Subunit organization of the membrane-bound HIV-1 envelope glycoprotein trimer

The trimeric human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) spike is a molecular machine that mediates virus entry into host cells and is the sole target for virus-neutralizing antibodies. The mature Env spike results from cleavage of a trimeric gp160 precursor into three gp120 and three gp41 subunits. Here we describe an ~11-Å cryo-EM structure of the trimeric HIV-1 Env precursor in its unliganded state. The three gp120 and three gp41 subunits form a cage-like structure with an interior void surrounding the trimer axis. Interprotomer contacts are limited to the gp41 transmembrane region, the torus-like gp41 ectodomain, and a gp120 trimer association domain composed of the V1/V2 and V3 variable regions. The cage-like architecture, which is unique among characterized viral envelope proteins, restricts antibody access, reflecting requirements imposed by HIV-1 persistence in the host

Neutralization Efficiency Is Greatly Enhanced by Bivalent Binding of an Antibody to Epitopes in the V4 Region and the Membrane-Proximal External Region within One Trimer of Human Immunodeficiency Virus Type 1 Glycoproteins▿

Most antibodies are multivalent, with the potential to bind with high avidity. However, neutralizing antibodies commonly bind to virions monovalently. Bivalent binding of a monoclonal antibody (MAb) to a virion has been documented only in a single case. Thus, the role of high avidity in antibody-mediated neutralization of viruses has not been defined clearly. In this study, we demonstrated that when an artificial 2F5 epitope was inserted in the gp120 V4 region so that an HIV-1 envelope glycoprotein (Env) trimer contains a natural 2F5 epitope in the gp41 membrane-proximal envelope region (MPER) and an artificially engineered 2F5 epitope in the gp120 V4 region, bivalent 2F5 IgG achieved greatly enhanced neutralization efficiency, with a 50% inhibitory concentration (IC50) decrease over a 2-log scale. In contrast, the monovalent 2F5 Fab fragment did not exhibit any appreciable change in neutralization efficiency in the same context. These results demonstrate that bivalent binding of 2F5 IgG to a single HIV-1 Env trimer results in dramatic enhancement of neutralization, probably through an increase in binding avidity. Furthermore, we demonstrated that bivalent binding of MAb 2F5 to the V4 region and MPER of an HIV-1 Env trimer can be achieved only in a specific configuration, providing an important insight into the structure of a native/infectious HIV-1 Env trimer. This specific binding configuration also establishes a useful standard that can be applied to evaluate the biological relevance of structural information on the HIV-1 Env trimer

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

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

Stoichiometry of Antibody Neutralization of Human Immunodeficiency Virus Type 1

The human immunodeficiency virus envelope glycoproteins function as trimers on the viral surface, where they are targeted by neutralizing antibodies. Different monoclonal antibodies neutralize human immunodeficiency virus type 1 (HIV-1) infectivity by binding to structurally and functionally distinct moieties on the envelope glycoprotein trimer. By measuring antibody neutralization of viruses with mixtures of neutralization-sensitive and neutralization-resistant envelope glycoproteins, we demonstrate that the HIV-1 envelope glycoprotein trimer is inactivated by the binding of a single antibody molecule. Virus neutralization requires essentially all of the functional trimers to be occupied by at least one antibody. This model applies to antibodies differing in neutralizing potency and to virus isolates with various neutralization sensitivities. Understanding these requirements for HIV-1 neutralization by antibodies will assist in establishing goals for an effective AIDS vaccine

Antibody Binding Is a Dominant Determinant of the Efficiency of Human Immunodeficiency Virus Type 1 Neutralization

Primary and laboratory-adapted variants of human immunodeficiency virus type 1 (HIV-1) exhibit a wide range of sensitivities to neutralization by antibodies directed against the viral envelope glycoproteins. An antibody directed against an artificial FLAG epitope inserted into the envelope glycoproteins of three HIV-1 isolates with vastly different neutralization sensitivities inhibited all three viruses equivalently. Thus, naturally occurring HIV-1 isolates that are neutralization resistant are not necessarily more impervious to the inhibitory consequences of bound antibody. Moreover, the binding affinity of the anti-FLAG antibody correlated with neutralizing potency, underscoring the dominant impact on neutralization of antibody binding to the envelope glycoproteins

Elicitation of Neutralizing Antibodies with DNA Vaccines Expressing Soluble Stabilized Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Trimers Conjugated to C3d

DNA vaccines expressing the envelope (Env) of human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting immune responses. Oligomeric or trimeric (gp140) forms of Env that more closely mimic the native proteins on the virion are often more effective immunogens than monomeric (gp120) envelopes. In this study, several forms of Env constructed from the HIV-1 isolate YU-2 (HIV-1(YU-2)) were tested for their immunogenic potential: a trimeric form of uncleaved (−) Env stabilized with a synthetic trimer motif isolated from the fibritin (FT) protein of the T4 bacteriophage, sgp140(YU-2)(−/FT), was compared to sgp140(YU-2)(−) without a synthetic trimerization domain, as well as to monomeric gp120(YU-2). DNA plasmids were constructed to express Env alone or fused to various copies of murine C3d (mC3d). BALB/c mice were vaccinated (day 1 and week 4) with DNA expressing a codon-optimized envelope gene insert, alone or fused to mC3d. Mice were subsequently boosted (week 8) with the DNA or recombinant Env protein. All mice had high anti-Env antibody titers regardless of the use of mC3d. Sera from mice vaccinated with DNA expressing non-C3d-fused trimers elicited neutralizing antibodies against homologous HIV-1(YU-2) virus infection in vitro. In contrast, sera from mice inoculated with DNA expressing Env-C3d protein trimers elicited antibody that neutralized both homologous HIV-1(YU-2) and heterologous HIV-1(ADA), albeit at low titers. Therefore, DNA vaccines expressing trimeric envelopes coupled to mC3d, expressed in vivo from codon-optimized sequences, elicit low titers of neutralizing antibodies against primary isolates of HIV-1

Stoichiometry of Envelope Glycoprotein Trimers in the Entry of Human Immunodeficiency Virus Type 1

The human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Envs) function as a trimer, mediating virus entry by promoting the fusion of the viral and target cell membranes. HIV-1 Env trimers induce membrane fusion through a pH-independent pathway driven by the interaction between an Env trimer and its cellular receptors, CD4 and CCR5/CXCR4. We studied viruses with mixed heterotrimers of wild-type and dominant-negative Envs to determine the number (T) of Env trimers required for HIV-1 entry. To our surprise, we found that a single Env trimer is capable of supporting HIV-1 entry; i.e., T = 1. A similar approach was applied to investigate the entry stoichiometry of envelope glycoproteins from amphotropic murine leukemia virus (A-MLV), avian sarcoma/leukosis virus type A (ASLV-A), and influenza A virus. When pseudotyped on HIV-1 virions, the A-MLV and ASLV-A Envs also exhibit a T = 1 entry stoichiometry. In contrast, eight to nine influenza A virus hemagglutinin trimers function cooperatively to achieve membrane fusion and virus entry, using a pH-dependent pathway. The different entry requirements for cooperativity among Env trimers for retroviruses and influenza A virus may influence viral strategies for replication and evasion of the immune system

Subunit Stoichiometry of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Trimers during Virus Entry into Host Cells

The envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) function as a homotrimer of gp120/gp41 heterodimers to support virus entry. During the process of virus entry, an individual HIV-1 envelope glycoprotein trimer binds the cellular receptors CD4 and CCR5/CXCR4 and mediates the fusion of the viral and the target cellular membranes. By studying the function of heterotrimers between wild-type and nonfunctional mutant envelope glycoproteins, we found that two wild-type subunits within an envelope glycoprotein trimer are required to support virus entry. Complementation between HIV-1 envelope glycoprotein mutants defective in different functions to allow virus entry was not evident. These results assist our understanding of the mechanisms whereby the HIV-1 envelope glycoproteins mediate virus entry and membrane fusion and guide attempts to inhibit these processes