Perturbing HIV-1 ribosomal frameshifting frequency reveals a cis preference for Gag-Pol incorporation into assembling virions

HIV-1 virion production is driven by Gag and Gag-Pol (GP) proteins, with Gag forming the bulk of the capsid and driving budding while GP binds Gag to deliver the essential virion enzymes Protease, Reverse Transcriptase, and Integrase. Virion GP levels are traditionally thought to reflect the relative abundances of GP and Gag in cells (∼1:20), dictated by the frequency of a -1 programmed ribosomal frameshifting (PRF) event occurring in gag-pol mRNAs. Herein we exploited a panel of PRF mutant viruses to show that mechanisms in addition to PRF regulate GP incorporation into virions. First, we show that GP is enriched ∼3-fold in virions relative to cells, with viral infectivity better maintained at subphysiological levels of GP compared to when GP levels are too high. Second, we report that GP is more efficiently incorporated into virions when Gag and GP are synthesized in cis ( i.e., from the same gag-pol mRNA) relative to trans, suggesting that Gag/GP translation and assembly are spatially coupled processes. Third, we show that, surprisingly, virions exhibit a strong upper limit to trans -delivered GP incorporation; an adaptation that appears to allow the virus to temper defects to GP/Gag cleavage that may negatively impact reverse transcription. Taken together, we propose a “weighted Goldilocks” scenario for HIV-1 GP incorporation, wherein combined mechanisms of GP enrichment and exclusion buffer virion infectivity over a broad range of local GP concentrations. These results provide new insights into the HIV-1 virion assembly pathway relevant to the anticipated efficacy of PRF-targeted antiviral strategies. Importance HIV-1 infectivity requires incorporation of the Gag-Pol (GP) precursor polyprotein into virions during the process of virus particle assembly. Mechanisms dictating GP incorporation into assembling virions are poorly defined, with GP levels in virions traditionally thought to solely reflect relative levels of Gag and GP expressed in cells; dictated by the frequency of a -1 programmed ribosomal frameshifting (PRF) event that occurs in gag-pol mRNAs. Herein we provide experimental support for a “weighted Goldilocks” scenario for GP incorporation, wherein the virus exploits both random and non-random mechanisms to buffer infectivity over a wide range of GP expression levels. These mechanistic data are relevant to ongoing efforts to develop antiviral strategies targeting PRF frequency and/or HIV-1 virion maturation.

Download Full-text

Structural determinants of virion assembly and release in the C-terminus of the M-PMV capsid protein

Journal of Virology ◽

10.1128/jvi.00615-21 ◽

2021 ◽

Author(s):

Marlene V. Buckmaster ◽

Kaneil K. Zadrozny ◽

Barbie K. Ganser-Pornillos ◽

Owen Pornillos ◽

Stephen P. Goff

Keyword(s):

Capsid Protein ◽

Conformational Changes ◽

Structural Determinants ◽

Particle Assembly ◽

Gag Protein ◽

Virion Assembly ◽

C Terminus ◽

Hiv 1

The transition from an immature to a fully infectious mature retrovirus particle is associated with molecular switches that trigger dramatic conformational changes in the structure of the Gag proteins. A dominant maturation switch that stabilizes the immature capsid lattice is located downstream of the capsid (CA) protein in many retroviral Gags. The HIV-1 Gag contains a stretch of five amino acid residues termed the ‘clasp motif’, important for the organization of the hexameric subunits that provide stability to the overall immature HIV-1 shell. Sequence alignment of the CA C-terminal domains (CTDs) of the HIV-1 and Mason-Pfizer Monkey Virus (M-PMV) highlighted a spacer-like domain in M-PMV that may provide comparable function. The importance of the sequences spanning the CA-NC cleavage has been demonstrated by mutagenesis, but the specific requirements for the clasp motif in several steps of M-PMV particle assembly and maturation have not been determined in detail. In the present study we report an examination of the role of the clasp motif in the M-PMV life cycle. We generated a series of M-PMV Gag mutants and assayed for assembly of the recombinant protein in vitro , and for the assembly, maturation, release, genomic RNA packaging, and infectivity of the mutant virus in vivo . The mutants revealed major defects in virion assembly and release in 293T and HeLa cells, and even larger defects in infectivity. Our data identifies the clasp motif as a fundamental contributor to CA-CTD interactions necessary for efficient viral infection. Importance The C-terminal domain of the capsid protein of many retroviruses has been shown to be critical for virion assembly and maturation, but the functions of this region of M-PMV are uncertain. We show that a short ‘clasp’ motif in the capsid domain of the M-PMV Gag protein plays a key role in M-PMV virion assembly, genome packaging, and infectivity.

Download Full-text

Ex VivoBioactivity and HIV-1 Latency Reversal by Ingenol Dibenzoate and Panobinostat in Resting CD4+T Cells from Aviremic Patients

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01077-15 ◽

2015 ◽

Vol 59 (10) ◽

pp. 5984-5991 ◽

Cited By ~ 53

Author(s):

Adam M. Spivak ◽

Alberto Bosque ◽

Alfred H. Balch ◽

David Smyth ◽

Laura Martins ◽

...

Keyword(s):

T Cells ◽

Protein Kinase C ◽

Protein Kinase ◽

Ex Vivo ◽

Viral Reactivation ◽

Major Barrier ◽

Kinase C ◽

Wide Range ◽

Hiv 1 ◽

In Cells

ABSTRACTThe human immunodeficiency virus type 1 (HIV-1) latent reservoir in resting CD4+T cells represents a major barrier to viral eradication. Small compounds capable of latency reversal have not demonstrated uniform responses acrossin vitroHIV-1 latency cell models. Characterizing compounds that demonstrate latency-reversing activity in resting CD4+T cells from aviremic patientsex vivowill help inform pilot clinical trials aimed at HIV-1 eradication. We have optimized a rapidex vivoassay using resting CD4+T cells from aviremic HIV-1+patients to evaluate both the bioactivity and latency-reversing potential of candidate latency-reversing agents (LRAs). Using this assay, we characterize the properties of two candidate compounds from promising LRA classes, ingenol 3,20-dibenzoate (a protein kinase C agonist) and panobinostat (a histone deacetylase inhibitor), in cells from HIV-1+antiretroviral therapy (ART)-treated aviremic participants, including the effects on cellular activation and cytotoxicity. Ingenol induced viral release at levels similar to those of the positive control (CD3/28 receptor stimulation) in cells from a majority of participants and represents an exciting LRA candidate, as it combines a robust viral reactivation potential with a low toxicity profile. At concentrations that blocked histone deacetylation, panobinostat displayed a wide range of potency among participant samples and consistently induced significant levels of apoptosis. The protein kinase C agonist ingenol 3,20-dibenzoate demonstrated significant promise in a rapidex vivoassay using resting CD4+T cells from treated HIV-1-positive patients to measure latent HIV-1 reactivation.

Download Full-text

Subcellular Localization of HIV-1 gag-pol mRNAs Regulates Sites of Virion Assembly

Journal of Virology ◽

10.1128/jvi.02315-16 ◽

2017 ◽

Vol 91 (6) ◽

Cited By ~ 20

Author(s):

Jordan T. Becker ◽

Nathan M. Sherer

Keyword(s):

Particle Production ◽

Full Length ◽

Viral Mrnas ◽

Viral Rnas ◽

Virion Assembly ◽

Virion Production ◽

Immunodeficiency Virus ◽

Hiv 1 ◽

Virus Particle Production

ABSTRACT Full-length unspliced human immunodeficiency virus type 1 (HIV-1) RNAs serve dual roles in the cytoplasm as mRNAs encoding the Gag and Gag-Pol capsid proteins as well as genomic RNAs (gRNAs) packaged by Gag into virions undergoing assembly at the plasma membrane (PM). Because Gag is sufficient to drive the assembly of virus-like particles even in the absence of gRNA binding, whether viral RNA trafficking plays an active role in the native assembly pathway is unknown. In this study, we tested the effects of modulating the cytoplasmic abundance or distribution of full-length viral RNAs on Gag trafficking and assembly in the context of single cells. Increasing full-length viral RNA abundance or distribution had little-to-no net effect on Gag assembly competency when provided in trans. In contrast, artificially tethering full-length viral RNAs or surrogate gag-pol mRNAs competent for Gag synthesis to non-PM membranes or the actin cytoskeleton severely reduced net virus particle production. These effects were explained, in large part, by RNA-directed changes to Gag's distribution in the cytoplasm, yielding aberrant subcellular sites of virion assembly. Interestingly, RNA-dependent disruption of Gag trafficking required either of two cis-acting RNA regulatory elements: the 5′ packaging signal (Psi) bound by Gag during genome encapsidation or, unexpectedly, the Rev response element (RRE), which regulates the nuclear export of gRNAs and other intron-retaining viral RNAs. Taken together, these data support a model for native infection wherein structural features of the gag-pol mRNA actively compartmentalize Gag to preferred sites within the cytoplasm and/or PM. IMPORTANCE The spatial distribution of viral mRNAs within the cytoplasm can be a crucial determinant of efficient translation and successful virion production. Here we provide direct evidence that mRNA subcellular trafficking plays an important role in regulating the assembly of human immunodeficiency virus type 1 (HIV-1) virus particles at the plasma membrane (PM). Artificially tethering viral mRNAs encoding Gag capsid proteins (gag-pol mRNAs) to distinct non-PM subcellular locales, such as cytoplasmic vesicles or the actin cytoskeleton, markedly alters Gag subcellular distribution, relocates sites of assembly, and reduces net virus particle production. These observations support a model for native HIV-1 assembly wherein HIV-1 gag-pol mRNA localization helps to confine interactions between Gag, viral RNAs, and host determinants in order to ensure virion production at the right place and right time. Direct perturbation of HIV-1 mRNA subcellular localization may represent a novel antiviral strategy.

Download Full-text

Interactions between HIV-1 Gag and Viral RNA Genome Enhance Virion Assembly

Journal of Virology ◽

10.1128/jvi.02319-16 ◽

2017 ◽

Vol 91 (16) ◽

Cited By ~ 13

Author(s):

Kari A. Dilley ◽

Olga A. Nikolaitchik ◽

Andrea Galli ◽

Ryan C. Burdick ◽

Louis Levine ◽

...

Keyword(s):

Particle Production ◽

High Efficiency ◽

Virus Assembly ◽

Viral Rna ◽

Structural Protein ◽

Genome Packaging ◽

Virion Assembly ◽

Rna Genome ◽

Hiv 1 ◽

In Cells

ABSTRACT Most HIV-1 virions contain two copies of full-length viral RNA, indicating that genome packaging is efficient and tightly regulated. However, the structural protein Gag is the only component required for the assembly of noninfectious viruslike particles, and the viral RNA is dispensable in this process. The mechanism that allows HIV-1 to achieve such high efficiency of genome packaging when a packageable viral RNA is not required for virus assembly is currently unknown. In this report, we examined the role of HIV-1 RNA in virus assembly and found that packageable HIV-1 RNA enhances particle production when Gag is expressed at levels similar to those in cells containing one provirus. However, such enhancement is diminished when Gag is overexpressed, suggesting that the effects of viral RNA can be replaced by increased Gag concentration in cells. We also showed that the specific interactions between Gag and viral RNA are required for the enhancement of particle production. Taken together, these studies are consistent with our previous hypothesis that specific dimeric viral RNA-Gag interactions are the nucleation event of infectious virion assembly, ensuring that one RNA dimer is packaged into each nascent virion. These studies shed light on the mechanism by which HIV-1 achieves efficient genome packaging during virus assembly. IMPORTANCE Retrovirus assembly is a well-choreographed event, during which many viral and cellular components come together to generate infectious virions. The viral RNA genome carries the genetic information to new host cells, providing instructions to generate new virions, and therefore is essential for virion infectivity. In this report, we show that the specific interaction of the viral RNA genome with the structural protein Gag facilitates virion assembly and particle production. These findings resolve the conundrum that HIV-1 RNA is selectively packaged into virions with high efficiency despite being dispensable for virion assembly. Understanding the mechanism used by HIV-1 to ensure genome packaging provides significant insights into viral assembly and replication.

Download Full-text

Importance of Ribosomal Frameshifting for Human Immunodeficiency Virus Type 1 Particle Assembly and Replication

Journal of Virology ◽

10.1128/jvi.72.6.4819-4824.1998 ◽

1998 ◽

Vol 72 (6) ◽

pp. 4819-4824 ◽

Cited By ~ 68

Author(s):

Magdeleine Hung ◽

Pratiksha Patel ◽

Susan Davis ◽

Simon R. Green

Keyword(s):

Human Immunodeficiency Virus ◽

Combination Therapy ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Particle Assembly ◽

Ribosomal Frameshifting ◽

Immunodeficiency Virus ◽

Viral Particle Assembly ◽

Hiv 1

ABSTRACT The recent development and use of protease inhibitors have demonstrated the essential role that combination therapy will play in the treatment of individuals infected with the human immunodeficiency virus type 1 (HIV-1). Past clinical experience suggests that due to the appearance of resistant HIV-1 variants, additional therapeutics will be required in the future. To identify new options for combination therapy, it is of paramount importance to pursue novel targets for drug development. Ribosomal frameshifting is one potential target that has not been fully explored. Data presented here demonstrate that small molecules can stimulate frameshifting, leading to an imbalance in the ratio of Gag to Gag-Pol and inhibiting HIV-1 replication at what appears to be the point of viral particle assembly. Thus, we propose that frameshifting represents a new target for the identification of novel anti-HIV-1 therapeutics.

Download Full-text

1592U89, a novel carbocyclic nucleoside analog with potent, selective anti-human immunodeficiency virus activity.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.5.1082 ◽

1997 ◽

Vol 41 (5) ◽

pp. 1082-1093 ◽

Cited By ~ 276

Author(s):

S M Daluge ◽

S S Good ◽

M B Faletto ◽

W H Miller ◽

M H St Clair ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Oral Bioavailability ◽

Human Peripheral Blood ◽

Cross Resistance ◽

Immunodeficiency Virus ◽

Carbocyclic Nucleoside ◽

Hiv 1 ◽

In Cells

1592U89, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo pentene-1-methanol, is a carbocyclic nucleoside with a unique biological profile giving potent, selective anti-human immunodeficiency virus (HIV) activity. 1592U89 was selected after evaluation of a wide variety of analogs containing a cyclopentene substitution for the 2'-deoxyriboside of natural deoxynucleosides, optimizing in vitro anti-HIV potency, oral bioavailability, and central nervous system (CNS) penetration. 1592U89 was equivalent in potency to 3'-azido-3'-deoxythymidine (AZT) in human peripheral blood lymphocyte (PBL) cultures against clinical isolates of HIV type 1 (HIV-1) from antiretroviral drug-naive patients (average 50% inhibitory concentration [IC50], 0.26 microM for 1592U89 and 0.23 microM for AZT). 1592U89 showed minimal cross-resistance (approximately twofold) with AZT and other approved HIV reverse transcriptase (RT) inhibitors. 1592U89 was synergistic in combination with AZT, the nonnucleoside RT inhibitor nevirapine, and the protease inhibitor 141W94 in MT4 cells against HIV-1 (IIIB). 1592U89 was anabolized intracellularly to its 5'-monophosphate in CD4+ CEM cells and in PBLs, but the di- and triphosphates of 1592U89 were not detected. The only triphosphate found in cells incubated with 1592U89 was that of the guanine analog (-)-carbovir (CBV). However, the in vivo pharmacokinetic, distribution, and toxicological profiles of 1592U89 were distinct from and improved over those of CBV, probably because CBV itself was not appreciably formed from 1592U89 in cells or animals (<2%). The 5'-triphosphate of CBV was a potent, selective inhibitor of HIV-1 RT, with Ki values for DNA polymerases (alpha, beta, gamma, and epsilon which were 90-, 2,900-, 1,200-, and 1,900-fold greater, respectively, than for RT (Ki, 21 nM). 1592U89 was relatively nontoxic to human bone marrow progenitors erythroid burst-forming unit and granulocyte-macrophage CFU (IC50s, 110 microM) and human leukemic and liver tumor cell lines. 1592U89 had excellent oral bioavailability (105% in the rat) and penetrated the CNS (rat brain and monkey cerebrospinal fluid) as well as AZT. Having demonstrated an excellent preclinical profile, 1592U89 has progressed to clinical evaluation in HIV-infected patients.

Download Full-text

HIV-1 Vpr activates host CRL4-DCAF1 E3 ligase to degrade histone deacetylase SIRT7

Virology Journal ◽

10.1186/s12985-021-01514-2 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Xiaohong Zhou ◽

Christina Monnie ◽

Maria DeLucia ◽

Jinwoo Ahn

Keyword(s):

Cell Cycle ◽

Dna Repair ◽

Cell Cycle Arrest ◽

Histone Deacetylase ◽

E3 Ligase ◽

Cycle Arrest ◽

Wide Range ◽

In Vitro Reconstitution ◽

Hiv 1

Abstract Background Vpr is a virion-associated protein that is encoded by lentiviruses and serves to counteract intrinsic immunity factors that restrict infection. HIV-1 Vpr mediates proteasome-dependent degradation of several DNA repair/modification proteins. Mechanistically, Vpr directly recruits cellular targets onto DCAF1, a substrate receptor of Cullin 4 RING E3 ubiquitin ligase (CRL4) for poly-ubiquitination. Further, Vpr can mediate poly-ubiquitination of DCAF1-interacting proteins by the CRL4. Because Vpr-mediated degradation of its known targets can not explain the primary cell-cycle arrest phenotype that Vpr expression induces, we surveyed the literature for DNA-repair-associated proteins that interact with the CRL4-DCAF1. One such protein is SIRT7, a deacetylase of histone 3 that belongs to the Sirtuin family and regulates a wide range of cellular processes. We wondered whether Vpr can mediate degradation of SIRT7 via the CRL4-DCAF1. Methods HEK293T cells were transfected with cocktails of plasmids expressing DCAF1, DDB1, SIRT7 and Vpr. Ectopic and endogeneous levels of SIRT7 were monitered by immunoblotting and protein–protein interactions were assessed by immunoprecipitation. For in vitro reconstitution assays, recombinant CRL4-DCAF1-Vpr complexes and SIRT7 were prepared and poly-ubiqutination of SIRT7 was monitored with immunoblotting. Results We demonstrate SIRT7 polyubiquitination and degradation upon Vpr expression. Specifically, SIRT7 is shown to interact with the CRL4-DCAF1 complex, and expression of Vpr in HEK293T cells results in SIRT7 degradation, which is partially rescued by CRL inhibitor MNL4924 and proteasome inhibitor MG132. Further, in vitro reconstitution assays show that Vpr induces poly-ubiquitination of SIRT7 by the CRL4-DCAF1. Importantly, we find that Vpr from several different HIV-1 strains, but not HIV-2 strains, mediates SIRT7 poly-ubiquitination in the reconstitution assay and degradation in cells. Finally, we show that SIRT7 degradation by Vpr is independent of the known, distinctive phenotype of Vpr-induced cell cycle arrest at the G2 phase, Conclusions Targeting histone deacetylase SIRT7 for degradation is a conserved feature of HIV-1 Vpr. Altogether, our findings reveal that HIV-1 Vpr mediates down-regulation of SIRT7 by a mechanism that does not involve novel target recruitment to the CRL4-DCAF1 but instead involves regulation of the E3 ligase activity.

Download Full-text

Analysis of Human Immunodeficiency Virus Type 1 Gag Dimerization-Induced Assembly

Journal of Virology ◽

10.1128/jvi.79.23.14498-14506.2005 ◽

2005 ◽

Vol 79 (23) ◽

pp. 14498-14506 ◽

Cited By ~ 32

Author(s):

Ayna Alfadhli ◽

Tenzin Choesang Dhenub ◽

Amelia Still ◽

Eric Barklis

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Protein Assembly ◽

Particle Assembly ◽

Gag Protein ◽

Immunodeficiency Virus ◽

Hiv 1 ◽

Terminal Domains

ABSTRACT The nucleocapsid (NC) domains of retrovirus precursor Gag (PrGag) proteins play an essential role in virus assembly. Evidence suggests that NC binding to viral RNA promotes dimerization of PrGag capsid (CA) domains, which triggers assembly of CA N-terminal domains (NTDs) into hexamer rings that are interconnected by CA C-terminal domains. To examine the influence of dimerization on human immunodeficiency virus type 1 (HIV-1) Gag protein assembly in vitro, we analyzed the assembly properties of Gag proteins in which NC domains were replaced with cysteine residues that could be linked via chemical treatment. In accordance with the model that Gag protein pairing triggers assembly, we found that cysteine cross-linking or oxidation reagents induced the assembly of virus-like particles. However, efficient assembly also was observed to be temperature dependent or required the tethering of NTDs. Our results suggest a multistep pathway for HIV-1 Gag protein assembly. In the first step, Gag protein pairing through NC-RNA interactions or C-terminal cysteine linkage fosters dimerization. Next, a conformational change converts assembly-restricted dimers or small oligomers into assembly-competent ones. At the final stage, final particle assembly occurs, possibly through a set of larger intermediates.

Download Full-text