Distribution and Redistribution of HIV-1 Nucleocapsid Protein in Immature, Mature, and Integrase-Inhibited Virions: a Role for Integrase in Maturation

ABSTRACTDuring virion maturation, HIV-1 capsid protein assembles into a conical core containing the viral ribonucleoprotein (vRNP) complex, thought to be composed mainly of the viral RNA and nucleocapsid protein (NC). After infection, the viral RNA is reverse transcribed into double-stranded DNA, which is then incorporated into host chromosomes by integrase (IN) catalysis. Certain IN mutations (class II) and antiviral drugs (allosteric IN inhibitors [ALLINIs]) adversely affect maturation, resulting in virions that contain “eccentric condensates,” electron-dense aggregates located outside seemingly empty capsids. Here we demonstrate that in addition to this mislocalization of electron density, a class II IN mutation and ALLINIs each increase the fraction of virions with malformed capsids (from ∼12% to ∼53%). Eccentric condensates have a high NC content, as demonstrated by “tomo-bubblegram” imaging, a novel labeling technique that exploits the susceptibility of NC to radiation damage. Tomo-bubblegrams also localized NC inside wild-type cores and lining the spherical Gag shell in immature virions. We conclude that eccentric condensates represent nonpackaged vRNPs and that either genetic or pharmacological inhibition of IN can impair vRNP incorporation into mature cores. Supplying IN intransas part of a Vpr-IN fusion protein partially restored the formation of conical cores with internal electron density and the infectivity of a class II IN deletion mutant virus. Moreover, the ability of ALLINIs to induce eccentric condensate formation required both IN and viral RNA. Based on these observations, we propose a role for IN in initiating core morphogenesis and vRNP incorporation into the mature core during HIV-1 maturation.IMPORTANCEMaturation, a process essential for HIV-1 infectivity, involves core assembly, whereby the viral ribonucleoprotein (vRNP, composed of vRNA and nucleocapsid protein [NC]) is packaged into a conical capsid. Allosteric integrase inhibitors (ALLINIs) affect multiple viral processes. We have characterized ALLINIs and integrase mutants that have the same phenotype. First, by comparing the effects of ALLINIs on several steps of the viral cycle, we show that inhibition of maturation accounts for compound potency. Second, by using cryoelectron tomography, we find that ALLINIs impair conical capsid assembly. Third, by developing tomo-bubblegram imaging, which specifically labels NC protein, we find that ALLINIs block vRNP packaging; instead, vRNPs form “eccentric condensates” outside the core. Fourth, malformed cores, typical of integrase-deleted virus, are partially replaced by conical cores when integrase is supplied intrans. Fifth, vRNA is necessary for ALLINI-induced eccentric condensate formation. These observations suggest that integrase is involved in capsid morphogenesis and vRNP packaging.

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Zinc Finger Structures in the Human Immunodeficiency Virus Type 1 Nucleocapsid Protein Facilitate Efficient Minus- and Plus-Strand Transfer

Journal of Virology ◽

10.1128/jvi.74.19.8980-8988.2000 ◽

2000 ◽

Vol 74 (19) ◽

pp. 8980-8988 ◽

Cited By ~ 143

Author(s):

Jianhui Guo ◽

Tiyun Wu ◽

Jada Anderson ◽

Bradley F. Kane ◽

Donald G. Johnson ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Zinc Finger ◽

Nucleocapsid Protein ◽

Zinc Fingers ◽

Strand Transfer ◽

Minus Strand ◽

Wild Type ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) has two zinc fingers, each containing the invariant metal ion binding residues CCHC. Recent reports indicate that mutations in the CCHC motifs are deleterious for reverse transcription in vivo. To identify reverse transcriptase (RT) reactions affected by such changes, we have probed zinc finger functions in NC-dependent RT-catalyzed HIV-1 minus- and plus-strand transfer model systems. Our approach was to examine the activities of wild-type NC and a mutant in which all six cysteine residues were replaced by serine (SSHS NC); this mutation severely disrupts zinc coordination. We find that the zinc fingers contribute to the role of NC in complete tRNA primer removal from minus-strand DNA during plus-strand transfer. Annealing of the primer binding site sequences in plus-strand strong-stop DNA [(+) SSDNA] to its complement in minus-strand acceptor DNA is not dependent on NC zinc fingers. In contrast, the rate of annealing of the complementary R regions in (−) SSDNA and 3′ viral RNA during minus-strand transfer is approximately eightfold lower when SSHS NC is used in place of wild-type NC. Moreover, unlike wild-type NC, SSHS NC has only a small stimulatory effect on minus-strand transfer and is essentially unable to block TAR-induced self-priming from (−) SSDNA. Our results strongly suggest that NC zinc finger structures are needed to unfold highly structured RNA and DNA strand transfer intermediates. Thus, it appears that in these cases, zinc finger interactions are important components of NC nucleic acid chaperone activity.

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Integrase-RNA interactions underscore the critical role of integrase in HIV-1 virion morphogenesis

10.1101/2019.12.18.881649 ◽

2019 ◽

Cited By ~ 1

Author(s):

Jennifer Elliott ◽

Jenna E. Eschbach ◽

Pratibha C. Koneru ◽

Wen Li ◽

Maritza Puray Chavez ◽

...

Keyword(s):

Rna Binding ◽

Critical Role ◽

Underlying Mechanism ◽

Class Ii ◽

Viral Rna ◽

Target Cells ◽

Ribonucleoprotein Complexes ◽

Virion Morphogenesis ◽

Hiv 1

ABSTRACTA large number of HIV-1 integrase (IN) alterations, referred to as class II substitutions, exhibit pleotropic effects during virus replication. However, the underlying mechanism for the class II phenotype is not known. Here we demonstrate that all tested class II IN substitutions compromised IN-RNA binding in virions by one of three distinct mechanisms: i) markedly reducing IN levels thus precluding formation of IN complexes with viral RNA; ii) adversely affecting functional IN multimerization and consequently impairing IN binding to viral RNA; iii) directly compromising IN-RNA interactions without substantially affecting IN levels or functional IN multimerization. Inhibition of IN-RNA interactions resulted in mislocalization of the viral ribonucleoprotein complexes outside the capsid lattice, which led to premature degradation of the viral genome and IN in target cells. Collectively, our studies uncover causal mechanisms for the class II phenotype and highlight an essential role of IN-RNA interactions for accurate virion maturation.

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Allosteric HIV-1 Integrase Inhibitors Lead to Premature Degradation of the Viral RNA Genome and Integrase in Target Cells

Journal of Virology ◽

10.1128/jvi.00821-17 ◽

2017 ◽

Vol 91 (17) ◽

Cited By ~ 15

Author(s):

Michaela K. Madison ◽

Dana Q. Lawson ◽

Jennifer Elliott ◽

Ayşe Naz Ozantürk ◽

Pratibha C. Koneru ◽

...

Keyword(s):

Reverse Transcriptase ◽

Reverse Transcription ◽

Hexagonal Lattice ◽

Viral Rna ◽

Productive Infection ◽

Target Cells ◽

Integrase Inhibitors ◽

Ribonucleoprotein Complexes ◽

Rna Genome ◽

Hiv 1

ABSTRACT Recent evidence indicates that inhibition of HIV-1 integrase (IN) binding to the viral RNA genome by allosteric integrase inhibitors (ALLINIs) or through mutations within IN yields aberrant particles in which the viral ribonucleoprotein complexes (vRNPs) are eccentrically localized outside the capsid lattice. These particles are noninfectious and are blocked at an early reverse transcription stage in target cells. However, the basis of this reverse transcription defect is unknown. Here, we show that the viral RNA genome and IN from ALLINI-treated virions are prematurely degraded in target cells, whereas reverse transcriptase remains active and stably associated with the capsid lattice. The aberrantly shaped cores in ALLINI-treated particles can efficiently saturate and be degraded by a restricting TRIM5 protein, indicating that they are still composed of capsid proteins arranged in a hexagonal lattice. Notably, the fates of viral core components follow a similar pattern in cells infected with eccentric particles generated by mutations within IN that inhibit its binding to the viral RNA genome. We propose that IN-RNA interactions allow packaging of both the viral RNA genome and IN within the protective capsid lattice to ensure subsequent reverse transcription and productive infection in target cells. Conversely, disruption of these interactions by ALLINIs or mutations in IN leads to premature degradation of both the viral RNA genome and IN, as well as the spatial separation of reverse transcriptase from the viral genome during early steps of infection. IMPORTANCE Recent evidence indicates that HIV-1 integrase (IN) plays a key role during particle maturation by binding to the viral RNA genome. Inhibition of IN-RNA interactions yields aberrant particles with the viral ribonucleoprotein complexes (vRNPs) eccentrically localized outside the conical capsid lattice. Although these particles contain all of the components necessary for reverse transcription, they are blocked at an early reverse transcription stage in target cells. To explain the basis of this defect, we tracked the fates of multiple viral components in infected cells. Here, we show that the viral RNA genome and IN in eccentric particles are prematurely degraded, whereas reverse transcriptase remains active and stably associated within the capsid lattice. We propose that IN-RNA interactions ensure the packaging of both vRNPs and IN within the protective capsid cores to facilitate subsequent reverse transcription and productive infection in target cells.

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Emergence of compensatory mutations reveal the importance of electrostatic interactions between HIV-1 integrase and genomic RNA

10.1101/2021.12.16.472884 ◽

2021 ◽

Author(s):

Christian Shema Mugisha ◽

Tung Dinh ◽

Kasyap Tenneti ◽

Jenna Eve Eschbach ◽

Keanu Davis ◽

...

Keyword(s):

Electrostatic Interactions ◽

Rna Binding ◽

Class Ii ◽

Integrase Inhibitors ◽

Ribonucleoprotein Complexes ◽

Compensatory Mutations ◽

Insight Into ◽

Hiv 1 ◽

Molecular Nature

Independent of its catalytic activity, HIV-1 integrase (IN) enzyme regulates proper particle maturation by binding to and packaging the viral RNA genome (gRNA) inside the mature capsid lattice. Allosteric integrase inhibitors (ALLINIs) and class II IN substitutions inhibit the binding of IN to the gRNA and cause the formation of non-infectious virions characterized by mislocalization of the viral ribonucleoprotein complexes between the translucent conical capsid lattice and the viral lipid envelope. To gain insight into the molecular nature of IN-gRNA interactions, we have isolated compensatory substitutions in the background of a class II IN (R269A/K273A) variant that directly inhibits IN binding to the gRNA. We found that additional D256N and D270N substitutions in the C-terminal domain (CTD) of IN restored its ability to bind gRNA and led to the formation of infectious particles with correctly matured morphology. Furthermore, reinstating the overall positive electrostatic potential of the CTD through individual D256R or D256K substitutions was sufficient to restore IN-RNA binding and infectivity for the R269A/K273A as well as the R262A/R263A class II IN mutants. The compensatory mutations did not impact functional IN oligomerization, suggesting that they directly contributed to IN binding to the gRNA. Interestingly, HIV-1 IN R269A/K273A, but not IN R262A/R263A, bearing compensatory mutations was more sensitive to ALLINIs providing key genetic evidence that specific IN residues required for RNA binding also influence ALLINI activity. Structural modeling provided further insight into the molecular nature of IN-gRNA interactions and ALLINI mechanism of action. Taken together, our findings highlight an essential role of IN-gRNA interactions for proper virion maturation and reveal the importance of electrostatic interactions between the IN CTD and the gRNA.

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Integrase-RNA interactions underscore the critical role of integrase in HIV-1 virion morphogenesis

eLife ◽

10.7554/elife.54311 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 2

Author(s):

Jennifer L Elliott ◽

Jenna E Eschbach ◽

Pratibha C Koneru ◽

Wen Li ◽

Maritza Puray-Chavez ◽

...

Keyword(s):

Rna Binding ◽

Critical Role ◽

Underlying Mechanism ◽

Class Ii ◽

Viral Rna ◽

Target Cells ◽

Ribonucleoprotein Complexes ◽

Immunodeficiency Virus ◽

Hiv 1

A large number of human immunodeficiency virus 1 (HIV-1) integrase (IN) alterations, referred to as class II substitutions, exhibit pleiotropic effects during virus replication. However, the underlying mechanism for the class II phenotype is not known. Here we demonstrate that all tested class II IN substitutions compromised IN-RNA binding in virions by one of the three distinct mechanisms: (i) markedly reducing IN levels thus precluding the formation of IN complexes with viral RNA; (ii) adversely affecting functional IN multimerization and consequently impairing IN binding to viral RNA; and (iii) directly compromising IN-RNA interactions without substantially affecting IN levels or functional IN multimerization. Inhibition of IN-RNA interactions resulted in the mislocalization of viral ribonucleoprotein complexes outside the capsid lattice, which led to premature degradation of the viral genome and IN in target cells. Collectively, our studies uncover causal mechanisms for the class II phenotype and highlight an essential role of IN-RNA interactions for accurate virion maturation.

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Identification of Capsid Mutations That Alter the Rate of HIV-1 Uncoating in Infected Cells

Journal of Virology ◽

10.1128/jvi.03043-14 ◽

2014 ◽

Vol 89 (1) ◽

pp. 643-651 ◽

Cited By ~ 32

Author(s):

Amy E. Hulme ◽

Z Kelley ◽

Eneniziaogochukwu A. Okocha ◽

Thomas J. Hope

Keyword(s):

Cell Lines ◽

Reverse Transcription ◽

Viral Rna ◽

Wild Type Virus ◽

Wild Type ◽

Cellular Environment ◽

Cellular Factors ◽

Infected Cells ◽

Kinetics Of ◽

Hiv 1

ABSTRACTAfter viral fusion with the cell membrane, the conical capsid of HIV-1 disassembles by a process called uncoating. We recently utilized the cyclosporine (CsA) washout assay, in which TRIM-CypA-mediated restriction of viral replication is used to detect the state of the viral capsid, to study the kinetics of uncoating in HIV-1-infected cells. Here we have extended this analysis to examine the effects of p24 capsid protein (p24CA) mutations and cellular environment on the kinetics of uncoating in infected cells. We found that p24CAmutations can significantly increase (A92E), delay (E45A and N74D), or have no effect (G94D) on the rate of uncoating and that these alterations are not due to changes in reverse transcription. Inhibition of reverse transcription delayed uncoating kinetics to an extent similar to that of the wild-type virus with all the p24CAmutant viruses tested. In addition, we observed differences in uncoating in two cell lines, which suggests that the cellular environment can differentially impact the disassembly of wild-type and mutant capsids. Collectively, these experiments suggest that viral and cellular factors are important for the process of uncoating. Finally, these data support the model whereby early steps in reverse transcription facilitate HIV-1 uncoating.IMPORTANCEThe HIV-1 capsid is a cone-shaped structure, composed of the HIV-1-encoded protein p24CA, which contains the viral RNA and other proteins needed for infection. After the virus enters a target cell, this capsid must disassemble by a process called uncoating. Uncoating is required for HIV-1 infection to progress, but the details of how this process occurs is not known. In this study, we used anin vivoassay to examine the uncoating process in HIV-1-infected cells. We determined that p24CAmutations could increase or decrease the rate of uncoating and that this rate varied in different cell lines. We also found that reverse transcription of the viral RNA altered the process of uncoating before the p24CAmutations. Collectively, these experiments provide a better understanding of how viral and cellular factors are involved with a poorly understood step in HIV-1 infection.

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Mutations in Human Immunodeficiency Virus Type 1 Nucleocapsid Protein Zinc Fingers Cause Premature Reverse Transcription

Journal of Virology ◽

10.1128/jvi.00583-08 ◽

2008 ◽

Vol 82 (19) ◽

pp. 9318-9328 ◽

Cited By ~ 32

Author(s):

James A. Thomas ◽

William J. Bosche ◽

Teresa L. Shatzer ◽

Donald G. Johnson ◽

Robert J. Gorelick

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Reverse Transcription ◽

Nucleocapsid Protein ◽

Productive Infection ◽

Wild Type ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) requires that its genome be reverse transcribed into double-stranded DNA for productive infection of cells. This process requires not only reverse transcriptase but also the nucleocapsid protein (NC), which functions as a nucleic acid chaperone. Reverse transcription generally begins once the core of the virion enters the cytoplasm of a newly infected cell. However, some groups have reported the presence of low levels of viral DNA (vDNA) within particles prior to infection, the significance and function of which is controversial. We report here that several HIV-1 NC mutants, which we previously identified as being replication defective, contain abnormally high levels of intravirion DNA. These findings were further reinforced by the inability of these NC mutants to perform endogenous reverse transcription (ERT), in contrast to the readily measurable ERT activity in wild-type HIV-1. When either of the NC mutations is combined with a mutation that inactivates the viral protease, we observed a significant reduction in the amount of intravirion DNA. Interestingly, we also observed high levels of intravirion DNA in the context of wild-type NC when we delayed budding by means of a PTAP(−) (Pro-Thr-Ala-Pro) mutation. Premature reverse transcription is most probably occurring before these mutant virions bud from producer cells, but we fail to see any evidence that the NC mutations alter the timing of Pr55Gag processing. Critically, our results also suggest that the presence of intravirion vDNA could serve as a diagnostic for identifying replication-defective HIV-1.

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Structure-Based Identification of HIV-1 Nucleocapsid Protein Inhibitors Active against Wild-Type and Drug-Resistant HIV-1 Strains

ACS Chemical Biology ◽

10.1021/acschembio.7b00907 ◽

2017 ◽

Vol 13 (1) ◽

pp. 253-266 ◽

Cited By ~ 7

Author(s):

Mattia Mori ◽

Lesia Kovalenko ◽

Savina Malancona ◽

Francesco Saladini ◽

Davide De Forni ◽

...

Keyword(s):

Nucleocapsid Protein ◽

Drug Resistant ◽

Wild Type ◽

Protein Inhibitors ◽

Hiv 1

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Molecular and genetic characterization of natural variants of HIV-1 Nef gene from North India and its functional implication in down-regulation of MHC-I and CD-4

Current HIV Research ◽

10.2174/1570162x18666200925160755 ◽

2020 ◽

Vol 18 ◽

Author(s):

J. Singh ◽

L. Ronsard ◽

M. Pandey ◽

R. Kapoor ◽

V.G. Ramachandran ◽

...

Keyword(s):

Biological Activities ◽

Sequence Similarity ◽

Eukaryotic Expression ◽

North India ◽

Cell Surface Expression ◽

Functional Domains ◽

Wild Type ◽

Down Regulation ◽

Subtype B ◽

Hiv 1

Background: HIV-1 Nef is an important accessory protein with multiple effector functions. Genetic studies of HIV-1 Nef gene shows extensive genetic diversity and the functional studies have been carried out mostly with Nef derived from regions dominated by subtype B (North America & Europe). Objective: This study was carried out to characterize genetic variations of the Nef gene from HIV-1 infected individuals from North-India and to find out their functional implications. Methods: The unique representative variants were sub-cloned in eukaryotic expression vector and further characterized with respect to their ability to down regulate cell surface expression of CD4 and MHC-1molecules. Results: The phylogenetic analysis of Nef variants revealed sequence similarity with either consensus subtype B or B/C recombinants. Boot scan analysis of some of our variants showed homology to B/C recombinant and some to wild type Nef B. Extensive variations were observed in most of the variants. The dN/dS ratio revealed 80% purifying selection and 20% diversifying selection implying the importance of mutations in Nef variants. Intracellular stability of Nef variants differed greatly when compared with wild type Nef B and C. There were some variants that possessed mutations in the functional domains of Nef and responsible for its differential CD4 and MHC-1 down regulation activity. Conclusion: We observed enhanced biological activities in some of the variants, perhaps arising out of amino acid substitutions in their functional domains. The CD4 and MHC-1 down-regulation activity of Nef is likely to confer immense survival advantage allowing the most rare genotype in a population to become the most abundant after a single selection event.

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