Shilei Ding

The Interferon-Inducible MxB Protein Inhibits HIV-1 Infection

The interferon-inducible myxovirus resistance (Mx) proteins play important roles in combating a wide range of virus infections. MxA inhibits many RNA and DNA viruses, whereas the antiviral activity of MxB is less well established. We find that human MxB inhibits HIV-1 infection by reducing the level of integrated viral DNA. Passaging HIV-1 through MxB-expressing cells allowed the evolution of a mutant virus that escapes MxB restriction. HIV-1 escapes MxB restriction by mutating the alanine residue at position 88 in the viral capsid protein (CA), with a consequent loss of CA interaction with the host peptidylprolyl isomerase cyclophilin A (CypA), suggesting a role for CypA in MxB restriction. Consistent with this, MxB associates with CypA, and shRNA-mediated CypA depletion or cyclosporine A treatment resulted in the loss of MxB inhibition of HIV-1. Taken together, we conclude that human MxB protein inhibits HIV-1 DNA integration by a CypA-dependent mechanism.

IFITM Proteins Restrict Viral Membrane Hemifusion

The interferon-inducible transmembrane (IFITM) protein family represents a new class of cellular restriction factors that block early stages of viral replication; the underlying mechanism is currently not known. Here we provide evidence that IFITM proteins restrict membrane fusion induced by representatives of all three classes of viral membrane fusion proteins. IFITM1 profoundly suppressed syncytia formation and cell-cell fusion induced by almost all viral fusion proteins examined; IFITM2 and IFITM3 also strongly inhibited their fusion, with efficiency somewhat dependent on cell types. Furthermore, treatment of cells with IFN also markedly inhibited viral membrane fusion and entry. By using the Jaagsiekte sheep retrovirus envelope and influenza A virus hemagglutinin as models for study, we showed that IFITM-mediated restriction on membrane fusion is not at the steps of receptor- and/or low pH-mediated triggering; instead, the creation of hemifusion was essentially blocked by IFITMs. Chlorpromazine (CPZ), a chemical known to promote the transition from hemifusion to full fusion, was unable to rescue the IFITM-mediated restriction on fusion. In contrast, oleic acid (OA), a lipid analog that generates negative spontaneous curvature and thereby promotes hemifusion, virtually overcame the restriction. To explore the possible effect of IFITM proteins on membrane molecular order and fluidity, we performed fluorescence labeling with Laurdan, in conjunction with two-photon laser scanning and fluorescence-lifetime imaging microscopy (FLIM). We observed that the generalized polarizations (GPs) and fluorescence lifetimes of cell membranes expressing IFITM proteins were greatly enhanced, indicating higher molecularly ordered and less fluidized membranes. Collectively, our data demonstrated that IFITM proteins suppress viral membrane fusion before the creation of hemifusion, and suggested that they may do so by reducing membrane fluidity and conferring a positive spontaneous curvature in the outer leaflets of cell membranes. Our study provides novel insight into the understanding of how IFITM protein family restricts viral membrane fusion and infection.

The N-Terminal Region of IFITM3 Modulates Its Antiviral Activity by Regulating IFITM3 Cellular Localization

ABSTRACT Interferon-inducible transmembrane (IFITM) protein family members IFITM1, -2, and -3 restrict the infection of multiple enveloped viruses. Significant enrichment of a minor IFITM3 allele was recently reported for patients who were hospitalized for seasonal and 2009 H1N1 pandemic flu. This IFITM3 allele lacks the region corresponding to the first amino-terminal 21 amino acids and is unable to inhibit influenza A virus. In this study, we found that deleting this 21-amino-acid region relocates IFITM3 from the endosomal compartments to the cell periphery. This finding likely underlies the lost inhibition of influenza A virus that completes its entry exclusively within endosomes at low pH. Yet, wild-type IFITM3 and the mutant with the 21-amino-acid deletion inhibit HIV-1 replication equally well. Given the pH-independent nature of HIV-1 entry, our results suggest that IFITM3 can inhibit viruses that enter cells via different routes and that its N-terminal region is specifically required for controlling pH-dependent viruses.

IFITM Proteins Restrict HIV-1 Infection by Antagonizing the Envelope Glycoprotein

Summary The interferon-induced transmembrane (IFITM) proteins have been recently shown to restrict HIV-1 and other viruses. Here, we provide evidence that IFITM proteins, particularly IFITM2 and IFITM3, specifically antagonize the HIV-1 envelope glycoprotein (Env), thereby inhibiting viral infection. IFITM proteins interact with HIV-1 Env in viral producer cells, leading to impaired Env processing and virion incorporation. Notably, the level of IFITM incorporation into HIV-1 virions does not strictly correlate with the extent of inhibition. Prolonged passage of HIV-1 in IFITM-expressing T lymphocytes leads to emergence of Env mutants that overcome IFITM restriction. The ability of IFITMs to inhibit cell-to-cell infection can be extended to HIV-1 primary isolates, HIV-2 and SIVs; however, the extent of inhibition appears to be virus-strain dependent. Overall, our study uncovers a mechanism by which IFITM proteins specifically antagonize HIV-1 Env to restrict HIV-1 infection and provides insight into the specialized role of IFITMs in HIV infection.

A Highly Conserved Residue of the HIV-1 gp120 Inner Domain Is Important for Antibody-Dependent Cellular Cytotoxicity Responses Mediated by Anti-cluster A Antibodies

ABSTRACT Previous studies have shown that sera from HIV-1-infected individuals contain antibodies able to mediate antibody-dependent cellular cytotoxicity (ADCC). These antibodies preferentially recognize envelope glycoprotein (Env) epitopes induced upon CD4 binding. Here, we show that a highly conserved tryptophan at position 69 of the gp120 inner domain is important for ADCC mediated by anti-cluster A antibodies and sera from HIV-1-infected individuals.

Small CD4 Mimetics Prevent HIV-1 Uninfected Bystander CD4 + T Cell Killing Mediated by Antibody-dependent Cell-mediated Cytotoxicity

Human immunodeficiency virus type 1 (HIV-1) infection causes a progressive depletion of CD4 + T cells. Despite its importance for HIV-1 pathogenesis, the precise mechanisms underlying CD4 + T-cell depletion remain incompletely understood. Here we make the surprising observation that antibody-dependent cell-mediated cytotoxicity (ADCC) mediates the death of uninfected bystander CD4 + T cells in cultures of HIV-1-infected cells. While HIV-1-infected cells are protected from ADCC by the action of the viral Vpu and Nef proteins, uninfected bystander CD4 + T cells bind gp120 shed from productively infected cells and are efficiently recognized by ADCC-mediating antibodies. Thus, gp120 shedding represents a viral mechanism to divert ADCC responses towards uninfected bystander CD4 + T cells. Importantly, CD4-mimetic molecules redirect ADCC responses from uninfected bystander cells to HIV-1-infected cells; therefore, CD4-mimetic compounds might have therapeutic utility in new strategies aimed at specifically eliminating HIV-1-infected cells.

HIV-1 Mutates to Evade IFITM1 Restriction

Abstract Interferon-induced transmembrane (IFITM) proteins inhibit the infection of a wide range of viruses including human immunodeficiency virus type 1 (HIV-1). At present, little is known about how viruses overcome IFITM restriction. In this study, we have utilized HIV-1 as a model and selected IFITM1-resistant viruses after multiple passages of HIV-1 in IFITM1-expressing SupT1 cells. Sequencing the entire viral genome revealed several mutations in the vpu and envelope genes, among which mutations Vpu34 and EnvG367E together enable efficient HIV-1 replication in IFITM1-expressing cells. Vpu34 introduces a stop codon at amino acid position 35 of Vpu, whereas EnvG367E changes the G367 residue at the CD4-binding site of gp120. These two mutations do not appear to overcome the downregulation of viral p24 expression caused by IFITM1, but rather enhance HIV-1 replication by promoting cell-to-cell virus transmission. Altogether, our data demonstrate that HIV-1 can mutate to evade IFITM1 restriction by increasing cell-to-cell transmission.

Co-receptor Binding Site Antibodies Enable CD4-Mimetics to Expose Conserved Anti-cluster A ADCC Epitopes on HIV-1 Envelope Glycoproteins

Human immunodeficiency virus type 1 (HIV-1) has evolved a sophisticated strategy to conceal conserved epitopes of its envelope glycoproteins (Env) recognized by antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies. These antibodies, which are present in the sera of most HIV-1-infected individuals, preferentially recognize Env in its CD4-bound conformation. Accordingly, recent studies showed that small CD4-mimetics (CD4mc) able to “push” Env into this conformation sensitize HIV-1-infected cells to ADCC mediated by HIV + sera. Here we test whether CD4mc also expose epitopes recognized by anti-cluster A monoclonal antibodies such as A32, thought to be responsible for the majority of ADCC activity present in HIV + sera and linked to decreased HIV-1 transmission in the RV144 trial. We made the surprising observation that CD4mc are unable to enhance recognition of HIV-1-infected cells by this family of antibodies in the absence of antibodies such as 17b, which binds a highly conserved CD4-induced epitope overlapping the co-receptor binding site (CoRBS). Our results indicate that CD4mc initially open the trimeric Env enough to allow the binding of CoRBS antibodies but not anti-cluster A antibodies. CoRBS antibody binding further opens the trimeric Env, allowing anti-cluster A antibody interaction and sensitization of infected cells to ADCC. Therefore, ADCC responses mediated by cluster A antibodies in HIV-positive sera involve a sequential opening of the Env trimer on the surface of HIV-1-infected cells. The understanding of the conformational changes required to expose these vulnerable Env epitopes might be important in the design of new strategies aimed at fighting HIV-1.

Nef Proteins from HIV-1 Elite Controllers Are Inefficient at Preventing Antibody-Dependent Cellular Cytotoxicity

ABSTRACT Impairment of Nef function, including reduced CD4 downregulation, was described in a subset of HIV-1-infected individuals that control viral replication without antiretroviral treatment (elite controllers [EC]). Elimination of HIV-1-infected cells by antibody-dependent cellular cytotoxicity (ADCC) requires the presence of envelope glycoproteins (Env) in the CD4-bound conformation, raising the possibility that accumulating CD4 at the surface of virus-infected cells in EC could interact with Env and thereby sensitize these cells to ADCC. We observed a significant increase in the exposure of Env epitopes targeted by ADCC-mediating antibodies at the surface of cells expressing Nef isolates from EC; this correlated with enhanced susceptibility to ADCC. Altogether, our results suggest that enhanced susceptibility of HIV-1-infected cells to ADCC may contribute to the EC phenotype. IMPORTANCE Nef clones derived from elite controllers (EC) have been shown to be attenuated for CD4 downregulation; how this contributes to the nonprogressor phenotype of these infected individuals remains uncertain. Increasing evidence supports a role for HIV-specific antibody-dependent cellular cytotoxicity (ADCC) in controlling viral infection and replication. Here, we show that residual CD4 left at the surface of cells expressing Nef proteins isolated from ECs are sufficient to allow Env-CD4 interaction, leading to increased exposure of Env CD4-induced epitopes and increased susceptibility of infected cells to ADCC. Our results suggest that ADCC might be an active immune mechanism in EC that helps to maintain durable suppression of viral replication and low plasma viremia level in this rare subset of infected individuals. Therefore, targeting Nefs ability to downregulate CD4 could render HIV-1-infected cells susceptible to ADCC and thus have therapeutic utility.

Primate lentiviruses are differentially inhibited by interferon-induced transmembrane proteins

Abstract Interferon-induced transmembrane (IFITM) proteins inhibit the entry of a large number of viruses. Not surprisingly, many viruses are refractory to this inhibition. In this study, we report that different strains of HIV and SIV are inhibited by human IFITM proteins to various degrees, with SIV of African green monkeys (SIVAGM) being mostly restricted by human IFITM2. Interestingly, SIVAGM is as much inhibited by human IFITM2 as by IFITM3 of its own host African green monkeys. Our data further demonstrate that the entry of SIVAGM is impaired by human IFITM2 and that this inhibition is overcome by the cholesterol-binding compound amphotericin B that also overcomes IFITM inhibition of influenza A viruses. These results suggest that IFITM proteins exploit similar mechanisms to inhibit the entry of both pH-independent primate lentiviruses and the pH-dependent influenza A viruses.