Inter- and intraclade neutralization of human immunodeficiency virus type 1: genetic clades do not correspond to neutralization serotypes but partially correspond to gp120 antigenic serotypes.

We have studied genetic variation among clades A through E of human immunodeficiency virus type 1 (HIV-1) at the levels of antibody binding to gp120 molecules and virus neutralization. We are unable to identify neutralization serotypes that correspond to the genetic clades. Instead, we observe that inter- and intraclade neutralization of primary isolates by HIV-1-positive sera is generally weak and sporadic; some sera show a reasonable degree of neutralization breadth and potency whereas others are relatively sensitive to neutralization, but no consistent pattern was found. However, a few sera were able to neutralize across clades with significant potency, an observation which may have implications for the feasibility of a broadly effective HIV-1 vaccine involving humoral immunity. Serological assays measuring anti-gp120 antibody binding also failed to identify serotypes that correspond precisely to the genetic clades, but some indications of clade-specific binding were observed, notably with sera from clades B and E. A representative protein for each clade (A through E) was selected on the basis of its specificity, defined as high seroreactivity with sera from individuals infected with virus of that clade and lower reactivity with sera from individuals infected with viruses from other clades. The seroreactivity patterns against these five proteins could be used to predict the genotype of the infecting virus with moderate success.     

Virion envelope content, infectivity, and neutralization sensitivity of simian immunodeficiency virus

A truncating E767stop mutation was introduced into the envelope glycoprotein of simian immunodeficiency virus (SIV) strain SIV239-M5 (moderately sensitive to antibody-mediated neutralization and lacking five sites for N-linked carbohydrate attachment) and strain SIV316 (very sensitive to neutralization, with eight amino acid changes from the neutralization-resistant parental molecular clone, SIV239). The truncating mutation increased Env content in virions, increased infectivity, and decreased sensitivity to antibody-mediated neutralization in both strains. However, the magnitude of the effect on infectivity and neutralization sensitivity differed considerably between the two strains. In the context of strain SIV239-M5, truncation increased Env content in virions approximately 10-fold and infectivity in a reporter cell assay 24-fold. The truncated SIV239-M5 was only slightly more resistant to neutralization by polyclonal monkey sera and by monoclonal antibodies than SIV239-M5 with a full-length envelope glycoprotein. In the context of strain SIV316, truncation increased infectivity a dramatic 480-fold, while envelope content in virions was increased only about 14-fold. This dramatic increase in infectivity cannot be simply explained by the increase in envelope content and is likely due to an increase in inherent infectivity, i.e., infectivity per spike, that results from truncation. The truncated SIV316 was extremely resistant to antibody-mediated neutralization. In fact, it was not neutralized by any of the antibodies tested. When increasing amounts of SIV316 envelope glycoprotein (full length) were provided in trans to SIV316, infectivity was increased and sensitivity to neutralization was decreased, but to nowhere near the degree that was obtained when truncated SIV316 envelope glycoprotein was used. Truncated forms of SIV239 and SIV239-M5 required higher levels of soluble CD4 for inhibition of infection than their nontruncated forms; truncated SIV316 did not. Our results suggest that envelope content in SIV virions, infectivity, and resistance to antibody-mediated neutralization can be increased not only by truncation of the cytoplasmic domain but also by provision of excess envelope in trans. The striking increase in infectivity that results from truncation in the context of SIV316 appears to be due principally to an increase in inherent infectivity per spike.     

Mutations in the principal neutralization determinant of human immunodeficiency virus type 1 affect syncytium formation, virus infectivity, growth kinetics, and neutralization.

The principal neutralization determinant (PND) of human immunodeficiency virus type 1 envelope glycoprotein gp120 contains a conserved GPG sequence. The effects of a 29-amino-acid deletion of most of the PND, a 3-amino-acid deletion in the GPG sequence, and 16 single-amino-acid substitutions in the GPG sequence were determined in a transient expression assay. All mutant envelope glycoproteins were expressed at levels comparable to that of the wild-type envelope, and mutations in the GPG sequence did not affect processing to gp120 or, except for the 29-amino-acid deletion, binding to CD4. Of all of the mutants, only the GHG and GFG mutants induced formation of syncytia similar in size and number to those induced by the wild-type envelope. When the envelope expression level was increased 10-fold or more, several additional mutants (APG, GAG, GSG, GQG, GVG, and GPF) also induced syncytium formation. Transfection with infectious proviral molecular clones containing the GHG, GFG, APG, GAG, GSG, or GPF mutations induced production of viral particles; however, only the GPG, GHG, and GFG viruses produced active infections in CD4-bearing cells. Furthermore, whereas the wild-type virus was efficiently neutralized by PND polyclonal and monoclonal antibodies, the GHG- and GFG-containing viruses were not. These results show that mutations in the GPG sequence found within the PND do not affect envelope expression and do not significantly affect CD4 binding or production of viral particles but that they do affect the ability of the envelope to induce syncytia and those of the viral particles to infect CD4 cells and be neutralized by PND antibodies.     

Dissociation of the CD4 downregulation and viral infectivity enhancement functions of human immunodeficiency virus type 1 Nef.

Recent evidence indicates that the nef gene of human immunodeficiency virus type 1 augments rather than inhibits viral replication in both cell culture and in vivo models. In addition, nef alters various normal cellular processes, including the display of CD4 on the cell surface. However, it remains unknown whether the enhancement of infectivity and the downregulation of CD4 represent linked or independent biologic properties of this single protein. In the present studies, mutational analyses were performed to define structure-function relationships within the Nef protein that mediate these effects. To assess the functional consequences of these mutations, sensitive and reliable assays were developed to quantitate the viral infectivity enhancement and CD4 downregulation functions of Nef. The results indicate that membrane-targeting sequences at the N terminus of Nef are important for both functions of Nef, while certain other conserved regions are dispensable for both functions. A conserved proline-X-X repeat segment in the central core of the protein, which is reminiscent of an SH3-binding domain, is critical for the enhancement of infectivity function but is dispensable for CD4 downregulation. However, the downregulation of CD4 by Nef appears to involve a two-step process requiring the initial dissociation of p56lck from CD4 to permit engagement of the endocytic apparatus by CD4. Together, these findings demonstrate that the infectivity enhancement and CD4 downregulation activities of human immunodeficiency virus type 1 Nef can be dissociated. Thus, these processes may be independent of one another in the viral replication cycle.     

Inefficient enhancement of viral infectivity and CD4 downregulation by human immunodeficiency virus type 1 Nef from Japanese long-term nonprogressors

It has been reported that patients infected with nef-defective human immunodeficiency virus type 1 (HIV-1) do not progress to AIDS; however, mutations that abrogate Nef expression are not common in long-term nonprogressors (LTNPs). We postulated that Nef function might be impaired in LTNPs, irrespective of the presence or absence of detectable amino acid sequence anomalies. To challenge this hypothesis we compared in vitro function of nef alleles that were derived from three groups of Japanese patients: LTNPs, progressors, and asymptomatic carriers (ACs). The patient-derived nef alleles were subcloned into a nef-defective infectious HIV-1 molecular clone and an expression vector. We first examined Nef-dependent enhancement of infection in a single-round infectivity assay by the use of MAGNEF cells, in which Nef is required more strictly for the infection than in the parent MAGI cells. All nef alleles from LTNPs showed reduced enhancement in the infectivity of nef-defective HIV-1 mutants compared to the nef alleles of progressors or ACs. Second, we found that nef alleles from LTNPs were less efficient in CD4 downregulation than those of progressors or ACs. Third, all nef alleles from LTNPs, progressors, and ACs reduced the cell surface expression of major histocompatibility complex class I to a similar level. Last, there was no correlation between Hck-binding activity of Nef and clinical grouping. In conclusion, we detected inefficient enhancement of HIV-1 infectivity and CD4 downregulation by HIV-1 nef alleles of LTNPs. It awaits further study to conclude that these characteristics of nef alleles are the cause or the consequence of the long-term nonprogression after HIV-1 infection.     

Purification, properties, and evidence for two subtypes of human placenta hexokinase type I

In human placenta 85% of total hexokinase activity (EC 2.7.1.1) was found in a soluble form. Of this, 70% is hexokinase type I while the remaining 30% is hexokinase type II. All the bound hexokinase is type I. Soluble hexokinase I was purified 11,000-fold by a combination of ion-exchange chromatography, affinity chromatography, and dye-ligand chromatography. The specific activity was 190 units/mg protein with a 75% yield. The enzyme shows only one band in nondenaturing polyacrylamide gel electrophoresis that stains for protein and enzymatic activity; however, two components (with Mr 112,000 and 103,000) were constantly seen in sodium dodecyl sulfate-gel electrophoresis. Many attempts were made to separate these two proteins under native conditions; however, only one peak of activity was obtained when the enzyme was submitted to gel filtration (Mr 118,000), preparative isoelectric focusing (pI 5.9), anion-exchange chromatography, hydroxylapatite chromatography, and affinity chromatography on immobilized dyes and immobilized glucosamine. The high and low molecular weight hexokinases show the same isoelectric point under denaturing conditions as determined by two-dimensional gel electrophoresis. Each hexokinase subtype was obtained by preparative sodium dodecyl sulfate electrophoresis followed by electroelution. Monospecific antibodies raised in rabbits against electroeluted high and low molecular weight hexokinases were not able to recognize the native enzymes but each of them detected both hexokinases on immunoblots. Amino acid compositions and peptide mapping by limited proteolysis of the high and low molecular weight hexokinases were also performed and suggested a strong homology between these two subtypes of human hexokinase I.     

Pseudopackaging of adenovirus type 5 genomes into capsids containing the hexon proteins of adenovirus serotypes B, D, or E

Adenoviruses (Ad) show promise as a vector system for gene delivery in vivo. However, a major challenge in the development of Ad vectors is the circumvention of the host immune responses to Ad infection, including both the host cytotoxic T-cell response and the humoral response resulting in neutralizing antibodies. One method to circumvent the effect of neutralizing antibodies against an Ad vector is to use different Ad serotypes to deliver the transgene of interest. This approach has been demonstrated with Ad genomes of highly related members of subgroup C. However, it is not known whether an Ad5-based vector DNA molecule can be packaged into capsids of evolutionarily more divergent adenoviruses. The aim of these studies was to determine if capsids containing hexon proteins from other Ad subgroups could package the Ad5 genome. A genetic approach utilizing an Ad5 temperature-sensitive (ts) mutant with a mutation in the hexon protein was used. When grown at the nonpermissive temperature, Ad5 ts147 replicates normally, providing a source of Ad5 DNA for virus assembly, but does not produce virus particles due to the hexon protein mutation. Coinfection of Ad5 ts147 with a wild-type virus of other Ad serotypes (Ad3, Ad4, or Ad9), which supply functional hexon proteins, resulted in the pseudopackaging of the Ad5 DNA genome. Furthermore, the pseudopackaged Ad5 DNA virions obtained in the coinfections were infectious. Therefore, switching hexons did not impair the infectivity or uncoating process of the pseudopackaged virion. Since hexon protein is a major antigenic determinant of the Ad capsid, this approach may prove useful to reduce the antigenicity of therapeutic Ad vectors and allow repeated vector administration.     

Role of invariant Thr80 in human immunodeficiency virus type 1 protease structure, function, and viral infectivity

Sequence variability associated with human immunodeficiency virus type 1 (HIV-1) is useful for inferring structural and/or functional constraints at specific residues within the viral protease. Positions that are invariant even in the presence of drug selection define critically important residues for protease function. While the importance of conserved active-site residues is easily understood, the role of other invariant residues is not. This work focuses on invariant Thr80 at the apex of the P1 loop of HIV-1, HIV-2, and simian immunodeficiency virus protease. In a previous study, we postulated, on the basis of a molecular dynamics simulation of the unliganded protease, that Thr80 may play a role in the mobility of the flaps of protease. In the present study, both experimental and computational methods were used to study the role of Thr80 in HIV protease. Three protease variants (T80V, T80N, and T80S) were examined for changes in structure, dynamics, enzymatic activity, affinity for protease inhibitors, and viral infectivity. While all three variants were structurally similar to the wild type, only T80S was functionally similar. Both T80V and T80N had decreased the affinity for saquinavir. T80V significantly decreased the ability of the enzyme to cleave a peptide substrate but maintained infectivity, while T80N abolished both activity and viral infectivity. Additionally, T80N decreased the conformational flexibility of the flap region, as observed by simulations of molecular dynamics. Taken together, these data indicate that HIV-1 protease functions best when residue 80 is a small polar residue and that mutations to other amino acids significantly impair enzyme function, possibly by affecting the flexibility of the flap domain.     

Alternative splicing of human immunodeficiency virus type 1 mRNA modulates viral protein expression, replication, and infectivity.

Multiple RNA splicing sites exist within human immunodeficiency virus type 1 (HIV-1) genomic RNA, and these sites enable the synthesis of many mRNAs for each of several viral proteins. We evaluated the biological significance of the alternatively spliced mRNA species during productive HIV-1 infections of peripheral blood lymphocytes and human T-cell lines to determine the potential role of alternative RNA splicing in the regulation of HIV-1 replication and infection. First, we used a semiquantitative polymerase chain reaction of cDNAs that were radiolabeled for gel analysis to determine the relative abundance of the diverse array of alternatively spliced HIV-1 mRNAs. The predominant rev, tat, vpr, and env RNAs contained a minimum of noncoding sequence, but the predominant nef mRNAs were incompletely spliced and invariably included noncoding exons. Second, the effect of altered RNA processing was measured following mutagenesis of the major 5' splice donor and several cryptic, constitutive, and competing 3' splice acceptor motifs of HIV-1NL4-3. Mutations that ablated constitutive splice sites led to the activation of new cryptic sites; some of these preserved biological function. Mutations that ablated competing splice acceptor sites caused marked alterations in the pool of virus-derived mRNAs and, in some instances, in virus infectivity and/or the profile of virus proteins. The redundant RNA splicing signals in the HIV-1 genome and alternatively spliced mRNAs provides a mechanism for regulating the relative proportions of HIV-1 proteins and, in some cases, viral infectivity.     

A global neutralization resistance phenotype of human immunodeficiency virus type 1 is determined by distinct mechanisms mediating enhanced infectivity and conformational change of the envelope complex

We have described previously genetic characterization of neutralization-resistant, high-infectivity, and neutralization-sensitive, low-infectivity mutants of human immunodeficiency virus type 1 (HIV-1) MN envelope. The distinct phenotypes of these clones are attributable to six mutations affecting functional interactions between the gp120 C4-V5 regions and the gp41 leucine zipper. In the present study we examined mechanisms responsible for the phenotypic differences between these envelopes using neutralization and immunofluorescence assays (IFA). Most monoclonal antibodies (MAbs) tested against gp120 epitopes (V3, CD4 binding site, and CD4-induced) were 20 to 100 times more efficient at neutralizing pseudovirus expressing sensitive rather than resistant envelope. By IFA cells expressing neutralization sensitive envelope bound MAbs to gp120 epitopes more, but gp41 epitopes less, than neutralization-resistant envelope. This binding difference appeared to reflect conformational change, since it did not correlate with the level of protein expression or gp120-gp41 dissociation. This conformational change was mostly attributable to one mutation, L544P, which contributes to neutralization resistance but not to infectivity enhancement. The V420I mutation, which contributes a major effect to both high infectivity and neutralization resistance, had no apparent effect on conformation. Notably, a conformation-dependent V3 neutralization epitope remained sensitive to neutralization and accessible to binding by MAbs on neutralization-resistant HIV-1 envelope. Sensitivity to sCD4 did not distinguish the clones, suggesting that the phenotypes may be related to post-CD4-binding effects. The results demonstrate that neutralization resistance can be determined by distinguishable effects of mutations, which cause changes in envelope conformation and/or function(s) related to infectivity. A conformation-dependent V3 epitope may be an important target for neutralization of resistant strains of HIV-1.     

Comparing the ex vivo fitness of CCR5-tropic human immunodeficiency virus type 1 isolates of subtypes B and C

Continual human immunodeficiency virus type 1 (HIV-1) evolution and expansion within the human population have led to unequal distribution of HIV-1 group M subtypes. In particular, recent outgrowth of subtype C in southern Africa, India, and China has fueled speculation that subtype C isolates may be more fit in vivo. In this study, nine subtype B and six subtype C HIV-1 isolates were added to peripheral blood mononuclear cell cultures for a complete pairwise competition experiment. All subtype C HIV-1 isolates were less fit than subtype B isolates (P < 0.0001), but intrasubtype variations in HIV-1 fitness were not significant. Increased fitness of subtype B over subtype C was also observed in primary CD4(+) T cells and macrophages from different human donors but not in skin-derived human Langerhans cells. Detailed analysis of the retroviral life cycle during several B and C virus competitions indicated that the efficiency of host cell entry may have a significant impact on relative fitness. Furthermore, phyletic analyses of fitness differences suggested that, for a recombined subtype B/C HIV-1 isolate, higher fitness mapped to the subtype B env gene rather than the subtype C gag and pol genes. These results suggest that subtype B and C HIV-1 may be transmitted with equal efficiency (Langerhans cell data) but that subtype C isolates may be less fit following initial infection (T-cell and macrophage data) and may lead to slower disease progression.     

Quantitative model of antibody- and soluble CD4-mediated neutralization of primary isolates and T-cell line-adapted strains of human immunodeficiency virus type 1.

Primary isolates (PI) of human immunodeficiency virus type 1 (HIV-1) are considerably less sensitive than T-cell line-adapted strains to neutralization by soluble CD4 and by most cross-reactive monoclonal antibodies to the viral envelope (Env) glycoprotein, as well as by postinfection and postvaccination sera (J. P. Moore and D. D. Ho, AIDS 9 [suppl. A]:5117-5136, 1995). We developed a quantitative model to explain the neutralization resistance of PI. The factors incorporated into the model are the dissociation constants for the binding of the neutralizing agent to native Env oligomers, the number of outer Env molecules on the viral surface (which decreases by shedding), and the minimum number of Env molecules required for attachment and fusion. We conclude that modest differences in all these factors can, when combined, explain a relative neutralization resistance of PI versus T-cell line-adapted strains that sometimes amounts to several orders of magnitude. The hypothesis that neutralization of HIV is due to the reduction below a minimum number of the Env molecules on a virion available for attachment and fusion is at odds with single- and few-hit neutralization theories. Our analysis of these ideas favors the hypothesis that neutralization of HIV is instead a competitive blocking of interactions with cellular factors, including adsorption receptors.     

Simian immunodeficiency virus engrafted with human immunodeficiency virus type 1 (HIV-1)-specific epitopes: replication, neutralization, and survey of HIV-1-positive plasma

To date, only a small number of anti-human immunodeficiency virus type 1 (HIV-1) monoclonal antibodies (MAbs) with relatively broad neutralizing activity have been isolated from infected individuals. Adequate techniques for defining how frequently antibodies of these specificities arise in HIV-infected people have been lacking, although it is generally assumed that such antibodies are rare. In order to create an epitope-specific neutralization assay, we introduced well-characterized HIV-1 epitopes into the heterologous context of simian immunodeficiency virus (SIV). Specifically, epitope recognition sequences for the 2F5, 4E10, and 447-52D anti-HIV-1 neutralizing monoclonal antibodies were introduced into the corresponding regions of SIVmac239 by site-directed mutagenesis. Variants with 2F5 or 4E10 recognition sequences in gp41 retained replication competence and were used for neutralization assays. The parental SIVmac239 and the neutralization-sensitive SIVmac316 were not neutralized by the 2F5 and 4E10 MAbs, nor were they neutralized significantly by any of the 96 HIV-1-positive human plasma samples that were tested. The SIV239-2F5 and SIV239-4E10 variants were specifically neutralized by the 2F5 and 4E10 MAbs, respectively, at concentrations within the range of what has been reported previously for HIV-1 primary isolates (J. M. Binley et al., J. Virol. 78:13232-13252, 2004). The SIV239-2F5 and SIV239-4E10 epitope-engrafted variants were used as biological screens for the presence of neutralizing activity of these specificities. None of the 92 HIV-1-positive human plasma samples that were tested exhibited significant neutralization of SIV239-2F5. One plasma sample exhibited >90% neutralization of SIV239-4E10, but this activity was not competed by a 4E10 target peptide and was not present in concentrated immunoglobulin G (IgG) or IgA fractions. We thus confirm by direct analysis that neutralizing activities of the 2F5 and 4E10 specificities are either rare among HIV-1-positive individuals or, if present, represent only a very small fraction of the total neutralizing activity in any given plasma sample. We further conclude that the structures of gp41 from SIVmac239 and HIV-1 are sufficiently similar such that epitopes engrafted into SIVmac239 can be readily recognized by the cognate anti-HIV-1 monoclonal antibodies.     

Cutting edge: enhancement of antibody responses through direct stimulation of B and T cells by type I IFN

Type I IFN (IFN-alphabeta) is induced rapidly by infection and plays a key role in innate antiviral defense. IFN-alphabeta also exerts stimulatory effects on the adaptive immune system and has been shown to enhance Ab and T cell responses. We have investigated the importance of B and T cells as direct targets of IFN-alphabeta during IFN-alpha-mediated augmentation of the Ab response against a soluble protein Ag. Strikingly, the ability of IFN-alpha to stimulate the Ab response and induce isotype switching was markedly reduced in mice in which B cells were selectively deficient for the IFN-alphabetaR. Moreover, IFN-alpha-mediated enhancement of the Ab response was also greatly impaired in mice in which T cells were selectively IFN-alphabetaR-deficient. These results indicate that IFN-alphabetaR signaling in both B and T cells plays an important role in the stimulation of Ab responses by IFN-alphabeta.     

Immunobiological properties of staphylococcal enterotoxins type A, B, C, D and E

Comparative study of mitogenic and interferonogenic properties of staphylococcal enterotoxins of different serotypes is done. It is revealed that preparations of enterotoxins are polyclonal mitogens and have interferon-inducing activity. It is stated that enterotoxin of D type has the highest mitogenic activity, which is shown by interferon-inducing activity of A type toxin.