Subtype Differences in the Interaction of HIV-1 Matrix with Calmodulin: Implications for Biological Functions

The HIV-1 Gag polyprotein plays essential roles during the late stage of the HIV-1 replication cycle, and has recently been identified as a promising therapeutic target. The N-terminal portion of the HIV-1 Gag polyprotein encodes the myristoylated matrix (MA) protein, which functions in the trafficking of the structural proteins to the plasma membrane (PM) and facilitation of envelope incorporation into budding virus. Numerous host cell proteins interact with the MA portion of the Gag polyprotein during this process. One such factor is the ubiquitous calcium-binding protein calmodulin (CaM), which interacts preferentially with myristoylated proteins, thereby regulating cell physiology. The exact role of this interaction is poorly understood to date. Atomic resolution structures revealed the nature of the CaM-MA interaction for clade B isolates. In this study, we expanded our knowledge and characterized biophysically and computationally the CaM interaction with MA from other HIV-1 clades and discovered differences in the CaM recognition as compared to the prototypical clade B MA., with significant alterations in the interaction with the MA protein from clade C. Structural investigation and in silico mutational analysis revealed that HIV-1 MA protein from clade C, which is responsible for the majority of global HIV-1 infections, interacts with lower affinity and altered kinetics as compared to the canonical clade B. This finding may have implications for additional altered interaction networks as compared to the well-studied clade B. Our analysis highlights the importance of expanding investigations of virus-host cell factor interaction networks to other HIV-1 clades.

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Griffithsin Retains Anti-HIV-1 Potency with Changes in gp120 Glycosylation and Complements Broadly Neutralizing Antibodies PGT121 and PGT126

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01084-19 ◽

2019 ◽

Vol 64 (1) ◽

Cited By ~ 1

Author(s):

Kathryn Fischer ◽

Kimberly Nguyen ◽

Patricia J. LiWang

Keyword(s):

Neutralizing Antibodies ◽

Glycosylation Site ◽

Significant Loss ◽

Broadly Neutralizing Antibodies ◽

Clade B ◽

Key Factor ◽

Clade C ◽

Cd4 Binding Site ◽

Gp120 Glycosylation ◽

Hiv 1

ABSTRACT Griffithsin (Grft) is an antiviral lectin that has been shown to potently inhibit HIV-1 by binding high-mannose N-linked glycosylation sites on HIV-1 gp120. A key factor for Grft potency is glycosylation at N295 of gp120, which is directly adjacent to N332, a target glycan for an entire class of broadly neutralizing antibodies (bNAbs). Here, we unify previous work on the importance of other glycans to Grft potency against HIV-1 and Grft’s role in mediating the conformational change of gp120 by mutating nearly every glycosylation site in gp120. In addition to a significant loss of Grft activity by the removal of glycosylation at N295, glycan absence at N332 or N448 was found to have moderate effects on Grft potency. Interestingly, in the absence of N295, Grft effectiveness could be improved by a mutation that results in the glycan at N448 shifting to N446, indicating that the importance of individual glycans may be related to their effect on glycosylation density. Grft’s ability to alter the structure of gp120, exposing the CD4 binding site, correlated with the presence of glycosylation at N295 only in clade B strains, not clade C strains. We further demonstrate that Grft can rescue the activity of the bNAbs PGT121 and PGT126 in the event of a loss or a shift of glycosylation at N332, where the bNAbs suffer a drastic loss of potency. Despite targeting the same region, Grft in combination with PGT121 and PGT126 produced additive effects. This indicates that Grft could be an important combinational therapeutic.

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Genetic identity, biological phenotype, and evolutionary pathways of transmitted/founder viruses in acute and early HIV-1 infection

Journal of Experimental Medicine ◽

10.1084/jem.20090378 ◽

2009 ◽

Vol 206 (6) ◽

pp. 1273-1289 ◽

Cited By ~ 526

Author(s):

Jesus F. Salazar-Gonzalez ◽

Maria G. Salazar ◽

Brandon F. Keele ◽

Gerald H. Learn ◽

Elena E. Giorgi ◽

...

Keyword(s):

Direct Analysis ◽

Cytotoxic T Cell ◽

Adaptive Mutations ◽

Clade B ◽

Single Genome ◽

Coreceptor Binding ◽

Clade C ◽

Biological Phenotype ◽

Cytotoxic T Cell Responses ◽

Hiv 1

Identification of full-length transmitted HIV-1 genomes could be instrumental in HIV-1 pathogenesis, microbicide, and vaccine research by enabling the direct analysis of those viruses actually responsible for productive clinical infection. We show in 12 acutely infected subjects (9 clade B and 3 clade C) that complete HIV-1 genomes of transmitted/founder viruses can be inferred by single genome amplification and sequencing of plasma virion RNA. This allowed for the molecular cloning and biological analysis of transmitted/founder viruses and a comprehensive genome-wide assessment of the genetic imprint left on the evolving virus quasispecies by a composite of host selection pressures. Transmitted viruses encoded intact canonical genes (gag-pol-vif-vpr-tat-rev-vpu-env-nef) and replicated efficiently in primary human CD4+ T lymphocytes but much less so in monocyte-derived macrophages. Transmitted viruses were CD4 and CCR5 tropic and demonstrated concealment of coreceptor binding surfaces of the envelope bridging sheet and variable loop 3. 2 mo after infection, transmitted/founder viruses in three subjects were nearly completely replaced by viruses differing at two to five highly selected genomic loci; by 12–20 mo, viruses exhibited concentrated mutations at 17–34 discrete locations. These findings reveal viral properties associated with mucosal HIV-1 transmission and a limited set of rapidly evolving adaptive mutations driven primarily, but not exclusively, by early cytotoxic T cell responses.

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Differential Induction of Interleukin-10 in Monocytes by HIV-1 Clade B and Clade C Tat Proteins

Journal of Biological Chemistry ◽

10.1074/jbc.m110.120840 ◽

2010 ◽

Vol 285 (24) ◽

pp. 18319-18325 ◽

Cited By ~ 24

Author(s):

Justine K. Wong ◽

Grant R. Campbell ◽

Stephen A. Spector

Keyword(s):

Interleukin 10 ◽

Clade B ◽

Clade C ◽

Differential Induction ◽

Hiv 1

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CCL2 mobilizes ALIX to facilitate Gag-p6 mediated HIV-1 virion release

eLife ◽

10.7554/elife.35546 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 4

Author(s):

David O Ajasin ◽

Vasudev R Rao ◽

Xuhong Wu ◽

Santhamani Ramasamy ◽

Mario Pujato ◽

...

Keyword(s):

Virus Replication ◽

Virus Production ◽

Escrt Machinery ◽

Virion Release ◽

Clade B ◽

Novel Variants ◽

Clade C ◽

Extracellular Stimuli ◽

First Time ◽

Hiv 1

Cellular ESCRT machinery plays pivotal role in HIV-1 budding and release. Extracellular stimuli that modulate HIV-1 egress are currently unknown. We found that CCL2 induced by HIV-1 clade B (HIV-1B) infection of macrophages enhanced virus production, while CCL2 immuno-depletion reversed this effect. Additionally, HIV-1 clade C (HIV-1C) was refractory to CCL2 levels. We show that CCL2-mediated increase in virus production requires Gag late motif LYPX present in HIV-1B, but absent in HIV-1C, and ALIX protein that recruits ESCRT III complex. CCL2 immuno-depletion sequestered ALIX to F-actin structures, while CCL2 addition mobilized it to cytoplasm facilitating Gag-ALIX binding. The LYPX motif improves virus replication and its absence renders the virus less fit. Interestingly, novel variants of HIV-1C with PYRE/PYKE tetrapeptide insertions in Gag-p6 conferred ALIX binding, CCL2-responsiveness and enhanced virus replication. These results, for the first time, indicate that CCL2 mediates ALIX mobilization from F-actin and enhances HIV-1 release and fitness.

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HIV-1 Envelope Glycoprotein Trafficking and Viral Transmission

Proceedings ◽

10.3390/proceedings2020050017 ◽

2020 ◽

Vol 50 (1) ◽

pp. 17

Author(s):

Melissa Victoria Fernandez ◽

Lwar N Naing ◽

David A Scheiblin ◽

Sherimay D Ablan ◽

Jennifer A Simmons ◽

...

Keyword(s):

T Cell ◽

Envelope Glycoprotein ◽

Viral Transmission ◽

Virological Synapse ◽

Glycoprotein Complex ◽

Viral Spread ◽

Clade B ◽

Clade C ◽

T Cell Lines ◽

Hiv 1

HIV-1 encodes an envelope glycoprotein complex (Env) containing a long cytoplasmic tail (CT) harboring trafficking motifs implicated in Env incorporation into virions. Although the requirement for the Env CT in viral transmission is known, the precise mechanism by which Env is incorporated into nascent virions and localizes to the virological synapse remains poorly defined. To further elucidate the mechanism of Env trafficking, we examined three HIV-1 strains: the lab-adapted clade B strain, NL4-3, and a transmitted/founder (T/F) clade C virus, K3016, and a T/F clade B virus, CH077. The HIV-1 Env CT contains two invariant trafficking motifs: tyrosine endocytosis motif, Y712SPL, and C-terminal dileucine motif, LL855. Virion Env incorporation analysis revealed that Y712SPL is necessary for efficient Env incorporation, while LL855 is dispensable. Spreading infection kinetics were analyzed in various T-cell lines and primary human PBMCs; the results indicated that both endocytic motifs contribute to efficient viral spread in culture. Analysis of Env localization to the T-cell uropod, the portion of the plasma membrane that forms a virological synapse with uninfected cells, was found to be dependent on the Env CT and the Y712SPL motif. Cell-to-cell and cell-free transmission assays using T cells infected with HIV-1 bearing Y712A or LL855AA Env CT mutations are ongoing to establish a role for these motifs in both modes of viral transmission. These studies will significantly enhance our understanding of Env trafficking and viral transmission, providing insights into viral Env–host interactions in physiologically relevant cells.

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Influence of HLA Class I and HLA-KIR Compound Genotypes on HIV-2 Infection and Markers of Disease Progression in a Manjako Community in West Africa

Journal of Virology ◽

10.1128/jvi.00116-10 ◽

2010 ◽

Vol 84 (16) ◽

pp. 8202-8208 ◽

Cited By ~ 34

Author(s):

Louis-Marie Yindom ◽

Aleksandra Leligdowicz ◽

Maureen P. Martin ◽

Xiaojiang Gao ◽

Ying Qi ◽

...

Keyword(s):

Immune Responses ◽

Hla Class I ◽

Gene Products ◽

Viral Loads ◽

Susceptibility To Infection ◽

Clade B ◽

Clade C ◽

Unique Nature ◽

First Time ◽

Hiv 1

ABSTRACT Overall, the time to AIDS after HIV-2 infection is longer than with HIV-1, and many individuals infected with HIV-2 virus remain healthy throughout their lives. Multiple HLA and KIR gene products have been implicated in the control of HIV-1, but the effect of variation at these loci on HIV-2 disease is unknown. We show here for the first time that HLA-B*1503 is associated significantly with poor prognosis after HIV-2 infection and that HLA-B*0801 is associated with susceptibility to infection. Interestingly, previous data indicate that HLA-B*1503 is associated with low viral loads in HIV-1 clade B infection but has no significant effect on viral load in clade C infection. In general, alleles strongly associated with HIV-1 disease showed no effect in HIV-2 disease. These data emphasize the unique nature of the effects of HLA and HLA/KIR combinations on HIV-2 immune responses relative to HIV-1, which could be related to their distinct clinical course.

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Regional Clustering of Shared Neutralization Determinants on Primary Isolates of Clade C Human Immunodeficiency Virus Type 1 from South Africa

Journal of Virology ◽

10.1128/jvi.76.5.2233-2244.2002 ◽

2002 ◽

Vol 76 (5) ◽

pp. 2233-2244 ◽

Cited By ~ 86

Author(s):

Renata Bures ◽

Lynn Morris ◽

Carolyn Williamson ◽

Gita Ramjee ◽

Mark Deers ◽

...

Keyword(s):

South Africa ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Serum Samples ◽

Clade B ◽

Immunodeficiency Virus ◽

Clade C ◽

Cross Neutralization ◽

Hiv 1

ABSTRACT Clade C is one of the most prevalent genetic subtypes of human immunodeficiency virus type 1 (HIV-1) in the world today and one of the least studied with respect to neutralizing antibodies. Most information on HIV-1 serology as it relates to neutralization is derived from clade B. Clade C primary isolates of HIV-1 from South Africa and Malawi were shown here to resemble clade B isolates in their resistance to inhibition by soluble CD4 and their sensitivity to neutralization by human monoclonal antibody immunoglobulin G1b12 and, to a lesser extent, 2F5. Unlike clade B isolates, however, all 16 clade C isolates examined resisted neutralization by 2G12. Infection with clade C HIV-1 in a cohort of female sex workers in South Africa generated antibodies that neutralized the autologous clade C isolate and T-cell-line-adapted (TCLA) strains of clade B. Neutralization of clade B TCLA strains was much more sensitive to the presence of autologous gp120 V3 loop peptides compared to the neutralization of clade C isolates in most cases. Thus, the native structure of gp120 on primary isolates of clade C will likely pose a challenge for neutralizing antibody induction by candidate HIV-1 vaccines much the same as it has for clade B. The autologous neutralizing antibody response following primary infection with clade C HIV-1 in South Africa matured slowly, requiring at least 4 to 5 months to become detectable. Once detectable, extensive cross-neutralization of heterologous clade C isolates from South Africa was observed, suggesting an unusual degree of shared neutralization determinants at a regional level. This high frequency of cross-neutralization differed significantly from the ability of South African clade C serum samples to neutralize clade B isolates but did not differ significantly from results of other combinations of clade B and C reagents tested in checkerboard assays. Notably, two clade C serum samples obtained after less than 2 years of infection neutralized a broad spectrum of clade B and C isolates. Other individual serum samples showed a significant clade preference in their neutralizing activity. Our results suggest that clades B and C are each comprised of multiple neutralization serotypes, some of which are more clade specific than others. The clustering of shared neutralization determinants on clade C primary HIV-1 isolates from South Africa suggests that neutralizing antibodies induced by vaccines will have less epitope diversity to overcome at a regional level.

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The C-Terminal End of HIV-1 Vpu Has a Clade-Specific Determinant That Antagonizes BST-2 and Facilitates Virion Release

Journal of Virology ◽

10.1128/jvi.02315-18 ◽

2019 ◽

Vol 93 (11) ◽

Cited By ~ 2

Author(s):

Shilpi Sharma ◽

Moein Jafari ◽

Amandip Bangar ◽

Karen William ◽

John Guatelli ◽

...

Keyword(s):

Plasma Membrane ◽

Chronic Infection ◽

High Surface ◽

Human Populations ◽

Accessory Proteins ◽

Virion Assembly ◽

Virion Release ◽

Clade B ◽

Clade C ◽

Hiv 1

ABSTRACTThe cellular protein bone marrow stromal antigen-2 (BST-2)/tetherin acts against a variety of enveloped viruses by restricting their release from the plasma membrane. The HIV-1 accessory protein Vpu counteracts BST-2 by downregulating it from the cell surface and displacing it from virion assembly sites. Previous comparisons of Vpus from transmitted/founder viruses and between viruses isolated during acute and chronic infection led to the identification of a tryptophan at position 76 in Vpu (W76) as a key determinant for the displacement of BST-2 from virion assembly sites. Although present in Vpus from clades B, D, and G, W76 is absent from Vpus from clades A, C, and H. Mutagenesis of the C-terminal region of Vpu from two clade C viruses led to the identification of a conserved LL sequence that is functionally analogous to W76 of clade B. Alanine substitution of these leucines partially impaired virion release. This impairment was even greater when the mutations were combined with mutations of the Vpu β-TrCP binding site, resulting in Vpu proteins that induced high surface levels of BST-2 and reduced the efficiency of virion release to less than that of virus lackingvpu. Microscopy confirmed that these C-terminal leucines in clade C Vpu, like W76 in clade B, contribute to virion release by supporting the displacement of BST-2 from virion assembly sites. These results suggest that although encoded differently, the ability of Vpu to displace BST-2 from sites of virion assembly on the plasma membrane is evolutionarily conserved among clade B and C HIV-1 isolates.IMPORTANCEAlthough targeted by a variety of restriction mechanisms, HIV-1 establishes chronic infection in most cases, in part due to the counteraction of these host defenses by viral accessory proteins. Using conserved motifs, the accessory proteins exploit the cellular machinery to degrade or mistraffic host restriction factors, thereby counteracting them. The Vpu protein counteracts the virion-tethering factor BST-2 in part by displacing it from virion assembly sites along the plasma membrane, but a previously identified determinant of that activity is clade specific at the level of protein sequence and not found in the clade C viruses that dominate the pandemic. Here, we show that clade C Vpu provides this activity via a leucine-containing sequence rather than the tryptophan-containing sequence found in clade B Vpu. This difference seems likely to reflect the different evolutionary paths taken by clade B and clade C HIV-1 in human populations.

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Dominant Negative MA-CA Fusion Protein Is Incorporated into HIV-1 Cores and Inhibits Nuclear Entry of Viral Preintegration Complexes

Journal of Virology ◽

10.1128/jvi.01118-19 ◽

2019 ◽

Vol 93 (21) ◽

Cited By ~ 2

Author(s):

Jordan Anderson-Daniels ◽

Parmit K. Singh ◽

Gregory A. Sowd ◽

Wen Li ◽

Alan N. Engelman ◽

...

Keyword(s):

Antiviral Activity ◽

Mutational Analysis ◽

Membrane Binding ◽

Dominant Negative ◽

Target Cells ◽

Virus Infectivity ◽

Nuclear Entry ◽

Gag Polyprotein ◽

Hiv 1 ◽

Ca Protein

ABSTRACT Particle maturation is a critical step in the HIV-1 replication cycle that requires proteolytic cleavage of the Gag polyprotein into its constitutive proteins: the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 proteins. The accurate and efficient cleavage of Gag is essential for virion infectivity; inhibitors of the viral protease are potent antivirals, and substitutions in Gag that prevent its cleavage result in reduced HIV-1 infectivity. In a previous study, a mutation inhibiting cleavage at the MA-CA junction was observed to potently inhibit virus infection: incorporation of small amounts of uncleaved MA-CA protein into HIV-1 particles inhibited infectivity by ∼95%, and the resulting viral particles exhibited aberrant capsids. Here we report a detailed mechanistic analysis of HIV-1 particles bearing uncleaved MA-CA protein. We show that the particles contain stable cores and can efficiently saturate host restriction by TRIMCyp in target cells. We further show that MA-CA associates with CA in particles without detectably affecting the formation of intermolecular CA interfaces. Incorporation of MA-CA did not markedly affect reverse transcription in infected cells, but nuclear entry was impaired and integration targeting was altered. Additionally, results from mutational analysis of Gag revealed that membrane-binding elements of MA contribute to the antiviral activity of uncleaved MA-CA protein. Our results suggest that small amounts of partially processed Gag subunits coassemble with CA during virion maturation, resulting in impaired capsid functions. IMPORTANCE To become infectious, newly formed HIV-1 particles undergo a process of maturation in which the viral polyproteins are cleaved into smaller components. A previous study demonstrated that inclusion of even small quantities of an uncleavable mutant Gag polyprotein results in a strong reduction in virus infectivity. Here we show that the mechanism of transdominant inhibition by uncleavable Gag involves inhibition of nuclear entry and alteration of viral integration sites. Additionally, the results of mutational analysis suggest that the membrane-binding activity of Gag is a major requirement for the antiviral activity. These results further define the antiviral mechanism of uncleavable Gag, which may be useful for exploiting this effect to develop new antivirals.

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Identification and Characterization of Positively Selected Mutations in Nef of Four HIV-1 Major Subtypes from Los Alamos National Laboratory

Current HIV Research ◽

10.2174/1570162x16666180330140807 ◽

2018 ◽

Vol 16 (2) ◽

pp. 130-142

Author(s):

Elnaz Shadabi ◽

Binhua Liang ◽

Frank Plummer ◽

Ma Luo

Keyword(s):

National Laboratory ◽

Alamos National Laboratory ◽

Los Alamos ◽

Evolutionary Patterns ◽

Los Alamos National Laboratory ◽

Immune Pressure ◽

Subtype B ◽

Clade B ◽

Clade C ◽

Hiv 1

Background:Human immunodeficiency virus-1 (HIV-1) mutates rapidly to escape host immune pressure. This results in the generation of positively selected mutations (PSM) throughout the viral genome. Escape mutations in Nef, one of the accessory proteins of HIV-1, which plays an important role in viral pathogenicity have previously been identified in several large cohort studies, but the evolution of PSMs overtime in various HIV-1 subtypes remains unknown.Methods:161 clade A1, 3093 clade B, 647 clade C and 115 clade D HIV-1 nef sequences were obtained from the HIV Database of Los Alamos National Laboratory and aligned using MEGA 6.0. The sequences from each clade were grouped based on the year of collection. Quasi analysis was used to identify PSMs and the number and locations of PSMs were compared among different subtypes.Results:PSMs for all four subtypes were distributed across the sequence of Nef, and conserved residues F90, W113, PxxPxR (a.a 72-77) remain unaltered overtime. The frequency of PSMs was stable among subtype B sequences but increased overtime for other subtypes. Phylogenetic analysis shows that sequences containing PSMs tend to cluster together at both inter and intra- subtype levels.Conclusion:Identification of PSMs and their changes overtime within various subtypes of HIV-1 is important in defining global viral evolutionary patterns that can provide insights for designing therapeutic strategies.

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