Sequence-specific interaction between HIV-1 matrix protein and viral genomic RNA revealed by in vitro genetic selection

Fullerene derivatives with hydrophilic substituents have been shown to exhibit a range of biological activities, including antiviral ones. For a long time, the anti-HIV activity of fullerene derivatives was believed to be due to their binding into the hydrophobic pocket of HIV-1 protease, thereby blocking its activity. Recent work, however, brought new evidence of a novel, protease-independent mechanism of fullerene derivatives’ action. We studied in more detail the mechanism of the anti-HIV-1 activity of N,N-dimethyl[70]fulleropyrrolidinium iodide fullerene derivatives. By using a combination of in vitro and cell-based approaches, we showed that these C70 derivatives inhibited neither HIV-1 protease nor HIV-1 maturation. Instead, our data indicate effects of fullerene C70 derivatives on viral genomic RNA packaging and HIV-1 cDNA synthesis during reverse transcription—without impairing reverse transcriptase activity though. Molecularly, this could be explained by a strong binding affinity of these fullerene derivatives to HIV-1 nucleocapsid domain, preventing its proper interaction with viral genomic RNA, thereby blocking reverse transcription and HIV-1 infectivity. Moreover, the fullerene derivatives’ oxidative activity and fluorescence quenching, which could be one of the reasons for the inconsistency among reported anti-HIV-1 mechanisms, are discussed herein.

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HIV-1 integrase binding to genomic RNA 5′-UTR induces local structural changes in vitro and in virio

Retrovirology ◽

10.1186/s12977-021-00582-0 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Shuohui Liu ◽

Pratibha C. Koneru ◽

Wen Li ◽

Chathuri Pathirage ◽

Alan N. Engelman ◽

...

Keyword(s):

Secondary Structure ◽

Structural Changes ◽

Genomic Rna ◽

Packaging Signal ◽

Virus Particles ◽

Structure Changes ◽

Apical Loop ◽

Viral Genomic Rna ◽

Hiv 1

Abstract Background During HIV-1 maturation, Gag and Gag-Pol polyproteins are proteolytically cleaved and the capsid protein polymerizes to form the honeycomb capsid lattice. HIV-1 integrase (IN) binds the viral genomic RNA (gRNA) and impairment of IN-gRNA binding leads to mis-localization of the nucleocapsid protein (NC)-condensed viral ribonucleoprotein complex outside the capsid core. IN and NC were previously demonstrated to bind to the gRNA in an orthogonal manner in virio; however, the effect of IN binding alone or simultaneous binding of both proteins on gRNA structure is not yet well understood. Results Using crosslinking-coupled selective 2′-hydroxyl acylation analyzed by primer extension (XL-SHAPE), we characterized the interaction of IN and NC with the HIV-1 gRNA 5′-untranslated region (5′-UTR). NC preferentially bound to the packaging signal (Psi) and a UG-rich region in U5, irrespective of the presence of IN. IN alone also bound to Psi but pre-incubation with NC largely abolished this interaction. In contrast, IN specifically bound to and affected the nucleotide (nt) dynamics of the apical loop of the transactivation response element (TAR) and the polyA hairpin even in the presence of NC. SHAPE probing of the 5′-UTR RNA in virions produced from allosteric IN inhibitor (ALLINI)-treated cells revealed that while the global secondary structure of the 5′-UTR remained unaltered, the inhibitor treatment induced local reactivity differences, including changes in the apical loop of TAR that are consistent with the in vitro results. Conclusions Overall, the binding interactions of NC and IN with the 5′-UTR are largely orthogonal in vitro. This study, together with previous probing experiments, suggests that IN and NC binding in vitro and in virio lead to only local structural changes in the regions of the 5′-UTR probed here. Accordingly, disruption of IN-gRNA binding by ALLINI treatment results in local rather than global secondary structure changes of the 5′-UTR in eccentric virus particles. Graphical Abstract

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Novel Intersubunit Interaction Critical for HIV-1 Core Assembly Defines a Potentially Targetable Inhibitor Binding Pocket

mBio ◽

10.1128/mbio.02858-18 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 6

Author(s):

Pierrick Craveur ◽

Anna T. Gres ◽

Karen A. Kirby ◽

Dandan Liu ◽

John A. Hammond ◽

...

Keyword(s):

Viral Replication ◽

Specific Interaction ◽

Binding Pocket ◽

Core Stability ◽

Host Cells ◽

Capsid Assembly ◽

Dynamic Simulations ◽

Virus Like Particle ◽

Hiv 1

ABSTRACTHIV-1 capsid protein (CA) plays critical roles in both early and late stages of the viral replication cycle. Mutagenesis and structural experiments have revealed that capsid core stability significantly affects uncoating and initiation of reverse transcription in host cells. This has led to efforts in developing antivirals targeting CA and its assembly, although none of the currently identified compounds are used in the clinic for treatment of HIV infection. A specific interaction that is primarily present in pentameric interfaces in the HIV-1 capsid core was identified and is reported to be important for CA assembly. This is shown by multidisciplinary characterization of CA site-directed mutants using biochemical analysis of virus-like particle formation, transmission electron microscopy ofin vitroassembly, crystallographic studies, and molecular dynamic simulations. The data are consistent with a model where a hydrogen bond between CA residues E28 and K30′ from neighboring N-terminal domains (CANTDs) is important for CA pentamer interactions during core assembly. This pentamer-preferred interaction forms part of anN-terminaldomaininterface (NDI) pocket that is amenable to antiviral targeting.IMPORTANCEPrecise assembly and disassembly of the HIV-1 capsid core are key to the success of viral replication. The forces that govern capsid core formation and dissociation involve intricate interactions between pentamers and hexamers formed by HIV-1 CA. We identified one particular interaction between E28 of one CA and K30′ of the adjacent CA that appears more frequently in pentamers than in hexamers and that is important for capsid assembly. Targeting the corresponding site could lead to the development of antivirals which disrupt this interaction and affect capsid assembly.

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Ebola Virus VP35-VP40 Interaction Is Sufficient for Packaging 3E-5E Minigenome RNA into Virus-Like Particles

Journal of Virology ◽

10.1128/jvi.01857-05 ◽

2006 ◽

Vol 80 (11) ◽

pp. 5135-5144 ◽

Cited By ~ 44

Author(s):

Reed F. Johnson ◽

Sarah E. McCarthy ◽

Peter J. Godlewski ◽

Ronald N. Harty

Keyword(s):

Confocal Microscopy ◽

Reverse Transcriptase ◽

Matrix Protein ◽

Ebola Virus ◽

Genomic Rna ◽

Transfected Cells ◽

Virus Like Particles ◽

Rna Complexes ◽

Viral Genomic Rna ◽

Two Hybrid

ABSTRACT The packaging of viral genomic RNA into nucleocapsids and subsequently into virions is not completely understood. Phosphoprotein (P) and nucleoprotein (NP) interactions link NP-RNA complexes with P-L (polymerase) complexes to form viral nucleocapsids. The nucleocapsid then interacts with the viral matrix protein, leading to specific packaging of the nucleocapsid into the virion. A mammalian two-hybrid assay and confocal microscopy were used to demonstrate that Ebola virus VP35 and VP40 interact and colocalize in transfected cells. VP35 was packaged into budding virus-like particles (VLPs) as observed by protease protection assays. Moreover, VP40 and VP35 were sufficient for packaging an Ebola virus minignome RNA into VLPs. Results from immunoprecipitation-reverse transcriptase PCR experiments suggest that VP35 confers specificity of the nucleocapsid for viral genomic RNA by direct VP35-RNA interactions.

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Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: Implications for viral genomic RNA packaging

RNA ◽

10.1261/rna.038869.113 ◽

2013 ◽

Vol 19 (8) ◽

pp. 1078-1088 ◽

Cited By ~ 52

Author(s):

J. A. Webb ◽

C. P. Jones ◽

L. J. Parent ◽

I. Rouzina ◽

K. Musier-Forsyth

Keyword(s):

Genomic Rna ◽

Rna Packaging ◽

Binding Interactions ◽

Viral Genomic Rna ◽

Hiv 1

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Anti-Human Immunodeficiency Virus Type 1 Activity of R-95288, a Phosphodiester Hexadeoxyribonucleotide Modified by Dibenzyloxybenzyl and Hydroxyethyl Residues at the 5′- and 3′-Ends

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632029700800505 ◽

1997 ◽

Vol 8 (5) ◽

pp. 429-438 ◽

Cited By ~ 4

Author(s):

T Agatsuma ◽

H Furukawa ◽

H Hotoda ◽

M Koizumi ◽

R Koga ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Specific Interaction ◽

V3 Loop ◽

Virus Envelope ◽

Immunodeficiency Virus ◽

Hiv 1

The phosphodiester hexadeoxyribonucleotide R-95288 is a potent anti-human immunodeficiency virus type 1 (HIV-1) agent in vitro which consists or a TGGGAG nucleoside sequence with dibenzyloxybenzyl and hydroxyethyl substituents at the 5′- and 3′-ends, respectively. In this study, the antiviral activity of R-95288 against various strains of HIV-1 in vitro was assessed and its mechanism of action was analysed. R-95288 inhibited replication of all strains of HIV-1 used including laboratory strains with the syncytium-inducing (SI) phenotype and clinical isolates with both SI and non-SI (NSI) phenotypes. The 50% inhibitory concentrations (IC50s) were 0.62–18 μg mL−1 (0.21–6.2 μM). R-95288 inhibited the binding and fusion of HIV-1-infected T cells with CD4+ cells. In addition, R-95288 specifically blocked the binding of monoclonal antibodies, recognizing the anti-V3 loop or the CD4-binding site of the virus envelope glycoprotein gp120. Furthermore, the target site of R-95288 within the V3 loop was found in the putative heparin-binding region by binding inhibition assays using various anti-V3 loop antibodies. These results suggest that R-95288 can inhibit various strains of HIV-1, possibly by specific interaction with gp120.

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Role of RNA in Facilitating Gag/Gag-Pol Interaction

Journal of Virology ◽

10.1128/jvi.76.8.4131-4137.2002 ◽

2002 ◽

Vol 76 (8) ◽

pp. 4131-4137 ◽

Cited By ~ 39

Author(s):

Ahmad Khorchid ◽

Rabih Halwani ◽

Mark A. Wainberg ◽

Lawrence Kleiman

Keyword(s):

Human Immunodeficiency Virus ◽

Genomic Rna ◽

Immunodeficiency Virus ◽

Membrane Bound ◽

Bulk Membrane ◽

Triton X ◽

Viral Genomic Rna ◽

Hiv 1

ABSTRACT We have examined the influence of RNA upon the interaction of Gag-Pol with Gag during human immunodeficiency virus type 1 (HIV-1) assembly. COS7 cells were transfected with protease-negative HIV-1 proviral DNA, and Gag/Gag-Pol complexes were detected by coimmunoprecipitation with anti-integrase. In COS7 cells, Gag/Gag-Pol is found almost entirely in pelletable, membrane-bound complexes. Exposure of cells to 1% Triton X-100 releases Gag/Gag-Pol from bulk membrane, but the complexes remain pelletable. The role of RNA in facilitating the interaction between Gag and Gag-Pol was examined in these bulk membrane-free, pelletable complexes. The specific presence of viral genomic RNA is not required to maintain the Gag/Gag-Pol interaction, but some type of RNA is, since exposure to RNase destabilized the Gag/Gag-Pol complex. When present only in Gag, the nucleocapsid mutation R7R10K11S, which inhibits Gag binding to RNA, inhibits the formation of both Gag and Gag/Gag-Pol complexes. When present only in Gag-Pol, this mutation has no effect upon complex formation. This result indicates that Gag-Pol may not interact directly with RNA but rather requires RNA-facilitated Gag multimerization for its interaction with Gag.

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Association of Human Immunodeficiency Virus Type 1 Vif with RNA and Its Role in Reverse Transcription

Journal of Virology ◽

10.1128/jvi.74.19.8938-8945.2000 ◽

2000 ◽

Vol 74 (19) ◽

pp. 8938-8945 ◽

Cited By ~ 83

Author(s):

Markus Dettenhofer ◽

Shan Cen ◽

Bradley A. Carlson ◽

Lawrence Kleiman ◽

Xiao-Fang Yu

Keyword(s):

Human Immunodeficiency Virus ◽

Reverse Transcriptase ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Reverse Transcription ◽

Genomic Rna ◽

Immunodeficiency Virus ◽

Viral Genomic Rna ◽

Hiv 1

ABSTRACT The vif gene of human immunodeficiency virus type 1 (HIV-1) is essential for viral replication, although the functional target of Vif remains elusive. HIV-1 vif mutant virions derived from nonpermissive H9 cells displayed no significant differences in the amount, ratio, or integrity of their protein composition relative to an isogenic wild-type virion. The amounts of the virion-associated viral genomic RNA and tRNA3 Lyswere additionally present at normal levels in vif mutant virions. We demonstrate that Vif associates with RNA in vitro as well as with viral genomic RNA in virus-infected cells. A functionally conserved lentivirus Vif motif was found in the double-stranded RNA binding domain of Xenopus laevis, Xlrbpa. The natural intravirion reverse transcriptase products were markedly reduced invif mutant virions. Moreover, purified vifmutant genomic RNA-primer tRNA complexes displayed severe defects in the initiation of reverse transcription with recombinant reverse transcriptase. These data point to a novel role for Vif in the regulation of efficient reverse transcription through modulation of the virion nucleic acid components.

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Human Immunodeficiency Virus Type 1 Vif Protein Is Packaged into the Nucleoprotein Complex through an Interaction with Viral Genomic RNA

Journal of Virology ◽

10.1128/jvi.75.16.7252-7265.2001 ◽

2001 ◽

Vol 75 (16) ◽

pp. 7252-7265 ◽

Cited By ~ 102

Author(s):

Mohammad A. Khan ◽

Claudia Aberham ◽

Sandra Kao ◽

Hirofumi Akari ◽

Robert Gorelick ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Genomic Rna ◽

Immunodeficiency Virus ◽

Nucleoprotein Complex ◽

Vif Protein ◽

Viral Genomic Rna ◽

Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) Vif protein plays a critical role in the production of infectious virions. Previous studies have demonstrated the presence of small amounts of Vif in virus particles. However, Vif packaging was assumed to be nonspecific, and its functional significance has been questioned. We now report that packaging of Vif is dependent on the packaging of viral genomic RNA in both permissive and restrictive HIV-1 target cells. Mutations in the nucleocapsid zinc finger domains that abrogate packaging of viral genomic RNA abolished packaging of Vif. Additionally, an RNA packaging-defective virus exhibited significantly reduced packaging of Vif. Finally, deletion of a putative RNA-interacting domain in Vif abolished packaging of Vif into virions. Virion-associated Vif was resistant to detergent extraction and copurified with components of the viral nucleoprotein complex and functional reverse transcription complexes. Thus, Vif is specifically packaged into virions as a component of the viral nucleoprotein complex. Our data suggest that the specific association of Vif with the viral nucleoprotein complex might be functionally significant and could be a critical requirement for infectivity of viruses produced from restrictive host cells.

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Hantavirus N Protein Exhibits Genus-Specific Recognition of the Viral RNA Panhandle

Journal of Virology ◽

10.1128/jvi.00820-06 ◽

2006 ◽

Vol 80 (22) ◽

pp. 11283-11292 ◽

Cited By ~ 42

Author(s):

M. A. Mir ◽

B. Brown ◽

B. Hjelle ◽

W. A. Duran ◽

A. T. Panganiban

Keyword(s):

Viral Rna ◽

Genomic Rna ◽

N Protein ◽

High Affinity ◽

The Andes ◽

The Family ◽

Viral Genomic Rna ◽

Negative Sense

ABSTRACT A key genomic characteristic that helps define Hantavirus as a genus of the family Bunyaviridae is the presence of distinctive terminal complementary nucleotides that promote the folding of the viral genomic segments into “panhandle” hairpin structures. The hantavirus nucleocapsid protein (N protein), which is encoded by the smallest of the three negative-sense genomic RNA segments, undergoes in vivo and in vitro trimerization. Trimeric hantavirus N protein specifically recognizes the panhandle structure formed by complementary base sequence of 5′ and 3′ ends of viral genomic RNA. N protein trimers from the Andes, Puumala, Prospect Hill, Seoul, and Sin Nombre viruses recognize their individual homologous panhandles as well as other hantavirus panhandles with high affinity. In contrast, these hantavirus N proteins bind with markedly reduced affinity to the panhandles from the genera Bunyavirus, Tospovirus, and Phlebovirus or Nairovirus. Interactions between most hantavirus N and heterologous hantavirus viral RNA panhandles are mediated by the nine terminal conserved nucleotides of the panhandle, whereas Sin Nombre virus N requires the first 23 nucleotides for high-affinity binding. Trimeric hantavirus N complexes undergo a prominent conformational change while interacting with panhandles from members of the genus Hantavirus but not while interacting with panhandles from viruses of other genera of the family Bunyaviridae. These data indicate that high-affinity interactions between trimeric N and hantavirus panhandles are conserved within the genus Hantavirus.

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