scholarly journals Short- and long-range interactions in the HIV-1 5’UTR regulate genome dimerization and packaging

2021 ◽  
Author(s):  
Redmond Smyth ◽  
Liqing Ye ◽  
Anne-Sophie Gribling ◽  
Patrick Bohn ◽  
Anuja Kibe ◽  
...  
Keyword(s):  
Life Cycle ◽  
Rna Structure ◽  
Mechanistic Explanation ◽  
Primer Binding Site ◽  
Rna Dimerization ◽  
Long Range Interactions ◽  
Retroviral Replication ◽  
Genome Dimerization ◽  
Late Stages ◽  
Hiv 1

Abstract Genome dimerization is a conserved feature of retroviral replication and a critical step in the HIV-1 life cycle, but how it is regulated is incompletely understood. Here, we developed FARS-seq (Functional Analysis of RNA Structure) to comprehensively identify sequences and structures within the HIV-1 5’UTR influencing dimerization. We found nucleotides important for dimerization throughout the HIV-1 5’UTR and identified distinct structural conformations in monomeric and dimeric RNA. The dimer displayed TAR, PolyA, PBS, and SL1-SL3 as stem-loops. In the monomer, SL1 was dramatically reconfigured into long- and short-range base-pairings with polyA and PBS, respectively. The polyA-SL1 interaction disrupts the major packaging motifs, and the PBS-SL1 interaction functionally couples the primer binding site with dimerization and Pr55Gag binding. Altogether, our data provide insights into late stages of HIV-1 life cycle and a mechanistic explanation for the link between RNA dimerization and packaging.

scholarly journals Impact of Human Immunodeficiency Virus Type 1 RNA Dimerization on Viral Infectivity and of Stem-Loop B on RNA Dimerization and Reverse Transcription and Dissociation of Dimerization from Packaging

2000 ◽  
Vol 74 (12) ◽  
pp. 5729-5735 ◽  
Author(s):  
Ni Shen ◽  
Louis Jetté ◽  
Chen Liang ◽  
Mark A. Wainberg ◽  
Michael Laughrea
Keyword(s):  
Reverse Transcription ◽  
Viral Infectivity ◽  
Stem Loop ◽  
Rna Dimerization ◽  
Reduced Genome ◽  
Immunodeficiency Virus ◽  
Genome Dimerization ◽  
Hiv 1 ◽  
Kissing Loop

ABSTRACT The kissing-loop domain (KLD) encompasses a stem-loop, named kissing-loop or dimerization initiation site (DIS) hairpin (nucleotides [nt] 248 to 270 in the human immunodeficiency virus type 1 strains HIV-1Lai and HIV-1Hxb2), seated on top of a 12-nt stem-internal loop called stem-loop B (nt 243 to 247 and 271 to 277). Destroying stem-loop B reduced genome dimerization by ∼50% and proviral DNA synthesis by ∼85% and left unchanged the dissociation temperature of dimeric genomic RNA. The most affected step of reverse transcription was plus-strand DNA transfer, which was reduced by ∼80%. Deleting nt 241 to 256 or 200 to 256 did not reduce genome dimerization significantly more than the destruction of stem-loop B or the DIS hairpin. We conclude that the KLD is nonmodular: mutations in stem-loop B and in the DIS hairpin have similar effects on genome dimerization, reverse transcription, and encapsidation and are also “nonadditive”; i.e., a larger deletion spanning both of these structures has the same effects on genome dimerization and encapsidation as if stem-loop B strongly impacted DIS hairpin function and vice versa. A C258G transversion in the palindrome of the kissing-loop reduced genome dimerization by ∼50% and viral infectivity by ∼1.4 log. Two mutations, CGCG261→UUAA261 (creating a weaker palindrome) and a Δ241–256 suppressor mutation, were each able to reduce genome dimerization but leave genome packaging unaffected.

scholarly journals 5′-Cap sequestration is an essential determinant of HIV-1 genome packaging

2021 ◽  
Vol 118 (37) ◽  
pp. e2112475118
Author(s):  
Pengfei Ding ◽  
Siarhei Kharytonchyk ◽  
Nansen Kuo ◽  
Emily Cannistraci ◽  
Hana Flores ◽  
...  
Keyword(s):  
Rna Processing ◽  
Binding Sites ◽  
Rna Structure ◽  
Virus Assembly ◽  
Wild Type ◽  
Rna Packaging ◽  
Genome Packaging ◽  
Rna Dimerization ◽  
Gag Polyprotein ◽  
Hiv 1

HIV-1 selectively packages two copies of its 5′-capped RNA genome (gRNA) during virus assembly, a process mediated by the nucleocapsid (NC) domain of the viral Gag polyprotein and encapsidation signals located within the dimeric 5′ leader of the viral RNA. Although residues within the leader that promote packaging have been identified, the determinants of authentic packaging fidelity and efficiency remain unknown. Here, we show that a previously characterized 159-nt region of the leader that possesses all elements required for RNA dimerization, high-affinity NC binding, and packaging in a noncompetitive RNA packaging assay (ΨCES) is unexpectedly poorly packaged when assayed in competition with the intact 5′ leader. ΨCES lacks a 5′-tandem hairpin element that sequesters the 5′ cap, suggesting that cap sequestration may be important for packaging. Consistent with this hypothesis, mutations within the intact leader that expose the cap without disrupting RNA structure or NC binding abrogated RNA packaging, and genetic addition of a 5′ ribozyme to ΨCES to enable cotranscriptional shedding of the 5′ cap promoted ΨCES-mediated RNA packaging to wild-type levels. Additional mutations that either block dimerization or eliminate subsets of NC binding sites substantially attenuated competitive packaging. Our studies indicate that packaging is achieved by a bipartite mechanism that requires both sequestration of the 5′ cap and exposure of NC binding sites that reside fully within the ΨCES region of the dimeric leader. We speculate that cap sequestration prevents irreversible capture by the cellular RNA processing and translation machinery, a mechanism likely employed by other viruses that package 5′-capped RNA genomes.

Inhibition of Replication of HIV-1 at Both Early and Late Stages of the Viral Life Cycle by Single-Chain Antibody Against Viral Integrase

1999 ◽  
Vol 20 (2) ◽  
pp. 105-114 ◽  
Author(s):  
Yoshihiro Kitamura ◽  
Tetsuya Ishikawa ◽  
Nobuo Okui ◽  
Noriko Kobayashi ◽  
Tadahito Kanda ◽  
...  
Keyword(s):  
Life Cycle ◽  
Viral Life Cycle ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Late Stages ◽  
Hiv 1

scholarly journals Role of Stem B, Loop B, and Nucleotides next to the Primer Binding Site and the Kissing-Loop Domain in Human Immunodeficiency Virus Type 1 Replication and Genomic-RNA Dimerization

2001 ◽  
Vol 75 (21) ◽  
pp. 10543-10549 ◽  
Author(s):  
Ni Shen ◽  
Louis Jetté ◽  
Mark A. Wainberg ◽  
Michael Laughrea
Keyword(s):  
Human Immunodeficiency Virus ◽  
Viral Replication ◽  
Base Pair ◽  
Complete Genome ◽  
Genomic Rna ◽  
Rna Dimerization ◽  
Immunodeficiency Virus ◽  
Genome Dimerization ◽  
Hiv 1

ABSTRACT Stem-loop B is a 12-nucleotide [nt]-long completely conserved sequence postulated to form a 4-bp stem and a 4-nt internal loop under the kissing-loop hairpin (klh) (nt 248 to 270) of human immunodeficiency virus type 1 (HIV-1) genomic RNA. We investigated its role in viral replication, genomic RNA dimerization, and dimerization of partial HIV-1 RNA transcripts. The putative CUCG246-CGAG277 duplex was replaced by nine alternative complementary sequences, five likely to base pair only in short RNAs and four likely to base pair in long (∼500-nt) RNAs, as assessed by the algorithm mfold. Among the five former sequences, none preserved genome dimerization and all reduced viral replication by 98 to 99.9%. Among the four latter sequences, three (MB6, -9, and -10) preserved genome dimerization, one (MB7) did not significantly inhibit it, and two (MB9 and -10) preserved viral replication. We conclude that duplex formation by stem B nucleotides is necessary for viral infectivity and complete genome dimerization. Deleting the 5′ or 3′ side of loop B or of stem B had little impact on dimerization of partial RNA transcript and no impact on klh folding (and, for loop B mutations, on stem B folding), but each deletion inhibited genome dimerization almost as much as klh destruction. This suggests that loop B is required for complete genome dimerization and that loop B and stem B stimulate dimerization only in very long RNAs and/or in the presence of unidentified viral and cellular factors. Finally, we asked if nine deletions or nucleotide substitutions within nt 200 to 242 and/or nt 282 to 335 could influence genome dimerization. These mutations had intermediate inhibitory impacts consistent with their predicted influence on stem B, loop B, and klh formation. Two exceptions were Δ200–226 and Δ236–242 genomic RNAs, which dimerized relatively poorly despite having neutral or positive influences on stem B, loop B, and klh folding.

scholarly journals Minimal Region Sufficient for Genome Dimerization in the Human Immunodeficiency Virus Type 1 Virion and Its Potential Roles in the Early Stages of Viral Replication

2007 ◽  
Vol 81 (15) ◽  
pp. 7985-7992 ◽  
Author(s):  
Jun-ichi Sakuragi ◽  
Sayuri Sakuragi ◽  
Tatsuo Shioda
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Primer Binding Site ◽  
Early Stages ◽  
Immunodeficiency Virus ◽  
Rna Interaction ◽  
Genome Dimerization ◽  
Hiv 1

ABSTRACT It has been suggested that the dimer initiation site/dimer linkage sequence (DIS/DLS) region of the human immunodeficiency virus type 1 (HIV-1) RNA genome plays an important role at various stages of the viral life cycle. Recently we found that the duplication of the DIS/DLS region on viral RNA caused the production of partially monomeric RNAs in virions, indicating that this region indeed mediates RNA-RNA interaction. In this report, we followed up on this finding to identify the necessary and sufficient region for RNA dimerization in the virion of HIV-1. The region thus identified was 144 bases in length, extending from the junction of R/U5 and U5/L stem-loops to the end of SL4. The trans-acting responsive element, polyadenylation signal, primer binding site, upper stem-loop of U5/L, and SL2 were not needed for the function of this region. The insertion of this region into the ectopic location of the viral genome did not affect the level of virion production by transfection. However, the resultant virions contained monomerized genomes and showed drastic reductions in infectivity. A reduction was observed especially in the reverse transcription process. An attempt to generate a replication-competent virus with monomerized genome was performed by the long-term culture of mutant virus-infected cells. All recovered viruses were wild-type revertants, indicating a fatal defect of the mutation. These results suggest that genome dimerization or DIS/DLS itself also plays an important role in the early stages of virus infection.

scholarly journals Visualizing Rous Sarcoma Virus Genomic RNA Dimerization in the Nucleus, Cytoplasm, and at the Plasma Membrane

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 903
Author(s):  
Eunice C. Chen ◽  
Rebecca J. Kaddis Maldonado ◽  
Leslie J. Parent
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Rous Sarcoma Virus ◽  
Genomic Rna ◽  
Virus Particles ◽  
Rna Dimerization ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Genome Dimerization ◽  
Hiv 1

Retroviruses are unique in that they package their RNA genomes as non-covalently linked dimers. Failure to dimerize their genomes results in decreased infectivity and reduced packaging of genomic RNA into virus particles. Two models of retrovirus genome dimerization have been characterized: in murine leukemia virus (MLV), genomic RNA dimerization occurs co-transcriptionally in the nucleus, resulting in the preferential formation of genome homodimers; whereas in human immunodeficiency virus (HIV-1), genomic RNA dimerization occurs in the cytoplasm and at the plasma membrane, with a random distribution of heterodimers and homodimers. Although in vitro studies have identified the genomic RNA sequences that facilitate dimerization in Rous sarcoma virus (RSV), in vivo characterization of the location and preferences of genome dimerization has not been performed. In this study, we utilized three single molecule RNA imaging approaches to visualize genome dimers of RSV in cultured quail fibroblasts. The formation of genomic RNA heterodimers within cells was dependent on the presence of the dimerization initiation site (DIS) sequence in the L3 stem. Subcellular localization analysis revealed that heterodimers were present the nucleus, cytoplasm, and at the plasma membrane, indicating that genome dimers can form in the nucleus. Furthermore, single virion analysis revealed that RSV preferentially packages genome homodimers into virus particles. Therefore, the mechanism of RSV genomic RNA dimer formation appears more similar to MLV than HIV-1.

scholarly journals NMR detection of intermolecular interaction sites in the dimeric 5′-leader of the HIV-1 genome

2016 ◽  
Vol 113 (46) ◽  
pp. 13033-13038 ◽  
Author(s):  
Sarah C. Keane ◽  
Verna Van ◽  
Heather M. Frank ◽  
Carly A. Sciandra ◽  
Sayo McCowin ◽  
...  
Keyword(s):  
Interface Structure ◽  
Base Pairing ◽  
Rna Dimerization ◽  
Interaction Sites ◽  
Intermolecular Contacts ◽  
Hiv Type 1 ◽  
Rna Chaperones ◽  
Genome Dimerization ◽  
Hiv 1

HIV type-1 (HIV-1) contains a pseudodiploid RNA genome that is selected for packaging and maintained in virions as a noncovalently linked dimer. Genome dimerization is mediated by conserved elements within the 5′-leader of the RNA, including a palindromic dimer initiation signal (DIS) that has been proposed to form kissing hairpin and/or extended duplex intermolecular contacts. Here, we have applied a2H-edited NMR approach to directly probe for intermolecular interactions in the full-length, dimeric HIV-1 5′-leader (688 nucleotides; 230 kDa). The interface is extensive and includes DIS:DIS base pairing in an extended duplex state as well as intermolecular pairing between elements of the upstream Unique-5′ (U5) sequence and those near thegagstart site (AUG). Other pseudopalindromic regions of the leader, including the transcription activation (TAR), polyadenylation (PolyA), and primer binding (PBS) elements, do not participate in intermolecular base pairing. Using a2H-edited one-dimensional NMR approach, we also show that the extended interface structure forms on a time scale similar to that of overall RNA dimerization. Our studies indicate that a kissing dimer-mediated structure, if formed, exists only transiently and readily converts to the extended interface structure, even in the absence of the HIV-1 nucleocapsid protein or other RNA chaperones.

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