Contribution of the clathrin adaptor AP-1 subunit µ1 to acidic cluster protein sorting

Acidic clusters act as sorting signals for packaging cargo into clathrin-coated vesicles (CCVs), and also facilitate down-regulation of MHC-I by HIV-1 Nef. To find acidic cluster sorting machinery, we performed a gene-trap screen and identified the medium subunit (µ1) of the clathrin adaptor AP-1 as a top hit. In µ1 knockout cells, intracellular CCVs still form, but acidic cluster proteins are depleted, although several other CCV components were either unaffected or increased, indicating that cells can compensate for long-term loss of AP-1. In vitro experiments showed that the basic patch on µ1 that interacts with the Nef acidic cluster also contributes to the binding of endogenous acidic cluster proteins. Surprisingly, µ1 mutant proteins lacking the basic patch and/or the tyrosine-based motif binding pocket could rescue the µ1 knockout phenotype completely. In contrast, these mutants failed to rescue Nef-induced down-regulation of MHC class I, suggesting a possible mechanism for attacking the virus while sparing the host cell.

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CD73+ CD127high Long-Term Memory CD4 T Cells Are Highly Proliferative in Response to Recall Antigens and Are Early Targets in HIV-1 Infection

International Journal of Molecular Sciences ◽

10.3390/ijms22020912 ◽

2021 ◽

Vol 22 (2) ◽

pp. 912

Author(s):

Nabila Seddiki ◽

John Zaunders ◽

Chan Phetsouphanh ◽

Vedran Brezar ◽

Yin Xu ◽

...

Keyword(s):

T Cells ◽

Peripheral Blood ◽

Cd4 T Cells ◽

Significant Proportion ◽

Long Term Memory ◽

Ki 67 ◽

Memory Cd4 T Cells ◽

Hiv 1

HIV-1 infection rapidly leads to a loss of the proliferative response of memory CD4+ T lymphocytes, when cultured with recall antigens. We report here that CD73 expression defines a subset of resting memory CD4+ T cells in peripheral blood, which highly express the α-chain of the IL-7 receptor (CD127), but not CD38 or Ki-67, yet are highly proliferative in response to mitogen and recall antigens, and to IL-7, in vitro. These cells also preferentially express CCR5 and produce IL-2. We reasoned that CD73+ memory CD4+ T cells decrease very early in HIV-1 infection. Indeed, CD73+ memory CD4+ T cells comprised a median of 7.5% (interquartile range: 4.5–10.4%) of CD4+ T cells in peripheral blood from healthy adults, but were decreased in primary HIV-1 infection to a median of 3.7% (IQR: 2.6–6.4%; p = 0.002); and in chronic HIV-1 infection to 1.9% (IQR: 1.1–3%; p < 0.0001), and were not restored by antiretroviral therapy. Moreover, we found that a significant proportion of CD73+ memory CD4+ T cells were skewed to a gut-homing phenotype, expressing integrins α4 and β7, CXCR3, CCR6, CD161 and CD26. Accordingly, 20% of CD4+ T cells present in gut biopsies were CD73+. In HIV+ subjects, purified CD73+ resting memory CD4+ T cells in PBMC were infected with HIV-1 DNA, determined by real-time PCR, to the same level as for purified CD73-negative CD4+ T cells, both in untreated and treated subjects. Therefore, the proliferative CD73+ subset of memory CD4+ T cells is disproportionately reduced in HIV-1 infection, but, unexpectedly, their IL-7 dependent long-term resting phenotype suggests that residual infected cells in this subset may contribute significantly to the very long-lived HIV proviral DNA reservoir in treated subjects.

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Effects of Mutations on Replicative Fitness and Major Histocompatibility Complex Class I Binding Affinity Are Among the Determinants Underlying Cytotoxic-T-Lymphocyte Escape of HIV-1 Gag Epitopes

mBio ◽

10.1128/mbio.01050-17 ◽

2017 ◽

Vol 8 (6) ◽

Cited By ~ 7

Author(s):

Yushen Du ◽

Tian-Hao Zhang ◽

Lei Dai ◽

Xiaojuan Zheng ◽

Aleksandr M. Gorin ◽

...

Keyword(s):

Binding Affinity ◽

High Throughput ◽

Peptide Binding ◽

Virus Evolution ◽

Mhc I ◽

Hla Alleles ◽

Ctl Escape ◽

Peptide Binding Affinity ◽

Hiv 1

ABSTRACT Certain “protective” major histocompatibility complex class I (MHC-I) alleles, such as B*57 and B*27, are associated with long-term control of HIV-1 in vivo mediated by the CD8+ cytotoxic-T-lymphocyte (CTL) response. However, the mechanism of such superior protection is not fully understood. Here we combined high-throughput fitness profiling of mutations in HIV-1 Gag, in silico prediction of MHC-peptide binding affinity, and analysis of intraperson virus evolution to systematically compare differences with respect to CTL escape mutations between epitopes targeted by protective MHC-I alleles and those targeted by nonprotective MHC-I alleles. We observed that the effects of mutations on both viral replication and MHC-I binding affinity are among the determinants of CTL escape. Mutations in Gag epitopes presented by protective MHC-I alleles are associated with significantly higher fitness cost and lower reductions in binding affinity with respect to MHC-I. A linear regression model accounting for the effect of mutations on both viral replicative capacity and MHC-I binding can explain the protective efficacy of MHC-I alleles. Finally, we found a consistent pattern in the evolution of Gag epitopes in long-term nonprogressors versus progressors. Overall, our results suggest that certain protective MHC-I alleles allow superior control of HIV-1 by targeting epitopes where mutations typically incur high fitness costs and small reductions in MHC-I binding affinity. IMPORTANCE Understanding the mechanism of viral control achieved in long-term nonprogressors with protective HLA alleles provides insights for developing functional cure of HIV infection. Through the characterization of CTL escape mutations in infected persons, previous researchers hypothesized that protective alleles target epitopes where escape mutations significantly reduce viral replicative capacity. However, these studies were usually limited to a few mutations observed in vivo. Here we utilized our recently developed high-throughput fitness profiling method to quantitatively measure the fitness of mutations across the entirety of HIV-1 Gag. The data enabled us to integrate the results with in silico prediction of MHC-peptide binding affinity and analysis of intraperson virus evolution to systematically determine the differences in CTL escape mutations between epitopes targeted by protective HLA alleles and those targeted by nonprotective HLA alleles. We observed that the effects of Gag epitope mutations on HIV replicative fitness and MHC-I binding affinity are among the major determinants of CTL escape. IMPORTANCE Understanding the mechanism of viral control achieved in long-term nonprogressors with protective HLA alleles provides insights for developing functional cure of HIV infection. Through the characterization of CTL escape mutations in infected persons, previous researchers hypothesized that protective alleles target epitopes where escape mutations significantly reduce viral replicative capacity. However, these studies were usually limited to a few mutations observed in vivo. Here we utilized our recently developed high-throughput fitness profiling method to quantitatively measure the fitness of mutations across the entirety of HIV-1 Gag. The data enabled us to integrate the results with in silico prediction of MHC-peptide binding affinity and analysis of intraperson virus evolution to systematically determine the differences in CTL escape mutations between epitopes targeted by protective HLA alleles and those targeted by nonprotective HLA alleles. We observed that the effects of Gag epitope mutations on HIV replicative fitness and MHC-I binding affinity are among the major determinants of CTL escape.

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Phosphoserine acidic cluster motifs in the cytoplasmic domains of transmembrane proteins bind distinct basic regions on the μ subunits of clathrin adaptor protein complexes

10.1101/286633 ◽

2018 ◽

Author(s):

Rajendra Singh ◽

Charlotte Stoneham ◽

Christopher Lim ◽

Xiaofei Jia ◽

Javier Guenaga ◽

...

Keyword(s):

Protein Trafficking ◽

Transmembrane Proteins ◽

Adaptor Protein ◽

Cellular Protein ◽

Membrane Lipid ◽

Dependent Manner ◽

Cytoplasmic Domains ◽

Clathrin Adaptor ◽

Hiv 1

AbstractProtein trafficking in the endosomal system involves the recognition of specific signals within the cytoplasmic domains (CDs) of transmembrane proteins by clathrin adaptors. One such signal is the phosphoserine acidic cluster (PSAC), the prototype of which is in the endoprotease Furin. How PSACs are recognized by clathrin adaptors has been controversial. We reported previously that HIV-1 Vpu, which modulates cellular immunoreceptors, contains a PSAC that binds to the µ subunits of clathrin adaptor protein (AP) complexes. Here, we show that the CD of Furin binds the µ subunits of AP-1 and AP-2 in a phosphorylation-dependent manner. Moreover, we identify a PSAC in a cytoplasmic loop of the cellular transmembrane Serinc3, an inhibitor of the infectivity of retroviruses. The two serines within the PSAC of Serinc3 are phosphorylated by casein kinase II and mediate interaction with the µ subunits in vitro. The sites of these serines vary among mammals in a manner consistent with host-pathogen conflict, yet the Serinc3-PSAC seems dispensible for anti-HIV activity and for counteraction by HIV-1 Nef. The CDs of Vpu, Furin, and the PSAC-containing loop of Serinc3 each bind the μ subunit of AP-2 (µ2) with similar affinities, but they appear to utilize different basic regions on µ2. The Serinc3 loop requires a region previously reported to bind the acidic plasma membrane lipid phosphatidylinositol-4,5-bisphosphate. These data suggest that the PSACs within different proteins recognize different basic regions on the µ surface, providing the potential to inhibit the activity of viral proteins without necessarily affecting cellular protein trafficking.

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Structure of HIV-2 Nef reveals unique features distinct from HIV-1 involved in immune regulation

10.1101/779439 ◽

2019 ◽

Author(s):

Kengo Hirao ◽

Sophie Andrews ◽

Kimiko Kuroki ◽

Hiroki Kusaka ◽

Takashi Tadokoro ◽

...

Keyword(s):

Immune Evasion ◽

Immune Regulation ◽

Titration Calorimetry ◽

Viral Control ◽

Mhc I ◽

Infected People ◽

Sorting Motif ◽

Α Helix ◽

Hiv 1

SummaryThe HIV accessory protein Nef plays a major role in establishing and maintaining infection, particularly through immune evasion. Many HIV-2 infected people experience long-term viral control and survival, resembling HIV-1 elite control. HIV-2 Nef has overlapping but also distinct functions from HIV-1 Nef. Here we report the crystal structure of HIV-2 Nef core. The dileucine sorting motif forms a helix bound to neighboring molecules, and moreover, isothermal titration calorimetry demonstrated that the CD3 endocytosis motif can directly bind to HIV-2 Nef, ensuring AP-2 mediated endocytosis for CD3. The highly-conserved C-terminal region forms a α-helix, absent from HIV-1. We further determined the structure of SIV Nef harboring this region, demonstrating similar C-terminal α-helix, which may contribute to AP-1 binding for MHC-I downregulation. These results provide new insights into the distinct pathogenesis of HIV-2 infection.

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Loss of primitive hematopoietic progenitors in patients with human immunodeficiency virus infection

Blood ◽

10.1182/blood.v88.12.4568.bloodjournal88124568 ◽

1996 ◽

Vol 88 (12) ◽

pp. 4568-4578 ◽

Cited By ~ 60

Author(s):

A Marandin ◽

A Katz ◽

E Oksenhendler ◽

M Tulliez ◽

F Picard ◽

...

Keyword(s):

Flow Cytometry ◽

Human Immunodeficiency Virus ◽

Long Term Culture ◽

Cd34 Cells ◽

Immunodeficiency Virus ◽

Cell Fractions ◽

Cd38 Antigen ◽

Hiv 1

A number of hematologic abnormalities, including cytopenias, have been observed in patients with human immunodeficiency virus (HIV) infection. To elucidate their mechanisms, primitive cells from bone marrow aspirates of 21 patients with HIV-1 infection were quantitated by flow cytometry. The mean percentage of CD34+ cells is not significantly altered in HIV-1-infected patients in comparison with HIV-1- seronegative controls. In contrast, two- and three-color immunofluorescence analysis showed that in all HIV-1 samples, most CD34+ cells coexpressed the CD38 antigen. The proportion of HIV-1- derived CD34+ cells that did not express the CD38 antigen was significantly lower (HIV-1+: mean, 1.73%; controls: mean, 14%; P < .0005) than in controls. Moreover, of Thy-1+ cells, the proportion of CD34+ cells was twofold lower in HIV-1-infected patients (HIV-1+: mean, 12%; controls, 25%, P < .0005), which suggests that phenotypically primitive cells are depleted in HIV-1 infection. In vitro functional analysis in long-term cultures of sorted CD34+ cells from seven HIV-1 patients showed that CD34+ cells from HIV-1 patients generated much fewer colonies both in the nonadherent and adherent layers than CD34+ cells from controls after 5 weeks of culture (10-fold and four-fold less, respectively). Precise long-term culture initiating cell (LTC-IC) frequency in the CD34+ cell population was determined in three patients by limiting dilution and was markedly decreased in comparison to that of normal controls (from twofold to > sevenfold decreased). To determine if primitive cells were infected by HIV-1, both methylcellulose colonies generated from long-term culture of CD34+ cells and various CD34+ cell fractions purified by flow cytometry were evaluated for the presence of HIV-1 by polymerase chain reaction (PCR). Progeny from long-term culture was HIV-1-negative in three samples. In addition, using a sensitive PCR technique, the HIV-1 genome could not be detected in CD34+, CD34+/CD38-, and CD34+/CD4+ cells. These data show that hematologic disorders in HIV disease may be the consequence of a deficit of primitive cells. However, direct infection of these cells by HIV-1 does not seem to be responsible for this defect.

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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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The Structure-Activity Relationships of 2,4(1H,3H)-pyrimidinedione Derivatives as potent HIV type 1 and type 2 inhibitors

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632020701800502 ◽

2007 ◽

Vol 18 (5) ◽

pp. 259-275 ◽

Cited By ~ 30

Author(s):

Robert W Buckheit ◽

Tracy L Hartman ◽

Karen M Watson ◽

Ho Seok Kwon ◽

Sun Hwan Lee ◽

...

Keyword(s):

Antiviral Activity ◽

Binding Pocket ◽

Target Cells ◽

Significant Activity ◽

In Vitro Test ◽

Structure Activity Relationships ◽

Structure Activity ◽

Test Concentration ◽

Hiv 1

Since the discovery of the 2,4 (1 H,3 H)-pyrimidinediones as potent non-nucleoside inhibitors of the HIV-1 reverse transcriptase (RT) this class of compounds has yielded a number of N-1 acyclic substituted pyrimidinediones with substantial antiviral activity, which is highly dependent upon their molecular fit into the binding pocket common to this inhibitory class. We have specifically examined the structure activity relationships of compounds with chemical modification made by substituting homocyclic rather than acyclic moieties at N-1 of the pyrimidinedione. Seventy-four compounds were synthesized and evaluated for antiviral activity against HIV-1 and HIV-2. The homocyclic modifications resulted in compounds with significant activity against both HIV-1 and HIV-2, suggesting these compounds represent a new class of non-nucleoside RT inhibitors. The structure-activity relationship (SAR) evaluations indicated that cyclopropyl, phenyl and 1- or 3-cyclopenten-1-yl substitutions at the N-1 of the pyrimidinedione, the addition of a methyl linker between the cyclic moiety and the N-1 and the addition of a benzoyl group at the C-6 of the pyrimidinedione had the greatest contribution to antiviral activity. Five pyrimidinedione analogues with therapeutic indexes (TIs)>450,000 and a specific analogue (1-cyclopropylmethyl-5-isopropyl-6-(3,5-dimethylbenzoyl)-2,4(1 H,3 H)-pyrimidinedione), which exhibited a TI of >2,000,000, were identified. None of the analogues were cytotoxic to target cells at the highest in vitro test concentration, which is the upper limit of compound solubility of the analogues in aqueous solution. Thus, we have identified a series of pyrimidinediones with substantially improved antiviral efficacy and range of action and with significantly reduced cellular cytotoxicity.

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Chemokine‐Independent In Vitro Resistance to Human Immunodeficiency Virus (HIV‐1) Correlating with Low Viremia in Long‐Term and Recently Infected HIV‐1‐Positive Persons

The Journal of Infectious Diseases ◽

10.1086/514109 ◽

1997 ◽

Vol 176 (5) ◽

pp. 1168-1174 ◽

Cited By ~ 11

Author(s):

David H. Schwartz ◽

Renan C. Castillo ◽

Silvio Arango‐Jaramillo ◽

Usha K. Sharma ◽

Hai Feng Song ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Immunodeficiency Virus ◽

Hiv 1

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Genetic Analyses of Conserved Residues in the Carboxyl-Terminal Domain of Human Immunodeficiency Virus Type 1 Integrase

Journal of Virology ◽

10.1128/jvi.79.16.10356-10368.2005 ◽

2005 ◽

Vol 79 (16) ◽

pp. 10356-10368 ◽

Cited By ~ 55

Author(s):

Richard Lu ◽

Hina Z. Ghory ◽

Alan Engelman

Keyword(s):

Human Immunodeficiency Virus ◽

Reverse Transcription ◽

Class Ii ◽

Class I ◽

In Vitro Assays ◽

Mutant Proteins ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Results of in vitro assays identified residues in the C-terminal domain (CTD) of human immunodeficiency virus type 1 (HIV-1) integrase (IN) important for IN-IN and IN-DNA interactions, but the potential roles of these residues in virus replication were mostly unknown. Sixteen CTD residues were targeted here, generating 24 mutant viruses. Replication-defective mutants were typed as class I (blocked at integration) or class II (additional reverse transcription and/or assembly defects). Most defective viruses (15 of 17) displayed reverse transcription defects. In contrast, replication-defective HIV-1E246K synthesized near-normal cDNA levels but processing of Pr55 g ag was largely inhibited in virus-producing cells. Because single-round HIV-1E246K.Luc(R-) transduced cells at approximately 8% of the wild-type level, we concluded that the late-stage processing defect contributed significantly to the overall replication defect of HIV-1E246K. Results of complementation assays revealed that the CTD could function in trans to the catalytic core domain (CCD) in in vitro assays, and we since determined that certain class I and class II mutants defined a novel genetic complementation group that functioned in cells independently of IN domain boundaries. Seven of eight novel Vpr-IN mutant proteins efficiently trans-complemented class I active-site mutant virus, demonstrating catalytically active CTD mutant proteins during infection. Because most of these mutants inefficiently complemented a class II CCD mutant virus, the majority of CTD mutants were likely more defective for interactions with cellular and/or viral components that affected reverse transcription and/or preintegration trafficking than the catalytic activity of the IN enzyme.

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Compensatory Substitutions in the HIV-1 Capsid Reduce the Fitness Cost Associated with Resistance to a Capsid-Targeting Small-Molecule Inhibitor

Journal of Virology ◽

10.1128/jvi.01411-14 ◽

2014 ◽

Vol 89 (1) ◽

pp. 208-219 ◽

Cited By ~ 18

Author(s):

Jiong Shi ◽

Jing Zhou ◽

Upul D. Halambage ◽

Vaibhav B. Shah ◽

Mallori J. Burse ◽

...

Keyword(s):

Amino Acid ◽

Small Molecule ◽

Therapeutic Target ◽

Binding Pocket ◽

Host Protein ◽

Viral Capsid ◽

Inhibitory Protein ◽

Hiv 1 ◽

Ca Protein

ABSTRACTThe HIV-1 capsid plays multiple roles in infection and is an emerging therapeutic target. The small-molecule HIV-1 inhibitor PF-3450074 (PF74) blocks HIV-1 at an early postentry stage by binding the viral capsid and interfering with its function. Selection for resistance resulted in accumulation of five amino acid changes in the viral CA protein, which collectively reduced binding of the compound to HIV-1 particles. In the present study, we dissected the individual and combinatorial contributions of each of the five substitutions Q67H, K70R, H87P, T107N, and L111I to PF74 resistance, PF74 binding, and HIV-1 infectivity. Q67H, K70R, and T107N each conferred low-level resistance to PF74 and collectively conferred strong resistance. The substitutions K70R and L111I impaired HIV-1 infectivity, which was partially restored by the other substitutions at positions 67 and 107. PF74 binding to HIV-1 particles was reduced by the Q67H, K70R, and T107N substitutions, consistent with the location of these positions in the inhibitor-binding pocket. Replication of the 5Mut virus was markedly impaired in cultured macrophages, reminiscent of the previously reported N74D CA mutant. 5Mut substitutions also reduced the binding of the host protein CPSF6 to assembled CA complexesin vitroand permitted infection of cells expressing the inhibitory protein CPSF6-358. Our results demonstrate that strong resistance to PF74 requires accumulation of multiple substitutions in CA to inhibit PF74 binding and compensate for fitness impairments associated with some of the sequence changes.IMPORTANCEThe HIV-1 capsid is an emerging drug target, and several small-molecule compounds have been reported to inhibit HIV-1 infection by targeting the capsid. Here we show that resistance to the capsid-targeting inhibitor PF74 requires multiple amino acid substitutions in the binding pocket of the CA protein. Three changes in CA were necessary to inhibit binding of PF74 while maintaining viral infectivity. Replication of the PF74-resistant HIV-1 mutant was impaired in macrophages, likely owing to altered interactions with host cell factors. Our results suggest that HIV-1 resistance to capsid-targeting inhibitors will be limited by functional constraints on the viral capsid protein. Therefore, this work enhances the attractiveness of the HIV-1 capsid as a therapeutic target.

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