A conserved hairpin motif in the R-U5 region of the human immunodeficiency virus type 1 RNA genome is essential for replication

Das, Atze T.; Klaver, Bep; Klasens, Bianca I.F.; Wamel, Jeroen L.B. van; Berkhout, Ben

doi:10.1128/jvi.71.3.2346-2356.1997

Cited by 119 publications

(60 citation statements)

References 62 publications

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“…Previously published reports from other laboratories suggest that additional packaging determinants other than TAR are present in R-U5 (16,47,48,63). The data suggest that at least two distinct stem loop structures exist in the R-U5 region and that both of these structures are needed for efficient encapsidation of HIV RNA.…”

Section: Discussionmentioning

confidence: 62%

An Intact TAR Element and Cytoplasmic Localization Are Necessary for Efficient Packaging of Human Immunodeficiency Virus Type 1 Genomic RNA

Helga-Maria

Hammarskjöld

Rekosh

1999

J Virol

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Although most reports defining the human immunodeficiency virus type 1 (HIV-1) genomic RNA packaging signal have focused on the region downstream of the major 5′ splice site, others have suggested that sequences upstream of the splice site may also play an important role. In this study we have directly examined the role played by the HIV-1 TAR region in RNA packaging. For these experiments we used a proviral expression system that is largely independent of Tat for transcriptional activation. This allowed us to create constructs that efficiently expressed RNAs carrying mutations in TAR and to determine the ability of these RNAs to be packaged. Our results indicate that loss of sequences in TAR significantly reduce the ability of a viral RNA to be packaged. The requirement for TAR sequences in RNA packaging was further examined by using a series of missense mutations positioned throughout the entire TAR structure. TAR mutations previously shown to influence Tat transactivation, such as G31U in the upper loop region or UCU to AAG in the bulge (nucleotides [nt] 22 to 24), failed to have any effect on RNA packaging. Mutations which disrupted the portion of the TAR stem immediately below the bulge also had little effect. In contrast, dramatic effects on RNA packaging were observed with constructs containing mutations in the lower portion of the TAR stem. Point mutations which altered nt 5 to 9, 10 to 15, 44 to 49, or 50 to 54 all reduced RNA packaging 11- to 25-fold. However, compensatory double mutations which restored the stem structure were able to restore packaging. These results indicate that an intact lower stem structure, rather than a specific sequence, is required for RNA packaging. Our results also showed that RNA molecules retained within the nucleus cannot be packaged, unless they are transported to the cytoplasm by either Rev/Rev response element or the Mason-Pfizer monkey virus constitutive transport element.

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Section: Discussionmentioning

confidence: 62%

An Intact TAR Element and Cytoplasmic Localization Are Necessary for Efficient Packaging of Human Immunodeficiency Virus Type 1 Genomic RNA

Helga-Maria

Hammarskjöld

Rekosh

1999

J Virol

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“…1, 2, 4, 5, 6, and 7, the GFP reporter HIV-1 AD8 strain was utilized. Plasmid DNA encoding an NL4.3 backbone with AD8 Env and enhanced GFP (EGFP) inserted one base downstream of the env open reading frame (15), followed by an IRES-Nef coding sequence, was transiently transfected into HEK293T cells using calcium phosphate as previously described (35). Briefly, virus-containing supernatant was collected after 2 days, filtered (0.45-m pore size), and concentrated by ultracentrifugation through 20% sucrose.…”

Section: Study Participantsmentioning

confidence: 99%

CD4- and Time-Dependent Susceptibility of HIV-1-Infected Cells to Antibody-Dependent Cellular Cytotoxicity

Lee

Prévost

Richard

et al. 2019

J Virol

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HIV-1-specific antibody-dependent cellular cytotoxicity (ADCC) antibodies within HIV-1-positive (HIV-1+) individuals predominantly target CD4-induced (CD4i) epitopes on HIV-1 envelope glycoprotein (Env). These CD4i epitopes are usually concealed on the surface of infected cells due to CD4 downregulation by the HIV-1 accessory proteins Nef and Vpu. We hypothesized that early-stage infected cells in the process of downregulating CD4 could be more susceptible to ADCC than late-stage infected cells that have fully downregulated CD4. There was significantly higher binding of antibodies within plasma from HIV-1-infected individuals to early-stage infected cells expressing intermediate levels of CD4 (CD4-intermediate cells) than in late-stage infected cells expressing low levels of CD4 (CD4-low cells). However, we noted that HIV-1-uninfected bystander cells and HIV-1-infected cells, at various stages of downregulating CD4, were all susceptible to NK cell-mediated ADCC. Importantly, we observed that the cytolysis of bystander cells and early infected cells in this culture system was driven by sensitization of target cells by inoculum-derived HIV-1 Env or virions. This phenomenon provided Env to target cells prior to de novo Env expression, resulting in artifactual ADCC measurements. Future studies should take into consideration the inherent caveats of in vitro infection systems and develop improved models to address the potential role for ADCC against cells with nascent HIV-1 infection. IMPORTANCE An increasing body of evidence suggests that ADCC contributes to protection against HIV-1 acquisition and slower HIV-1 disease progression. Targeting cells early during the infection cycle would be most effective in limiting virus production and spread. We hypothesized that there could be a time-dependent susceptibility of HIV-1-infected cells to ADCC in regard to CD4 expression. We observed NK cell-mediated ADCC of HIV-1-infected cells at multiple stages of CD4 downregulation. Importantly, ADCC of early infected cells appeared to be driven by a previously unappreciated problem of soluble Env and virions from the viral inoculum sensitizing uninfected cells to ADCC prior to de novo Env expression. These results have implications for studies examining ADCC against cells with nascent HIV-1 infection.

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“…After prolonged culturing (ϳ2 mo) on SupT1 T-cells, we identified a faster replicating virus in a culture of the C385A/C418A mutant virus. DNA was extracted from infected cells (Das et al, 1997), and proviral gp120 sequences were PCR amplified with primers A (5Ј-GCTCCATGGCT-TAGGGCAACATATATCTATG-3Ј) and B (5Ј-GTCTCGAGATGCTGCTCC-3Ј) and sequenced. Population sequencing revealed two reversions: a first-site pseudoreversion A418V and a second-site reversion at a nearby residue: T415I ( Figure 1).…”

Section: Virus Evolutionmentioning

confidence: 99%

Evolution Rescues Folding of Human Immunodeficiency Virus-1 Envelope Glycoprotein GP120 Lacking a Conserved Disulfide Bond

Sanders

Hsu

Anken

et al. 2008

MBoC

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The majority of eukaryotic secretory and membrane proteins contain disulfide bonds, which are strongly conserved within protein families because of their crucial role in folding or function. The exact role of these disulfide bonds during folding is unclear. Using virus-driven evolution we generated a viral glycoprotein variant, which is functional despite the lack of an absolutely conserved disulfide bond that links two antiparallel beta-strands in a six-stranded beta-barrel. Molecular dynamics simulations revealed that improved hydrogen bonding and side chain packing led to stabilization of the beta-barrel fold, implying that beta-sheet preference codirects glycoprotein folding in vivo. Our results show that the interactions between two beta-strands that are important for the formation and/or integrity of the beta-barrel can be supported by either a disulfide bond or beta-sheet favoring residues.

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A conserved hairpin motif in the R-U5 region of the human immunodeficiency virus type 1 RNA genome is essential for replication

Cited by 119 publications

References 62 publications

An Intact TAR Element and Cytoplasmic Localization Are Necessary for Efficient Packaging of Human Immunodeficiency Virus Type 1 Genomic RNA

An Intact TAR Element and Cytoplasmic Localization Are Necessary for Efficient Packaging of Human Immunodeficiency Virus Type 1 Genomic RNA

CD4- and Time-Dependent Susceptibility of HIV-1-Infected Cells to Antibody-Dependent Cellular Cytotoxicity

Evolution Rescues Folding of Human Immunodeficiency Virus-1 Envelope Glycoprotein GP120 Lacking a Conserved Disulfide Bond

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