genome is selected for packaging. The 5′ leader of the HIV-1 genome contains conserved elements that direct selective packaging of the unspliced, dimeric viral RNA into assembling particles. By using a 2H-edited nuclear magnetic resonance (NMR) approach, we determined the structure of a 155-nucleotide region of the leader that is independently capable of directing packaging (core encapsidation signal; ΨCES). The RNA adopts an unexpected tandem three-way junction structure, in which residues of the major splice donor and translation initiation sites are sequestered by long-range base pairing and guanosines essential for both packaging and high-affinity binding to the cognate Gag protein are exposed in helical junctions. The structure reveals how translation is attenuated, Gag binding promoted, and unspliced dimeric genomes selected, by the RNA conformer that directs packaging.

Assays for retroviral reverse transcriptase.

Publisher Summary This chapter summarizes some of the popular assays for reverse transcriptase (RT) and their use in quantifying levels of viral particles, viral RNA templates, and mutant RTs with altered polymerase and nuclease activities. The enzyme responsible for retroviral DNA synthesis, reverse transcriptase (RT) is both, a DNA polymerase and a nuclease. A simple homopolymer-based assay for RT DNA polymerase activity that can be used to detect retroviruses in the culture media of infected cells, or alternatively to detect or quantify RT during its purification is described. The chapter also describes endogenous assay, a method to analyze the products of reverse transcription in purified virions and presents a method for screening libraries of bacterially expressed RT mutants as a means of isolating particular variant enzymes displaying selected properties. An in situ method that allows RT RNase H activity to be assayed separately from contaminating cellular RNase H activities is also presented.

Human Immunodeficiency Virus Type 1 Genetic Recombination Is More Frequent Than That of Moloney Murine Leukemia Virus despite Similar Template Switching Rates

ABSTRACT Retroviral recombinants result from template switching between copackaged viral genomes. Here, marker reassortment between coexpressed vectors was measured during single replication cycles, and human immunodeficiency virus type 1 (HIV-1) recombination was observed six- to sevenfold more frequently than murine leukemia virus (MLV) recombination. Template switching was also assayed by using transduction-type vectors in which donor and acceptor template regions were joined covalently. In this situation, where RNA copackaging could not vary, MLV and HIV-1 template switching rates were indistinguishable. These findings argue that MLVs lower intermolecular recombination frequency does not reflect enzymological differences. Instead, these data suggest that recombination rates differ because coexpressed MLV RNAs are less accessible to the recombination machinery than are coexpressed HIV RNAs. This hypothesis provides a plausible explanation for why most gammaretrovirus recombinants, although relatively rare, display evidence of multiple nonselected crossovers. By implying that recombinogenic template switching occurs roughly four times on average during the synthesis of every MLV or HIV-1 DNA, these results suggest that virtually all products of retroviral replication are biochemical recombinants.

Retroviral RNA Dimerization and Packaging: The What, How, When, Where, and Why

Retroviral genomic RNAs (gRNAs) are packaged as dimers, joined near their 59 ends in non-covalent linkages that withstand modest heat treatment but dissociate at ,65uC. Determinants of gRNA dimerization and recruitment for packaging map to the same ,100 to ,300 base regions and are, for the most part, physically and genetically inseparable [1]. Synthetic RNAs containing these sequences dimerize in vitro. Because transplanting these sequences onto a cell mRNA confers selective packaging, and ablating them greatly reduces gRNA packaging, these sequences are known as Y (psi), for ‘‘packaging signal’’ (Figure 1A) [2]. Within virions, gRNAs are coated with a basic viral protein called nucleocapsid (NC) at a density of about one NC per five to eight RNA bases [3]. This nucleoprotein complex resides within the mature virion core. Total gRNA length, were it in an A-helix, exceeds the core inner diameter by more than 30-fold [4]. Thus, encapsidated gRNA is highly condensed. The co-packaged gRNAs likely are not aligned along their lengths because they are identical and cannot basepair in register, but the nature of their compaction is unknown. If Y is experimentally removed from gRNA but viral proteins are still expressed, morphologically normal virions can form, which are devoid of gRNA. These contain random samples of host mRNA [5]. Each Y+ or Y2 virion also contains several copies of certain host RNAs such as 7SL, the RNA scaffold of signal recognition particles. Other than the primer tRNA, any roles of these host non-coding RNAs in retroviruses are unknown. Usually, a virion’s two gRNAs are identical. However, if a producer cell contains two distinct dimer-compatible proviruses, virions can contain gRNA heterdimers. Both gRNAs are genetically complete but not intact, as they appear to contain nicks and run as smears on denaturing gels. Accordingly, it has been speculated that retroviruses’ dimeric genome organization may serve in part as defense against antiviral nucleases that would otherwise restrict replication [4]. RNA degradation during reverse transcription further limits provirus synthesis to one or fewer per virion [6]. Transmission of no more than one allele at each locus explains why, although they package two gRNAs, retroviruses are not truly diploid.

Nonrandom Packaging of Host RNAs in Moloney Murine Leukemia Virus

ABSTRACT Moloney murine leukemia virus (MLV) particles contain both viral genomic RNA and an assortment of host cell RNAs. Packaging of virus-encoded RNA is selective, with virions virtually devoid of spliced env mRNA and highly enriched for unspliced genome. Except for primer tRNA, it is unclear whether packaged host RNAs are randomly sampled from the cell or specifically encapsidated. To address possible biases in host RNA sampling, the relative abundances of several host RNAs in MLV particles and in producer cells were compared. Using 7SL RNA as a standard, some cellular RNAs, such as those of the Ro RNP, were found to be enriched in MLV particles in that their ratios relative to 7SL differed little, if at all, from their ratios in cells. Some RNAs were underrepresented, with ratios relative to 7SL several orders of magnitude lower in virions than in cells, while others displayed intermediate values. At least some enriched RNAs were encapsidated by genome-defective nucleocapsid mutants. Virion RNAs were not a random sample of the cytosol as a whole, since some cytoplasmic RNAs like tRNAMet were vastly underrepresented, while U6 spliceosomal RNA, which functions in the nucleus, was enriched. Real-time PCR demonstrated that env mRNA, although several orders of magnitude less abundant than unspliced viral RNA, was slightly enriched relative to actin mRNA in virions. These data demonstrate that certain host RNAs are nearly as enriched in virions as genomic RNA and suggest that Ψ− mRNAs and some other host RNAs may be specifically excluded from assembly sites.

Effects of varying sequence similarity on the frequency of repeat deletion during reverse transcription of a human immunodeficiency virus type 1 vector

ABSTRACT Genetic recombination contributes to human immunodeficiency virus type 1 (HIV-1) diversity, with homologous recombination being more frequent than nonhomologous recombination. In this study, HIV-1-based vectors were used to assay the effects of various extents of sequence divergence on the frequency of the recombination-related property of repeat deletion. Sequence variation, similar in degree to that which differentiates natural HIV-1 isolates, was introduced by synonymous substitutions into a gene segment. Repeated copies of this segment were then introduced into assay vectors. With the use of a phenotypic screen, the deletion frequency of identical repeats was compared to the frequencies of repeats that differed in sequence by various extents. During HIV-1 reverse transcription, the deletion frequency observed with repeats that differed by 5% was 65% of that observed with identical repeats. The deletion frequency decreased to 26% for repeats that differed by 9%, and when repeats differed by 18%, the deletion frequency was about 5% of the identical repeat value. Deletion frequencies fell to less than 0.3% of identical repeat values when genetic distances of 27% or more were examined. These data argue that genetic variation is not as inhibitory to HIV-1 repeat deletion as it is to the corresponding cellular process and suggest that, for sequences that differ by about 25% or more, HIV-1 recombination directed by sequence homology may be no more frequent than that which is homology independent.

Nonrandom Dimerization of Murine Leukemia Virus Genomic RNAs

ABSTRACT Retroviral genomes consist of two unspliced RNAs linked noncovalently in a dimer. Although these two RNAs are generally identical, two different RNAs can be copackaged when virions are produced by coinfected cells. It has been assumed, but not tested, that copackaging results from random RNA associations in the cytoplasm to yield encapsidated RNA homodimers and heterodimers in Hardy-Weinberg proportions. Here, virion RNA homo- and heterodimerization were examined for Moloney murine leukemia virus (MLV) using nondenaturing Northern blotting and a novel RNA dimer capture assay. The results demonstrated that coexpressed MLV RNAs preferentially self-associated, even when RNAs were identical in known packaging and dimerization sequences or when they differed overall by less than 0.1%. In contrast, HIV-1 RNAs formed homo- and heterodimers in random proportions. We speculate that these species-specific differences in RNA dimer partner selection may at least partially explain the higher frequency of genetic recombination observed for human immunodeficiency virus type 1 than for MLV.