Functional formation of domain V of the poliovirus noncoding region: significance of unpaired bases

The Env protein of human immunodeficiency virus type 1 is assembled into a stable trimer, and oligomerization is required for maintenance of viral infectivity. This property of Env suggests that Env mutants may have a dominant-negative effect on virus infectivity. To investigate this possibility, we established a packaging cell line in which both wild-type and mutant Env proteins could be expressed simultaneously in a single cell. We analyzed the effects of two types of Env mutants: cytoplasmic tail-truncated TM mutants and a mutant defective in gp120/gp41 cleavage. The cytoplasmic tail-truncated proteins were found to be incorporated into virions by forming an oligomer with wild-type TM, but could not inhibit the wild-type function. In contrast, phenotypic mixing of cleavage-defective Env with the wild-type protein caused dramatic inhibition of infectivity, indicating that this mutant has a strong dominant-negative phenotype.     

Dominant-negative effect of hetero-oligomerization on the function of the human immunodeficiency virus type 1 envelope glycoprotein complex

The human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein forms trimers that mediate interactions with the CD4 receptor and a co-receptor on the target cell surface, thereby triggering viral fusion with the cell membrane. Cleavage of Env into its surface, gp120, and transmembrane, gp41, moieties is necessary for activation of its fusogenicity. Here, we produced pseudoviruses with phenotypically mixed wild-type (Wt) and mutant, cleavage-incompetent Env in order to quantify the effects of incorporating uncleaved Env on virion infectivity, antigenicity and neutralization sensitivity. We modeled the relative infectivity of three such phenotypically mixed viral strains, JR-FL, HXBc2 and a derivative of the latter, 3.2P, as a function of the relative amount of Wt Env. The data were fit very closely (R(2) > 0.99) by models which assumed that only Wt homotrimers were functional, with different approximate thresholds of critical numbers of functional trimers per virion for the three strains. We also produced 3.2P pseudoviruses containing both a cleavage-competent Env that is defective for binding the neutralizing monoclonal antibody (NAb) 2G12, and a cleavage-incompetent Env that binds 2G12. The 2G12 NAb was not able to reduce the infectivity of these pseudoviruses detectably. Their neutralization by the CD4-binding site-directed agents CD4-IgG2 and NAb b12 was also unaffected by 2G12 binding to uncleaved Env. These results further strengthen the conclusion that only homotrimers consisting of cleaved Env are functional. They also imply that the function of a trimer is unaffected sterically by the binding of an antibody to an adjacent trimer.     

Longitudinal study of antibody reactivity against HIV-1 envelope and a peptide representing a conserved site on Gp41 in HIV-1-infected patients.

This study was designed to distinguish between antibodies in HIV-1-infected patients directed against epitopes accessible on the native HIV-1 envelope (Env) complex and non-native Env epitopes. Peptide p#13 (Env. aa642-673) containing the neutralising 2F5 epitope and recombinant soluble glycoprotein 160 (rsgp160) were used in ELISA to determine the antibody (Ab) reactivity in sera of 116 HIV-1-infected individuals and 18 HIV negative controls. The reactivity of sera classified CDC stage C against p#13 was significantly decreased in comparison to stage A sera, while staying constant against rsgp160. Accordingly, in 6 out of 8 individual patients tested over time the response against p#13 was declining at later time points of infection. The reactivity of patients' sera against p#13 corresponded directly to the recognition of infected T cells and largely also to the CD4 cell count. The causal relationships of these phenomena are not clear. It is conceivable that antibodies against epitopes on HIV are lost or escape mutants arise and consequently control of HIV is lost and virus load increases as it is known for CDC stage C. Alternatively, increasing virus load may affect B cells recognising native Env epitopes and turn antibody production down by some mechanism. In this latter scenario helper T cells might have a critical role.     

In vitro binding of simian immunodeficiency virus matrix protein to the cytoplasmic domain of the envelope glycoprotein

Incorporation of the envelope (Env) glycoprotein into budding virions is a key step in the replication cycle of lentiviruses. Previously, we provided genetic and biochemical evidence indicating that Env packaging into simian immunodeficiency virus (SIV) particles is mediated by the association of the Env cytoplasmic domain (CD) with the matrix (MA) domain of Gag. In this study, we developed an in vitro binding assay that, based on recombinant proteins expressed in bacteria, allowed us to demonstrate the physical interaction between the SIV Env CD and the MA in the absence of other viral or cellular proteins. We show that this association is blocked by mutations in each of the interacting domains that have been reported to interfere in vivo with the incorporation of Env into SIV virions. Moreover, we determined that the binding of SIV MA to the Env CD is saturable with a dissociation constant of 7x10(-7) M. Interestingly, the SIV MA is capable of specifically interacting in vitro with the human immunodeficiency virus type 1 Env CD, but not with that of the distantly related feline immunodeficiency virus. Our results strongly support the notion that the association between the SIV MA and Env CD plays a central role in the process of SIV Env incorporation into Gag-made particles.     

Specific amino acids in the N-terminus of the gp41 ectodomain contribute to the stabilization of a soluble, cleaved gp140 envelope glycoprotein from human immunodeficiency virus type 1

The HIV-1 envelope glycoprotein is expressed on the viral membrane as a trimeric complex, formed by three gp120 surface glycoproteins non-covalently associated with three membrane-anchored gp41 subunits. The labile nature of the association between gp120 and gp41 hinders the expression of soluble, fully cleaved, trimeric gp140 proteins for structural and immunization studies. Disruption of the primary cleavage site within gp160 allows the production of stable gp140 trimers, but cleavage-defective trimers are antigenically dissimilar from their cleaved counterparts. Soluble, stabilized, proteolytically cleaved, trimeric gp140 proteins can be generated by engineering an intermolecular disulfide bond between gp120 and gp41 (SOS), combined with a single residue change, I559P, within gp41 (SOSIP). We have found that SOSIP gp140 proteins based on the subtype A HIV-1 strain KNH1144 form particularly homogenous trimers compared to a prototypic strain (JR-FL, subtype B). We now show that the determinants of this enhanced stability are located in the N-terminal region of KNH11144 gp41 and that, when substituted into heterologous Env sequences (e.g., JR-FL and Ba-L) they have a similarly beneficial effect on trimer stability. The stabilized trimers retain the epitopes for several neutralizing antibodies (b12, 2G12, 2F5 and 4E10) and the CD4-IgG2 molecule, suggesting that the overall antigenic structure of the gp140 protein has not been adversely impaired by the trimer-stabilizing substitutions. The ability to increase the stability of gp140 trimers might be useful for neutralizing antibody-based vaccine strategies based on the use of this type of immunogen.     

Residues in the membrane-spanning domain core modulate conformation and fusogenicity of the HIV-1 envelope glycoprotein

The membrane-spanning domain (MSD) of human immunodeficiency virus type I (HIV-1) envelope glycoprotein (Env) is critical for its biological activity. Initial studies have defined an almost invariant “core” structure in the MSD and demonstrated that it is crucial for anchoring Env in the membrane and virus entry. We show here that amino acid substitutions in the MSD “core” do not influence specific virus-cell attachment, nor CD4 receptor and CXCR4 coreceptor recognition by Env. However, substitutions within the MSD “core” delayed the kinetics and reduced the efficiency of cell-cell fusion mediated by Env. Although we observed no evidence that membrane fusion mediated by the MSD core mutants was arrested at a hemifusion stage, impaired Env fusogenicity was correlated with minor conformational changes in the V2, C1, and C5 regions in gp120 and the immunodominant loop in gp41. These changes could delay initiation of the conformational changes required in the fusion process.     

Evolution of B cell analysis and Env trimer redesign

HIV-1 and its surface envelope glycoproteins (Env), gp120 and gp41, have evolved immune evasion strategies that render the elicitation of effective antibody responses to the functional Env entry unit extremely difficult. HIV-1 establishes chronic infection and stimulates vigorous immune responses in the human host; forcing selection of viral variants that escape cellular and antibody (Ab)-mediated immune pressure, yet possess contemporary fitness. Successful survival of fit variants through the gauntlet of the human immune system make this virus and these glycoproteins a formidable challenge to target by vaccination, requiring a systematic approach to Env mimetic immunogen design and evaluation of elicited responses. Here, we review key aspects of HIV-1 Env immunogenicity and immunogen re-design, based on experimental data generated by us and others over the past decade or more. We further provide rationale and details regarding the use of newly evolving tools to analyze B cell responses, including approaches to use next generation sequencing for antibody lineage tracing and B cell fate mapping. Together, these developments offer opportunities to address long-standing questions about the establishment of effective B cell immunity elicited by vaccination, not just against HIV-1.     

Two distinct mechanisms regulate recruitment of murine leukemia virus envelope protein to retroviral assembly sites

The cytoplasmic tail domain (CTD) of retroviral envelope (Env) proteins has been implicated in modulating Env incorporation into viral particles. We generated a panel of murine leukemia virus (MLV) Env mutants and analyzed their ability to be recruited to human immunodeficiency virus-1 (HIV-1) assembly sites. Surprisingly, the entire CTD was dispensable for recruitment to assembly sites, but a mutation that disrupted the furin cleavage site in Env abolished recruitment. To determine if MLV Env can show selectivity for homologous assembly sites, cells were co-transfected with both HIV-1 and MLV assembly components along with each MLV Env construct and assayed for infectious particle production. MLV Env selectively formed infectious particles with the MLV components at the expense of infectious HIV-1 infectious particle production, but truncation of the CTD progressively reduced this selectivity. Collectively these data suggest that there are two separable mechanisms that govern MLV Env recruitment to viral assembly sites.     

N-terminal substitutions in HIV-1 gp41 reduce the expression of non-trimeric envelope glycoproteins on the virus

The native, functional HIV-1 envelope glycoprotein (Env) complex is a trimer of two non-covalently associated subunits: the gp120 surface glycoprotein and the gp41 transmembrane glycoprotein. However, various non-functional forms of Env are present on virus particles and HIV-1-infected cells, some of which probably arise as the native complex decays. The aberrant forms include gp120-gp41 monomers and oligomers, as well as gp41 subunits from which gp120 has dissociated. The presence of non-functional Env creates binding sites for antibodies that do not recognize native Env complexes and that are, therefore, non-neutralizing. Non-native Env forms (monomers, dimers, tetramers and aggregates) can also arise when soluble gp140 proteins, lacking the cytoplasmic and transmembrane domains of gp41, are expressed for vaccine studies. We recently identified five amino acids in the gp41 N-terminal region (I535, Q543, S553, K567 and R588) that promote gp140 trimerization. We have now studied their influence on the function and antigenic properties of JR-FL Env expressed on the surfaces of pseudoviruses and Env-transfected cells. The 5 substitutions in gp41 reduce the expression of non-trimeric gp160s, without affecting trimer levels. Pseudovirions bearing the mutant Env are fully infectious with similar kinetics of Env-mediated fusion. Various non-neutralizing antibodies bind less strongly to the Env mutant, but neutralizing antibody binding is unaffected. Hence the gp41 substitutions do not adversely affect Env structure, supporting their use for making new Env-based vaccines. The mutant Env might also help in studies intended to correlate antibody binding to virus neutralization. Of note is that the 5 residues are much more frequent, individually or collectively, in viruses from subtypes other than B.     

Immunogenicity of full length and truncated SIV envelope proteins.

We have compared the immunogenicity of the full-length (FL) SIV envelope (Env) protein and a truncated (T) form of the Env protein which has a short cytoplasmic tail. The Env(T) protein was previously shown to be more fusogenic than Env(FL), has a higher level of incorporation into virus-like particles (VLPs) and membrane vesicles, and expands the viral host range. We have found that mice immunized with VLPs which contained an equal amount of Env(FL) or Env(T) produced similar titres of neutralizing antibody. Comparison of immune responses between animals that received DNA vaccines encoding Env(T) vs. Env (FL) by epidermal delivery demonstrated that a higher level of specific antibody was elicited by Env(T) than Env(FL). This result correlated with a higher level of expression of pCMVEnv(T) than pCMVEnv(FL) observed in vitro. DNA immunization combined with VLP boosting elicited a similar level of neutralizing antibody with both forms of Env proteins. These data indicate that the immunogenicity of Env(FL) and Env(T) is similar, and that either form of Env protein appears to be potentially suitable for use in further development of vaccine preparations.     

Generation of mucosal anti-human immunodeficiency virus type 1 T-cell responses by recombinant Mycobacterium smegmatis

A successful vaccine vector for human immunodeficiency virus type 1 (HIV-1) should induce anti-HIV-1 immune responses at mucosal sites. We have generated recombinant Mycobacterium smegmatis vectors that express the HIV-1 group M consensus envelope protein (Env) as a surface, intracellular, or secreted protein and have tested them in animals for induction of both anti-HIV-1 T-cell and antibody responses. Recombinant M. smegmatis engineered for expression of secreted protein induced optimal T-cell gamma interferon enzyme-linked immunospot assay responses to HIV-1 envelope in the spleen, female reproductive tract, and lungs. Unlike with the induction of T-cell responses, priming and boosting with recombinant M. smegmatis did not induce anti-HIV-1 envelope antibody responses, due primarily to insufficient protein expression of the insert. However, immunization with recombinant M. smegmatis expressing HIV-1 Env was able to prime for an HIV-1 Env protein boost for the induction of anti-HIV-1 antibody responses.     

Cross-reactivity of anti-HIV-1-p17-derivative peptide (P30-52) antibody to Env V3 peptide.

Strong antibody responses are often seen in human immunodeficiency virus type 1 (HIV-1) carriers, but it is not known whether these antibodies are effective in the inhibition of disease progression. In this study, we examined antigenic epitopes for anti-HIV-1 p17 antibody (p17 Ab) in an HIV-1 carrier's serum, and found that the residues of amino acid numbers 1 to 12 (P1-12), 12 to 29 (P12-29) and 30 to 52 (P30-52) of p17 were highly recognized in the serum. Our examination of purified antibodies from the patient using the p17-derivative-peptide-immunoaffinity columns showed that the reactivity of anti-p30-52 Ab (p30-52Ab) was high for p30-52 and the naive protein, p17. In addition, this P30-52Ab cross-reacted with the third variable region of the envelope glycoprotein (Env V3). To confirm this cross-reactivity, we immunized mice with P30-52, and established a monoclonal antibody (MAb), 8H10. We found that 8H10 was also reactive to Env V3. It is unclear whether this cross-reactivity of P30-52 Ab can function as the inhibitor of HIV-1, but these results will be of help in clarifying the interaction of Env protein with HIV-1 gag polyprotein and the relationship of the decline of the p17 antibody titer with the disease progression in HIV-1 carriers.     

HIV-1 resistance to the anti-HIV activity of a shRNA targeting a dual-coding region

We generated a lymphoid cell line (Sup-T1-Rev/Env) that stably expresses a 19-bp short hairpin RNA (shRNA) targeting a conserved region of HIV-1 encoding for the Envelope and Rev proteins, which potently inhibited viral replication. However, continuous passage of HIV-1 in Sup-T1-Rev/Env generated virus mutants able to overcome the RNAi restriction. Sequence analysis of the emerging viruses showed that mutations were located at positions 5 and 17 of the target sequence. Both mutations are silent in the Env frame, but the mutation 5 generated an amino acid change (V47M) in the Rev reading frame. We have analyzed the impact of these two mutations on the RNAi mechanism, showing a more crucial role of the mutation 17 in the resistance to RNAi. We show that even targeting a conserved region of the HIV-1 genome involved in the biosynthesis of two essential genes, env and rev, the virus could evolve to escape by single point mutations in the target sequence, without a significant fitness cost.     

HIV-1膜抗原部分糖基化位点改造对免疫原性及假病毒形成的影响

目的 研究HIV-1膜蛋白(Env)特定糖基化位点改造对Env免疫原性及功能性假病毒形成能力的影响.方法 采用环形诱变,DpnⅠ筛选的方法对Env进行定点突变,单周期感染试验检测功能性假病毒形成能力,免疫小鼠,利用假病毒中和试验与ELISPOT分别检测突变对中和抗体和T细胞分泌Env特异的IFN-γ的影响.结果 N197Q突变体使Env诱导中和抗体的能力提高而诱导T细胞分泌Env特异的IFN-γ的能力降低,并使Env不能形成功能性假病毒;G2突变体(N624Q,N637Q)诱导的中和抗体能更好地中和假病毒74-2,仉中和假病毒Wt的能力下降,对Env诱导T细胞分泌Env特异的IFN-γ的能力和功能性假病毒形成无明显影响.结论 特定糖基化位点的改造影响假病毒的形成及Env的免疫原性.     

Insertion of targeting domains into the envelope glycoprotein of Moloney murine leukemia virus (MoMLV)-based vectors modulates the route of mCAT-1-mediated viral entry

Several groups have inserted targeting domains into the envelope glycoprotein (Env) of Moloney murine leukemia virus (MoMLV) in an attempt to produce targeted retroviral vectors for human gene therapy. While binding of these modified Envs to the target molecule expressed on the surface of human cells was observed, specific high-titer infection of human cells expressing the target molecule was not achieved. Here we investigate the initial steps in the entry process of targeted MoMLV vectors both in murine and human cells expressing the MoMLV receptor, the mouse cationic amino acid transporter-1 (mCAT-1). We show that insertion of a small ligand targeted to E-selectin and of a single chain antibody (scFv) targeted to folate-binding protein (FBP) into the N-terminus of MoMLV Env results in the reduction of the infectivity and the kinetics of entry of the MoMLV vectors. The use of soluble receptor-binding domain (sRBD), bafilomycin A1 (BafA1) and methyl-beta-cyclodextrin (MbetaC) increase the infectivity of the MoMLV vectors targeted to FBP (MoMLV-FBP) suggesting that the scFv targeted to FBP increases the threshold for fusion and might re-route entry of the targeted MoMLV-FBP vector towards an endocytic, non-productive pathway.     

Neutralization sensitivity of HIV-1 Env-pseudotyped virus clones is determined by co-operativity between mutations which modulate the CD4-binding site and those that affect gp120-gp41 stability

Adaptation of antibody neutralization-resistant human immunodeficiency virus type I (HIV-1) to growth in vitro generally results in the acquisition of a neutralization-sensitive phenotype, an alteration of viral growth kinetics, and an array of amino acid substitutions associated with these changes. Here we examine a panel of Env chimeras and mutants derived from these neutralization-resistant and -sensitive parental Envs. A range of neutralization and infectivity phenotypes was observed. These included a modulation of the CD4 binding site (CD4bs) towards recognition by neutralizing and non-neutralizing CD4bs-directed antibodies, resulting in a globally neutralization-sensitive Env; alterations which affected Env complex stability; and interactions which resulted in differential infectivity and CCR5/CXCR4 usage. This range of properties resulted from the complex interactions of no more than three amino acids found in key Env locations. These data add to a growing body of evidence that dramatic functional alterations of the native oligomeric Env protein complex can result from relatively minor amino acid substitutions.     

Modified HIV envelope proteins with enhanced binding to neutralizing monoclonal antibodies

The target for neutralizing antibodies against human immunodeficiency virus (HIV) is the trimeric Env protein on the native virion. Conserved neutralizing epitopes of receptor binding sites are located in the recessed core of the Env protein, partially masked by glycosylations and variable loops. In this study, we have investigated the effects of modifications of the HIV Env protein by glycosylation site mutations, deletions of variable loops, or combinations of both types of mutations on their protein functions and reactivities with neutralizing antibodies. Modified Env proteins were expressed in insect or mammalian cells, and their reactivity with epitope-specific broadly neutralizing monoclonal antibodies (Mabs) was determined by flow cytometry. A unique mutant designated 3G with mutations in three glycosylation motifs within the V3/C3 domains surrounding the CD4 binding site showed higher levels of binding to most broadly neutralizing Mabs (b12 and 2F5) in both insect and mammalian expression systems. Mutants with a deletion of both V1 and V2 loop domains or with a unique combination of both types of mutations also bound to most neutralizing Mabs at higher levels compared to the wild-type control. Most mutants maintained the ability to bind CD4 and to induce syncytium formation at similar or higher levels as compared to that of the wild-type Env protein, except for a mutant with a combination of variable loop deletions and deglycosylation mutations. Our study suggests that modified HIV Env proteins with reduced glycosylation in domains surrounding the CD4 binding site or variable loop-deleted mutants expose important neutralizing epitopes at higher levels than wild type and may provide novel vaccine immunogens.     

Biochemical and biophysical comparison of cleaved and uncleaved soluble, trimeric HIV-1 envelope glycoproteins

Human immunodeficiency virus type 1 (HIV-1) entry into host cells is mediated by the trimeric envelope glycoprotein complex (Env). Accordingly, the Env proteins are the targets for neutralizing antibodies (NAbs) and are the focus of vaccines intended to induce NAbs. Because the Env complex is labile, soluble recombinant Env (gp140) trimers require engineering to stabilize them sufficiently for use as immunogens. Trimeric forms of gp140 trimers can be created that are either cleavage-competent or cleavage-defective at the junction between the gp120 and gp41 subunits. As functional trimers are cleaved at this site, the question arises as to whether cleavage affects the antigenic structure of the Env complex in a way that is relevant to vaccine design. Here, we present a comparative analysis of the antigenicity profiles of cleaved and uncleaved gp140 trimers derived from the KNH1144 (subtype A) virus that are otherwise closely sequence-matched. While cleavage did not affect the exposure of NAb epitopes on the gp140 trimers, non-neutralizing antibodies to gp41 epitopes bound much more strongly to uncleaved trimers. Hence cleavage does alter the structure of the HIV-1 Env complex.     

Mutational Analysis of the Conserved Cysteine Residues in the Simian Immunodeficiency Virus Matrix Protein

The matrix protein (MA) of human and simian immunodeficiency viruses (HIV and SIV) is encoded by the amino-terminal region of the Gag precursor and has been suggested to be involved in different processes during the early and rate stages of the virus life cycle. The MA protein of SIV contains three cysteine residues at positions 57, 83, and B7, which are also highly conserved among HIV-2 isolates. In order to study the functional significance of these residues in virus morphogenesis, a series of mutations affecting the cysteines of SIV MA were introduced into a gag-protease construct and expressed in the vaccinia vector system. The MA mutants were assayed for their ability to synthesize and process the Gag polyprotein precursor as well as to release particles into the culture medium. In addition, the incorporation of the envelope glycoprotein (Env) into the Gag-made particles was investigated. Substitution of alanine for cysteine 87 had little effect on particle release and Env glycoprotein association. By contrast, the individual replacement of cysteines 57 or 83 by alanine, as well as the simultaneous mutation of cysteines 83 and 87, significantly reduced the ability of Gag polypeptides to produce extracellular particles. Assembly into particles appeared to be also affected, albeit to a lesser extent, when both cysteines 57 and 83 were replaced by alanine. Furthermore, substitution of cysteine 83 in the SIV MA domain was found to be detrimental to Gag polyprotein processing. Analysis of the Env glycoprotein association with recombinant particles revealed that this process was moderately affected in the case of the double mutants lacking cysteines 57 and 83, or cysteines 57 and 87, and the cysteine-minus triple mutant. Our results suggest that the Conserved cysteines 57 and 83 in the MA domain are important for efficient SIV Gag particle production.     

Enhancement of immunogenicity of an HIV Env DNA vaccine by mutation of the Tyr-based endocytosis motif in the cytoplasmic domain

We investigated the effect of the conserved tyrosine-based endocytosis motif (YXXPhi) in the cytoplasmic domain of the human immunodeficiency viruses (HIV) envelope protein (Env) on its immunogenicity. Genes with codons optimized for mammalian expression were synthesized for the HIV 89.6 Env with a truncated cytoplasmic domain and a mutant Env in which the tyrosine residue in the YXXPhi motif was changed into a serine. Mutation of the Tyr residue enhanced surface expression of the Env protein. Analysis of immune responses induced by DNA immunization of mice showed that the DNA construct for the Tyr mutant Env induced moderately higher levels of T cell responses. More interestingly, the DNA construct for the mutant Env induced significantly higher levels of antibody responses against the Env protein in comparison to the construct for the wild type Env. Our results suggest that the YXXPhi motif in the HIV Env cytoplasmic domain may play a role in virus evasion of host immune responses through affecting its immunogenicity.