HIV-1 single-transcription start-site mutants display complementary replication functions that are restored by reversion.

HIV-1 transcription initiates at two positions, generating RNAs with either 1G or 3G 5' ends. The replication fates of these RNAs differ, with viral particles encapsidating almost exclusively 1G RNAs and 3G RNAs retained in cells where they are enriched on polysomes and among spliced viral RNAs. Here, we studied replication properties of virus promoter mutants that produced only one RNA 5' isoform or the other: separately, in combination, and during spreading infection.

HIV-1 single transcription start site mutants display complementary replication functions that are restored by reversion.

HIV-1 transcription initiates at two positions, generating RNAs with either 1G or 3G 5' ends. The replication fates of these RNAs di\er, with viral particles encapsidating almost exclusively 1G RNAs and 3G RNAs retained in cells where they are enriched on polysomes and among spliced viral RNAs. Here, we studied replication properties of virus promoter mutants that produced only one RNA 5' isoform or the other: separately, in combination, and during spreading infection.

Role of RNA structural plasticity in modulating HIV-1 genome packaging and translation.

HIV-1 transcript function is controlled in part by twinned transcriptional start site usage, where 5' capped RNAs beginning with a single guanosine (1G) are preferentially packaged into progeny virions as genomic RNA (gRNA) whereas those beginning with three sequential guanosines (3G) are retained in cells as mRNAs. In 3G transcripts, one of the additional guanosines base pairs with a cytosine located within a conserved 5' polyA element, resulting in formation of an extended 5' polyA structure as opposed to the hairpin structure formed in 1G RNAs. To understand how this remodeling influences overall transcript function, we applied in vitro biophysical studies with in-cell genome packaging and competitive translation assays to native and 5' polyA mutant transcripts generated with promoters that differentially produce 1G or 3G RNAs.

Transcription start site heterogeneity and its role in RNA fate determination distinguish HIV-1 from other retroviruses and are mediated by core promoter elements.

HIV-1 uses heterogeneous transcription start sites (TSSs) to generate two RNA 5´ isoforms that adopt radically different structures and perform distinct replication functions. Although these RNAs differ in length by only two bases, exclusively, the shorter RNA is encapsidated while the longer RNA is excluded from virions and provides intracellular functions. The current study examined TSS usage and packaging selectivity for a broad range of retroviruses and found that heterogeneous TSS usage was a conserved feature of all tested HIV-1 strains, but all other retroviruses examined displayed unique TSSs.

Transcription start site heterogeneity and its role in RNA fate determination distinguish HIV-1 from other retroviruses and are mediated by core promoter elements.

HIV-1 uses heterogeneous transcription start sites (TSSs) to generate two RNA 5' isoforms that adopt radically different structures and perform distinct replication functions. Although these RNAs differ in length by only two bases, exclusively the shorter RNA is encapsidated while the longer RNA is excluded from virions and provides intracellular functions. The current study examined TSS usage and packaging selectivity for a broad range of retroviruses and found that heterogenous TSS usage was a conserved feature of all tested HIV-1 strains, but all other retroviruses examined displayed unique TSSs.

Bimodal Expression Patterns, and Not Viral Burst Sizes, Predict the Effects of Vpr on HIV-1 Proviral Populations in Jurkat Cells.

Integration site landscapes, clonal dynamics, and latency reversal with or without were compared in HIV-1-infected Jurkat cell populations, and the properties of individual clones were defined. Clones differed in fractions of long terminal repeat (LTR)-active daughter cells, with some clones containing few to no LTR-active cells, while almost all cells were LTR active for others. Clones varied over 4 orders of magnitude in virus release per active cell.

Stability and conformation of the dimeric HIV-1 genomic RNA 5'UTR.

During HIV-1 assembly, the viral Gag polyprotein specifically selects the dimeric RNA genome for packaging into new virions. The 5' untranslated region (5'UTR) of the dimeric genome may adopt a conformation that is optimal for recognition by Gag. Further conformational rearrangement of the 5'UTR, promoted by the nucleocapsid (NC) domain of Gag, is predicted during virus maturation.

Identification of the initial nucleocapsid recognition element in the HIV-1 RNA packaging signal.

Selective packaging of the HIV-1 genome during virus assembly is mediated by interactions between the dimeric 5'-leader of the unspliced viral RNA and the nucleocapsid (NC) domains of a small number of assembling viral Gag polyproteins. Here, we show that the dimeric 5'-leader contains more than two dozen NC binding sites with affinities ranging from 40 nM to 1.4 μM, and that all high-affinity sites ( ≲ 400 nM) reside within a ∼150-nt region of the leader sufficient to promote RNA packaging (core encapsidation signal, Ψ).

Structural basis for transcriptional start site control of HIV-1 RNA fate.

Heterogeneous transcriptional start site usage by HIV-1 produces 5'-capped RNAs beginning with one, two, or three 5'-guanosines (1G, 2G, or 3G, respectively) that are either selected for packaging as genomes (1G) or retained in cells as translatable messenger RNAs (mRNAs) (2G and 3G). To understand how 5'-guanosine number influences fate, we probed the structures of capped HIV-1 leader RNAs by deuterium-edited nuclear magnetic resonance. The 1G transcript adopts a dimeric multihairpin structure that sequesters the cap, inhibits interactions with eukaryotic translation initiation factor 4E, and resists decapping.

HIV-1 spliced RNAs display transcription start site bias.

Human immunodeficiency virus type 1 (HIV-1) transcripts have three fates: to serve as genomic RNAs, unspliced mRNAs, or spliced subgenomic mRNAs. Recent structural studies have shown that sequences near the 5' end of HIV-1 RNA can adopt at least two alternate three-dimensional conformations, and that these structures dictate genome versus unspliced mRNA fates. HIV-1's use of alternate transcription start sites (TSS) can influence which RNA conformer is generated, and this choice, in turn, dictates the fate of the unspliced RNA.

Stable integrant-specific differences in bimodal HIV-1 expression patterns revealed by high-throughput analysis.

HIV-1 gene expression is regulated by host and viral factors that interact with viral motifs and is influenced by proviral integration sites. Here, expression variation among integrants was followed for hundreds of individual proviral clones within polyclonal populations throughout successive rounds of virus and cultured cell replication, with limited findings using CD4+ cells from donor blood consistent with observations in immortalized cells. Tracking clonal behavior by proviral "zip codes" indicated that mutational inactivation during reverse transcription was rare, while clonal expansion and proviral expression states varied widely.

Flexibility in Nucleic Acid Binding Is Central to APOBEC3H Antiviral Activity.

APOBEC3 proteins APOBEC3F (A3F), APOBEC3G (A3G), and APOBEC3H (A3H) are host restriction factors that inhibit HIV-1 through DNA cytidine deaminase-dependent and -independent mechanisms and have either one (A3H) or two (A3F and A3G) zinc-binding domains. A3H antiviral activity encompasses multiple molecular functions, all of which depend on recognition of RNA or DNA. A3H crystal structures revealed an unusual interaction with RNA wherein an RNA duplex mediates dimerization of two A3H proteins.

Multiple, Switchable Protein:RNA Interactions Regulate Human Immunodeficiency Virus Type 1 Assembly.

Human immunodeficiency virus type 1 (HIV-1) particle assembly requires several protein:RNA interactions that vary widely in their character, from specific recognition of highly conserved and structured viral RNA elements to less specific interactions with variable RNA sequences. Genetic, biochemical, biophysical, and structural studies have illuminated how virion morphogenesis is accompanied by dramatic changes in the interactions among the protein and RNA virion components. The 5' leader RNA element drives RNA recognition by Gag upon initiation of HIV-1 assembly and can assume variable conformations that influence translation, dimerization, and Gag recognition.

Influence of gag and RRE Sequences on HIV-1 RNA Packaging Signal Structure and Function.

The packaging signal (Ψ) and Rev-responsive element (RRE) enable unspliced HIV-1 RNAs' export from the nucleus and packaging into virions. For some retroviruses, engrafting Ψ onto a heterologous RNA is sufficient to direct encapsidation. In contrast, HIV-1 RNA packaging requires 5' leader Ψ elements plus poorly defined additional features.

Transcriptional start site heterogeneity modulates the structure and function of the HIV-1 genome.

The promoter in HIV type 1 (HIV-1) proviral DNA contains three sequential guanosines at the U3-R boundary that have been proposed to function as sites for transcription initiation. Here we show that all three sites are used in cells infected with HIV-1 and that viral RNAs containing a single 5' capped guanosine (1G) are specifically selected for packaging in virions, consistent with a recent report [Masuda et al. (2015) Sci Rep 5:17680].

The Host RNAs in Retroviral Particles.

As they assemble, retroviruses encapsidate both their genomic RNAs and several types of host RNA. Whereas limited amounts of messenger RNA (mRNA) are detectable within virion populations, the predominant classes of encapsidated host RNAs do not encode proteins, but instead include endogenous retroelements and several classes of non-coding RNA (ncRNA), some of which are packaged in significant molar excess to the viral genome. Surprisingly, although the most abundant host RNAs in retroviruses are also abundant in cells, unusual forms of these RNAs are packaged preferentially, suggesting that these RNAs are recruited early in their biogenesis: before associating with their cognate protein partners, and/or from transient or rare RNA populations.

Analysis of the human immunodeficiency virus-1 RNA packageome.

All retroviruses package cellular RNAs into virions. Studies of murine leukemia virus (MLV) revealed that the major host cell RNAs encapsidated by this simple retrovirus were LTR retrotransposons and noncoding RNAs (ncRNAs). Several classes of ncRNAs appeared to be packaged by MLV shortly after synthesis, as precursors to tRNAs, small nuclear RNAs, and small nucleolar RNAs were all enriched in virions.

Resolution of Specific Nucleotide Mismatches by Wild-Type and AZT-Resistant Reverse Transcriptases during HIV-1 Replication.

A key contributor to HIV-1 genetic variation is reverse transcriptase errors. Some mutations result because reverse transcriptase (RT) lacks 3' to 5' proofreading exonuclease and can extend mismatches. However, RT also excises terminal nucleotides to a limited extent, and this activity contributes to AZT resistance.

Host RNA Packaging by Retroviruses: A Newly Synthesized Story.

A fascinating aspect of retroviruses is their tendency to nonrandomly incorporate host cell RNAs into virions. In addition to the specific tRNAs that prime reverse transcription, all examined retroviruses selectively package multiple host cell noncoding RNAs (ncRNAs). Many of these ncRNAs appear to be encapsidated shortly after synthesis, before assembling with their normal protein partners.

RNA structure. Structure of the HIV-1 RNA packaging signal.

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 (2)H-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; Ψ(CES)). 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.

A retrovirus packages nascent host noncoding RNAs from a novel surveillance pathway.

Although all retroviruses recruit host cell RNAs into virions, both the spectrum of RNAs encapsidated and the mechanisms by which they are recruited remain largely unknown. Here, we used high-throughput sequencing to obtain a comprehensive description of the RNAs packaged by a model retrovirus, murine leukemia virus. The major encapsidated host RNAs are noncoding RNAs (ncRNAs) and members of the VL30 class of endogenous retroviruses.

Determinants of Moloney murine leukemia virus Gag-Pol and genomic RNA proportions.

Unlabelled: The Moloney murine leukemia virus (MoMLV) ribonucleoprotein complex is composed of an approximately 20:1 mixture of Gag and Gag-Pol polyproteins plus a single genomic RNA (gRNA) dimer. The mechanisms that regulate these proportions are unknown. Here, we examined whether virion proportions of Gag, Gag-Pol, and gRNA were determined by sampling (that is, if they reflected expression ratios or intracellular concentrations) or more specific recruitment.

Moloney murine leukemia virus genomic RNA packaged in the absence of a full complement of wild type nucleocapsid protein.

The current model for MLV genomic RNA (gRNA) packaging predicts that of the thousands of Gag proteins in a budding virion, only a small number (≤1%) may be necessary to recruit gRNA. Here, we examined the threshold limits of functional Gag required to package gRNA using wild-type (WT) and packaging deficient mutant nucleocapsid (NC) phenotypically mixed virions. Although gRNA packaging was severely diminished for the NC mutant, the residual encapsidated RNA dimer displayed motility on gels, thermostability, and integrity that was indistinguishable from that of WT.