Structural Determinants for the Selective Anti-HIV-1 Activity of the All-β Alternative Conformer of XCL1

ABSTRACTHIV-1 replication is regulatedin vivoby a complex network of cytokines and chemokines. XCL1/lymphotactin, a unique metamorphic chemokine, was recently identified as a broad-spectrum endogenous HIV-1 inhibitor that blocks viral entry via direct interaction with the gp120 envelope glycoprotein. HIV-1 inhibition by XCL1 requires access to the alternative all-β conformation, which interacts with glycosaminoglycans (GAGs) but not with the specific XCL1 receptor, XCR1. To investigate the structural determinants of the HIV-inhibitory function of XCL1, we performed a detailed structure-function analysis of a stabilized all-β variant, XCL1 W55D. Individual alanine substitutions of two basic residues within the 40s' loop, K42 and R43, abrogated the ability of XCL1 to bind to the viral envelope and block HIV-1 infection; moreover, a loss of HIV-inhibitory function, albeit less marked, was seen upon individual mutation of three additional basic residues: R18, R35, and K46. In contrast, mutation of K42 to arginine did not cause any loss of function, suggesting that the interaction with gp120 is primarily electrostatic in nature. Strikingly, four of these five residues cluster to form a large (∼350 Å2) positively charged surface in the all-β XCL1 conformation, whereas they are dissociated in the classic chemokine fold, which is inactive against HIV-1, providing a structural basis for the selective antiviral activity of the alternatively folded XCL1. Furthermore, we observed that changes to the N-terminal domain, which is proximal to the cluster of putative HIV-1 gp120-interacting residues, also affect the antiviral activity of XCL1. Interestingly, the complement of residues involved in HIV-1 blockade is partially overlapping, but distinct from those involved in the GAG-binding function of XCL1. These data identify key structural determinants of anti-HIV activity in XCL1, providing new templates for the development of HIV-1 entry inhibitors.IMPORTANCEThe host immune system controls HIV-1 infection through a wide array of inhibitory responses, including the induction of cytotoxic effector cells and the secretion of noncytolytic soluble antiviral factors such as cytokines and chemokines. We recently identified XCL1/lymphotactin, a chemokine primarily produced by CD8+T cells, as a novel endogenous factor with broad anti-HIV activity. Strikingly, only one of the two conformations that XCL1 can adopt in solution, the alternative all-β fold, mediates antiviral activity. At variance with the classic HIV-inhibitory chemokines such as CCL5/RANTES, XCL1 acts via direct interaction with the external viral envelope glycoprotein, gp120. Here, we identify the interactive surface of XCL1 that is implicated in binding to the HIV-1 envelope and HIV-1 inhibition, providing a structural basis to explain why only the all-β XCL1 conformer is effective against HIV-1. Our findings may be useful in guiding the rational design of new inhibitors of HIV-1 entry.

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Does the Cytoplasmic Tail Matter? Mechanism of Viral Envelope Glycoprotein Targeting by Membrane-Associated-RING-CH (MARCH) Proteins

Proceedings ◽

10.3390/proceedings2020050021 ◽

2020 ◽

Vol 50 (1) ◽

pp. 21

Author(s):

Cheng man Lun ◽

Abdul A. Waheed ◽

Eric O. Freed

Keyword(s):

Antiviral Activity ◽

Envelope Glycoprotein ◽

Protein Targeting ◽

Viral Envelope ◽

Surface Proteins ◽

Type I Interferons ◽

Type I ◽

Endogenous Expression ◽

Viral Glycoproteins ◽

Hiv 1

The MARCH family of RING-finger E3 ubiquitin ligases comprise 11 members that have been reported to play a variety of roles in the downregulation of cell-surface proteins involved in adaptive immunity. The RING-CH domain of MARCH proteins is thought to ubiquitinate the cytoplasmic tails (CTs) of target proteins, leading to protein degradation through either lysosomal or proteasomal pathways. Three MARCH proteins (MARCH1, 2, and 8) have recently been reported to target the HIV-1 envelope glycoprotein (Env) and vesicular stomatitis virus G glycoprotein (VSV-G), thereby impairing the infectivity of HIV-1 virions bearing HIV-1 Env or VSV-G. However, the mechanism of antiviral activity remains poorly defined. Our data show that MARCH proteins antagonize the full-length forms of HIV-1 Env, VSV-G, and Ebola glycoprotein (GP), and impair the infectivity of HIV-1 virions bearing these viral glycoproteins. This Env-targeting activity of the MARCH proteins requires the E3 ubiquitin ligase activity of the RING-CH domain. We observe that the MARCH protein targeting of VSV-G is, to a large extent, CT-dependent. In striking contrast, the MARCH-protein targeting of HIV-1 Env and Ebola GP does not require the CT. Confocal microscopy data demonstrate that MARCH proteins are able to trap the viral glycoproteins in an intracellular compartment. We observe that the endogenous expression of MARCH8 in T-cell lines and PBMCs is inducible by type I interferons (a and b) and is also upregulated by HIV-1 infection. Current studies are aimed at identifying the cellular target for MARCH-mediated ubiquitination in the context of their antiviral activity. These results will clarify the mechanism by which MARCH proteins antagonize viral glycoproteins and provide insights into the antiviral role of cellular inhibitory factors in Env biogenesis, trafficking, and virion incorporation.

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N4-Acyl-Modified D-2′,3′-Dideoxy-5-Fluorocytidine Nucleoside Analogues with Improved Antiviral Activity

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632020301400203 ◽

2003 ◽

Vol 14 (2) ◽

pp. 81-90 ◽

Cited By ~ 2

Author(s):

Junxing Shi ◽

Judy S Mathew ◽

Phillip M Tharnish ◽

Suguna Rachakonda ◽

S Balakrishna Pai ◽

...

Keyword(s):

Antiviral Activity ◽

Parent Compound ◽

Direct Interaction ◽

Fold Increase ◽

B Virus ◽

Immunodeficiency Virus ◽

Anti Hiv ◽

Hiv 1

A series of 2,3-dideoxy (D2) and 2,3-didehydro-2,3-dideoxy (D4) 5-fluorocytosine nucleosides modified with substituted benzoyl, heteroaromatic carbonyl, cycloalkylcarbonyl and alkanoyl at the N4-position were synthesized and evaluated for anti-human immunodeficiency virus type 1 (HIV-1) and anti-hepatitis B virus (HBV) activity in vitro. For most D2-nucleosides, N4-substitutions improved the anti-HIV-1 activity markedly without increasing the cytotoxicity. In the D4-nucleosides series, some of the substituents at the N4-position enhanced the anti-HIV-1 activity with a modest increase in the cytotoxicity. The most potent and selective N4-modified nucleoside for the D2-series was N4- p-iodobenzoyl-D2FC, which had a 46-fold increase in anti-HIV-1 potency in MT-2 cells compared to the parent nucleoside D-D2FC. In the D4-series, N4- p-bromobenzoyl-D4FC was 12-fold more potent in MT-2 cells compared to the parent nucleoside D-D4FC. All eight N4- p-halobenzoyl-substituted D2- and D4-nucleosides evaluated against HBV in HepAD38 cells demonstrated equal or greater potency than the two parental compounds, D-D2FC and D-D4FC. The N4-modification especially in the D2-nucleoside series containing the N4-nicotinoyl, o-nitrobenzoyl and n-butyryl showed a significant reduction in mitochondrial toxicity relative to the parent nucleoside analogue. Although the 5′-triphosphate of the parent compound (D-D4FC-TP) was formed from the N4-acyl-D4FC analogues in different cells, the levels of the 5′-triphosphate nucleotide did not correlate with the cell-derived 90% effective antiviral concentrations (EC90), suggesting that a direct interaction of the triphosphates of these N4-acyl nucleosides was involved in the antiviral activity.

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Structural insights into the conformational plasticity of the full-length trimeric HIV-1 envelope glycoprotein precursor

10.1101/288472 ◽

2018 ◽

Author(s):

Shijian Zhang ◽

Wei Li Wang ◽

Shuobing Chen ◽

Maolin Lu ◽

Eden P. Go ◽

...

Keyword(s):

Viral Entry ◽

Envelope Glycoprotein ◽

Structural Data ◽

Full Length ◽

Heptad Repeat ◽

Structural Basis ◽

Glycoprotein Precursor ◽

Helix Bundle ◽

Conformational Plasticity ◽

Hiv 1

SummaryThe human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer mediates viral entry into cells and is the major target for the host antibody response. In infected cells, the mature Env [(gp120/gp41)3] is produced by cleavage of a trimeric gp160 precursor. Proteolytic cleavage decreases Env conformational flexibility, allowing the mature Env to resist antibody binding to conserved elements. The conformational plasticity of the Env precursor skews the humoral immune response towards the elicitation of ineffectual antibodies, contributing to HIV-1 persistence in the infected host. The structural basis for the plasticity of the Env precursor remains elusive. Here we use cryo-electron microscopy to visualize two coexisting conformational states of the full-length Env precursor at nominal resolutions of 5.5 and 8.0 Å. The State-P2 conformation features a three-helix bundle of the gp41 heptad repeat region in the core, but has disordered membrane-interactive regions. State-P1 trimers lack the three-helix bundle and instead retain ordered transmembrane and membrane-proximal external regions embracing a central cavity. Our structural data shed light on the unusual plasticity of the Env precursor and provide new clues to Env immunogen discovery.

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Synthesis and Antiviral Activity of 1,3-Disubstituted Uracils against HIV-1 and HCMV

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632020301400506 ◽

2003 ◽

Vol 14 (5) ◽

pp. 271-279 ◽

Cited By ~ 15

Author(s):

Tokumi Maruyama ◽

Shigetada Kozai ◽

Tetsuo Yamasaki ◽

Myriam Witvrouw ◽

Christophe Pannecouque ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Antiviral Activity ◽

Human Cytomegalovirus ◽

Therapeutic Agents ◽

Reverse Transcriptase Inhibitors ◽

Uracil Derivatives ◽

Immunodeficiency Virus ◽

Anti Hiv Agents ◽

Anti Hiv ◽

Hiv 1

The development of new non-nucleoside reverse transcriptase inhibitors (NNRTIs) is an efficient strategy for finding new therapeutic agents against human immunodeficiency virus (HIV). A large number of 6-substituted uracil derivatives have been prepared in order to explore new NNRTIs. However, there are few approaches to anti-HIV agents from 1,3-disubstituted uracil derivatives. Therefore, we tried to prepare several 1,3-disubstituted uracils, which were easily obtainable from uracil by preparation under alkali and Mitsunobu conditions, and examined their antiviral activity against HIV-1 and human cytomegalovirus (HCMV). We found that 1-benzyl-3-(3,5-dimethylbenzyl)uracil and 1-cyanomethyl-3-(3,5-dimethylbenzyl)-4-thiouracil showed powerful inhibition against HCMV and HIV-1, respectively.

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An Unrelated Monoclonal Antibody Neutralizes Human Immunodeficiency Virus Type 1 by Binding to an Artificial Epitope Engineered in a Functionally Neutral Region of the Viral Envelope Glycoproteins

Journal of Virology ◽

10.1128/jvi.79.9.5616-5624.2005 ◽

2005 ◽

Vol 79 (9) ◽

pp. 5616-5624 ◽

Cited By ~ 41

Author(s):

Xinping Ren ◽

Joseph Sodroski ◽

Xinzhen Yang

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Envelope Glycoprotein ◽

Virus Entry ◽

Neutralizing Antibody ◽

Viral Envelope ◽

Epitope Tag ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Neutralizing antibodies often recognize regions of viral envelope glycoproteins that play a role in receptor binding or other aspects of virus entry. To address whether this is a necessary feature of a neutralizing antibody, we identified the V4 region of the gp120 envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) as a sequence that is tolerant of drastic change and thus appears to play a negligible role in envelope glycoprotein function. An artificial epitope tag was inserted into the V4 region without a significant effect on virus entry or neutralization by antibodies that recognize HIV-1 envelope glycoprotein sequences. An antibody directed against the artificial epitope tag was able to neutralize the modified, but not the wild-type, HIV-1. Thus, the specific target of a neutralizing antibody need not contribute functionally to the process of virus entry.

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In Vitro Antiviral Activity of the Novel, Tyrosyl-Based Human Immunodeficiency Virus (HIV) Type 1 Protease Inhibitor Brecanavir (GW640385) in Combination with Other Antiretrovirals and against a Panel of Protease Inhibitor-Resistant HIV

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00401-07 ◽

2007 ◽

Vol 51 (9) ◽

pp. 3147-3154 ◽

Cited By ~ 23

Author(s):

Richard Hazen ◽

Robert Harvey ◽

Robert Ferris ◽

Charles Craig ◽

Phillip Yates ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Antiviral Activity ◽

Protease Inhibitor ◽

Reverse Transcriptase ◽

In Vitro Assays ◽

Immunodeficiency Virus ◽

Anti Hiv ◽

Hiv 1

ABSTRACT Brecanavir, a novel tyrosyl-based arylsulfonamide, high-affinity, human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI), has been evaluated for anti-HIV activity in several in vitro assays. Preclinical assessment of brecanavir indicated that this compound potently inhibited HIV-1 in cell culture assays with 50% effective concentrations (EC50s) of 0.2 to 0.53 nM and was equally active against HIV strains utilizing either the CXCR4 or CCR5 coreceptor, as was found with other PIs. The presence of up to 40% human serum decreased the anti-HIV-1 activity of brecanavir by 5.2-fold, but under these conditions the compound retained single-digit nanomolar EC50s. When brecanavir was tested in combination with nucleoside reverse transcriptase inhibitors, the antiviral activity of brecanavir was synergistic with the effects of stavudine and additive to the effects of zidovudine, tenofovir, dideoxycytidine, didanosine, adefovir, abacavir, lamivudine, and emtricitabine. Brecanavir was synergistic with the nonnucleoside reverse transcriptase inhibitor nevirapine or delavirdine and was additive to the effects of efavirenz. In combination with other PIs, brecanavir was additive to the activities of indinavir, lopinavir, nelfinavir, ritonavir, amprenavir, saquinavir, and atazanavir. Clinical HIV isolates from PI-experienced patients were evaluated for sensitivity to brecanavir and other PIs in a recombinant virus assay. Brecanavir had a <5-fold increase in EC50s against 80% of patient isolates tested and had a greater mean in vitro potency than amprenavir, indinavir, lopinavir, atazanavir, tipranavir, and darunavir. Brecanavir is by a substantial margin the most potent and broadly active antiviral agent among the PIs tested in vitro.

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Human CD38 interferes with HIV‐1 fusion through a sequence homologous to the V3 loop of the viral envelope glycoprotein gp120.

The FASEB Journal ◽

10.1096/fj.02-0512fje ◽

2003 ◽

Vol 17 (3) ◽

pp. 1-20 ◽

Cited By ~ 23

Author(s):

Andrea Savarino ◽

Thea Bensi ◽

Annalisa Chiocchetti ◽

Flavia Bottarel ◽

Riccardo Mesturini ◽

...

Keyword(s):

Envelope Glycoprotein ◽

Viral Envelope ◽

V3 Loop ◽

Viral Envelope Glycoprotein ◽

Envelope Glycoprotein Gp120 ◽

Glycoprotein Gp120 ◽

Hiv 1

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Optimized and enhanced DNA plasmid vector based in vivo construction of a neutralizing anti-HIV-1 envelope glycoprotein Fab

Human Vaccines & Immunotherapeutics ◽

10.4161/hv.26498 ◽

2013 ◽

Vol 9 (10) ◽

pp. 2253-2262 ◽

Cited By ~ 32

Author(s):

Kar Muthumani ◽

Seleeke Flingai ◽

Megan Wise ◽

Colleen Tingey ◽

Kenneth E Ugen ◽

...

Keyword(s):

Envelope Glycoprotein ◽

Plasmid Vector ◽

Dna Plasmid ◽

Anti Hiv ◽

Hiv 1

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Rapid Prescreening for Antiviral Agents against HIV-1 Based on Their Inhibitory Activity in Site-Directed Immunoassays. Approaches Applicable to Epidemic HIV-1 Strains

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632029300400403 ◽

1993 ◽

Vol 4 (4) ◽

pp. 207-214 ◽

Cited By ~ 6

Author(s):

A. R. Neurath ◽

N. Strick ◽

S. Jiang

Keyword(s):

Antiviral Activity ◽

North American ◽

Antiviral Agents ◽

Envelope Glycoproteins ◽

V3 Loop ◽

European North ◽

Consensus Peptide ◽

Antiviral Chemotherapy ◽

Anti Hiv ◽

Hiv 1

Several compounds, including the triphenylmethane derivative aurintricarboxylic acid (ATA) and porphyrins, were reported to inhibit the binding of anti-V3 loop-specific antibodies to the V3 loop of gp120 from HIV-1 III-B and to have antiviral activity, probably due to interference with the biological function of the V3 loop. However, these compounds can be applied to antiviral chemotherapy only if they interact with envelope glycoproteins from a multitude of epidemic HIV-1 strains and inhibit their replication. Since recombinant envelope glycoproteins, synthetic peptides and anti-V3 monoclonal antibodies may not be available for these HIV-1 strains, alternative assays are needed to prescreen different compounds for potential antiviral activity against these viruses. Results presented here indicate that: (1) virions of HIV-1 MN, most closely related to primary HIV-1 isolates from European and North American countries, and human anti-HIV-1 antibodies, can also be used for rapid prescreening of antiviral agents, (2) compounds with antiviral activity against HIV-1 MN, discerned by site-directed immunoassays, inhibited the reaction of human anti-HIV-1 with a V3 loop consensus peptide corresponding to European/North American HIV-1 isolates, and (3) meso-tetra (4-carboxyphenyl) porphine (MTCPP), one of the most potent inhibitors of HIV-1 replication selected on the basis of site-directed immunoassays, preferentially attached to the V3 loop of gp120.

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