Differential membrane binding of the human immunodeficiency virus type 1 matrix protein

The human immunodeficiency virus type 1 matrix protein (p17MA) plays a central role at both the early and late stages of the virus life cycle. During viral assembly, the p17MA domain of Pr55gag promotes membrane association, which is essential for the formation of viral particles. When viral infection occurs, the mature p17MA dissociates from the plasma membrane and participates in the nuclear targeting process. Thus, p17MA contains a reversible membrane binding signal to govern its differential subcellular localization and biological functions. We previously identified a membrane binding signal within the amino-terminal 31 amino acids of the matrix domain of human immunodeficiency virus type 1 Gag, consisting of myristate and a highly basic region (W. Zhou, L. J. Parent, J. W. Wills, and M. D. Resh, J. Virol. 68:2556-2569, 1994). Here we show that exposure of this membrane binding signal is regulated in different Gag protein contexts. Within full-length Pr55gag, the membrane targeting signal is exposed and can direct Pr55gag as well as heterologous proteins to the plasma membrane. However, in the context of p17MA alone, this signal is hidden and unable to confer plasma membrane binding. To investigate the molecular mechanism for regulation of membrane binding, a series of deletions within p17MA was generated by sequentially removing alpha-helical regions defined by the nuclear magnetic resonance structure. Removal of the last alpha helix (amino acids 97 to 109) of p17MA was associated with enhancement of binding to biological membranes in vitro and in vivo. Liposome binding experiments indicated that the C-terminal region of p17MA exerts a negative effect on the N-terminal MA membrane targeting domain by sequestering the myristate signal. We propose that mature p17MA adopts a conformation different from that of the p17MA domain within Pr55gag and present evidence to support this hypothesis. It is likely that such a conformational change results in an N-terminal myristyl switch which governs differential membrane binding.     

Serotypes of the human immunodeficiency virus type 1 in Madrid

BACKGROUND: HIV-1 shows high genetic variability, mainly in the genomic region codifying the envelope proteins, which are the most immunogenic. This fact explains the high heterogeneity of antibodies against HIV-1 epitopes. Both genetic and serologic diversity has allowed to classify HIV-1 variants in several subtypes (genotypes and serotypes, respectively). The clinical and epidemiological significance of infection caused by each subtype remains to be clarified. PATIENTS AND METHODS: Serum samples from 154 HIV-seropositive individuals living in Madrid were studied. Serotyping was performed using 4 peptides belonging to the V3 env region. Epidemiological and clinical variables examined in these patients were the route of infection, the year in which HIV infection occurred, the country of birth, and the rate of disease progression (rapid versus slow). RESULTS: 148 (96.2%) samples could be serotyped, and the B class was recognized in 131 (88.5%) of them. Serotype A/C was found in 9 (6.1%). Two samples (1.3%) reacted to peptide E; however, both were also reactive against the B peptide, suggesting co-infection with B and E subtypes. Six samples were EIA-reactive for HIV-1/2 but were typed as HIV-2 alone. Infection with serotypes A/C was more frequent amongst immigrants, mainly in Africans. There was not association between any subtype and the route of infection neither a different rate of disease progression. CONCLUSION: HIV-1 serotype B is the most frequently found in HIV-seropositive individuals living in Madrid, without association with the route of infection or the clinical course of the disease. Serotypes A/C and E were found sporadically, mainly among immigrants.     

Cervical and vaginal shedding of human immunodeficiency virus type 1-infected cells throughout the menstrual cycle

Cervical and vaginal secretions from 17 women infected with human immunodeficiency virus type 1 (HIV-1) were evaluated daily through the course of one menstrual cycle for HIV-1 DNA (21-31 visits per woman). HIV-1-infected cells were detected in 207 (46%) of 450 endocervical swabs and 74 (16%) of 449 vaginal swabs. There was considerable variability in the percentage of positive swabs from each woman, ranging from 4% to 100% of endocervical swabs and from 0 to 71% of vaginal swabs. In multivariate analyses, plasma HIV-1 RNA was significantly associated with shedding of HIV-1-infected cells; each 1-unit increase in the log of plasma virus load was associated with a 5.6-fold increase in the odds of cervical shedding (95% confidence interval [CI], 2.1-14.8) and a 3.9-fold increase in the odds of vaginal shedding (95% CI, 2.1-7.2). There was no discernible pattern of genital tract shedding with phase of the menstrual cycle and no significant association with serum estradiol or progesterone levels.     

Vasectomy and human immunodeficiency virus type 1 in semen

PURPOSE: Human immunodeficiency virus type 1 (HIV) is cultured more often from seminal cells than seminal plasma. Because vasectomy causes dramatic reductions in seminal cells and also eliminates secretions from proximal sites in the male reproductive tract, vasectomy may change the potential infectiousness of semen. MATERIALS AND METHODS: We used polymerase chain reaction (PCR) assays to measure HIV ribonucleic acid (RNA) in seminal plasma and HIV deoxyribonucleic acid (DNA) in seminal cells from 46 asymptomatic, seropositive men before and after vasectomy. RESULTS: HIV RNA levels in semen correlated only weakly with blood levels (r = 0.22, p = 0.03). Of 183 semen specimens assayed for cell-free HIV RNA and proviral DNA 37 (20%) were positive for HIV RNA only, 41 (22%) were positive for HIV DNA only, and 18 (10%) were positive for RNA and DNA. Thus, detection of HIV RNA in seminal plasma was not associated with detection of HIV DNA in seminal cells. HIV RNA was present in 23 of 82 specimens (28%) (mean 2.87 log copies/ml.) before vasectomy and in 38 of 121 specimens (31%) after vasectomy (mean 2.81 log copies/ml.). CONCLUSIONS: These findings suggest that direct measurement of HIV levels in semen is necessary to assess the potential for sexual transmission, most cell-free HIV in seminal plasma arises distal to the vas deferens, and vasectomy may have minimal impact on the infectiousness of HIV seropositive men on sexual partners.     

Culture-positive tuberculosis in human immunodeficiency virus type 1-infected children

BACKGROUND: Adults infected by HIV have increased susceptibility to Mycobacterium tuberculosis and progress more rapidly to disease. HIV and tuberculosis (TB) coinfection in children has been reported but often lacks bacterial confirmation. We report on the clinical picture, special investigations, clinical course and outcome of 14 children with HIV infection and culture-confirmed TB from a developing country. METHODS: The clinical records of all children, from 1992 to 1997, with HIV infection and culture-proved TB were reviewed. RESULTS: Fourteen (10.4%) of 135 children with vertically transmitted HIV infection, 93% <2 years of age, fit the criteria. Nonresolving pneumonia (4) and otorrhoea (6) were common complaints. A Mantoux test was positive (> or =15 mm) in 6 of 11 children. Extrapulmonary TB was present in 5 cases. Ear swabs were the source of M. tuberculosis culture in 3. Chest radiographs were abnormal in all with hilar and paratracheal lymphadenopathy present in 7. A source case with pulmonary TB was identified for 10. Susceptibility tests were done on 9 strains of which 1 was drug-resistant. Four children were culture-positive 4 to 10 months after initiation of TB treatment. Mortality was 21% and 3 were lost to follow-up. CONCLUSIONS: In HIV-infected children the Mantoux skin test remains useful and culture specimens should be obtained from all sources. Response to treatment is unpredictable, and for this reason repeated cultures should be taken during treatment and a 9-month course of treatment considered.     

Function of human immunodeficiency virus type 1 nef gene product in virus replication

We comparatively analyzed the replication kinetics of wild-type (wt) and nef mutant human immunodeficiency virus type 1 (HIV-1) in several CD4-positive cell lines, in order to clarify the molecular function of Nef protein. The delayed growth of nef mutant virus was observed at the initial stage of replication in all cell lines examined. This phenomenon was greatly amplified in the absence of vpu gene. In order to determine the infection stage in viral replication cycle which is specifically affected on virus replication rate in the presence of the Nef protein, we first examined the difference between wt and nef mutant viruses in the virus production rate from transfected cells, and found that the both viruses were produced with equal efficiency. This result showed that Nef protein could be dispensable for virion production. Therefore, early infection stages were focused by single-round infection assay, and the nef mutant virus was found to be much less infectious than wt virus. This indicated that the effect of Nef protein was exhibited in the early phase of a virus replication cycle, during viral adsorption to integration. By entry assay using wt and nef mutant virions, it was revealed that the Nef protein was required for efficient viral entry. These data suggest that the Nef protein might play a role in efficient incorporation of the Env protein into the virions, leading to enhanced viral infectivity.     

Particle size determinants in the human immunodeficiency virus type 1 Gag protein

The retroviral Gag protein plays the central role in the assembly process and can form membrane-enclosed, virus-like particles in the absence of any other viral products. These particles are similar to authentic virions in density and size. Three small domains of the human immunodeficiency virus type 1 (HIV-1) Gag protein have been previously identified as being important for budding. Regions that lie outside these domains can be deleted without any effect on particle release or density. However, the regions of Gag that control the size of HIV-1 particles are less well understood. In the case of Rous sarcoma virus (RSV), the size determinant maps to the CA (capsid) and adjacent spacer sequences within Gag, but systematic mapping of the HIV Gag protein has not been reported. To locate the size determinants of HIV-1, we analyzed a large collection of Gag mutants. To our surprise, all mutants with defects in the MA (matrix), CA, and the N-terminal part of NC (nucleocapsid) sequences produced dense particles of normal size, suggesting that oncoviruses (RSV) and lentiviruses (HIV-1) have different size-controlling elements. The most important region found to be critical for determining HIV-1 particle size is the p6 sequence. Particles lacking all or small parts of p6 were uniform in size distribution but very large as measured by rate zonal gradients. Further evidence for this novel function of p6 was obtained by placing this sequence at the C terminus of RSV CA mutants that produce heterogeneously sized particles. We found that the RSV-p6 chimeras produced normally sized particles. Thus, we present evidence that the entire p6 sequence plays a role in determining the size of a retroviral particle.     

Antibody neutralization-resistant primary isolates of human immunodeficiency virus type 1

Although typical primary isolates of human immunodeficiency virus type 1 (HIV-1) are relatively neutralization resistant, three human monoclonal antibodies and a small number of HIV-1(+) human sera that neutralize the majority of isolates have been described. The monoclonal antibodies (2G12, 2F5, and b12) represent specificities that a putative vaccine should aim to elicit, since in vitro neutralization has been correlated with protection against primary viruses in animal models. Furthermore, a neutralization escape mutant to one of the antibodies (b12) selected in vitro remains sensitive to neutralization by the other two (2G12 and 2F5) (H. Mo, L. Stamatatos, J. E. Ip, C. F. Barbas, P. W. H. I. Parren, D. R. Burton, J. P. Moore, and D. D. Ho, J. Virol. 71:6869-6874, 1997), supporting the notion that eliciting a combination of such specificities would be particularly advantageous. Here, however, we describe a small subset of viruses, mostly pediatric, which show a high level of neutralization resistance to all three human monoclonal antibodies and to two broadly neutralizing sera. Such viruses threaten antibody-based antiviral strategies, and the basis for their resistance should be explored.     

Membrane binding of human immunodeficiency virus type 1 matrix protein in vivo supports a conformational myristyl switch mechanism

The interaction of the human immunodeficiency virus (HIV) Gag protein with the plasma membrane of a cell is a critical event in the assembly of HIV particles. The matrix protein region (MA) of HIV type 1 (HIV-1) Pr55Gag has previously been demonstrated to confer membrane-binding properties on the precursor polyprotein. Both the myristic acid moiety and additional determinants within MA are essential for plasma membrane binding and subsequent particle formation. In this study, we demonstrated the myristylation-dependent membrane interaction of MA in an in vivo membrane-binding assay. When expressed within mammalian cells, MA was found both in association with cellular membranes and in a membrane-free form. In contrast, the intact precursor Pr55Gag molecule analyzed in an identical manner was found almost exclusively bound to membranes. Both membrane-bound and membrane-free forms of MA were myristylated and phosphorylated. Differential membrane binding was not due to the formation of multimers, as dimeric and trimeric forms of MA were also found in both membrane-bound and membrane-free fractions. To define the requirements for membrane binding of MA, we analyzed the membrane binding of a series of MA deletion mutants. Surprisingly, deletions within alpha-helical regions forming the globular head of MA led to a dramatic increase in overall membrane binding. The stability of the MA-membrane interaction was not affected by these deletions, and no deletion eliminated membrane binding of the molecule. These results establish that myristic acid is a primary determinant of the stability of the Gag protein-membrane interaction and provide support for the hypothesis that a significant proportion of HIV-1 MA molecules may adopt a conformation in which myristic acid is hidden and unavailable for membrane interaction.     

Long-latency event-related potentials in asymptomatic human immunodeficiency virus type 1 infection

An ideal technique for following the development of the vertebrate nervous system would allow cells to be followed at the resolution of light microscopy at depths of several millimeters into the tissue. This would permit critical events to be followed at cellular or sub-cellular resolution even deep within the developing organism. To date, no technique has emerged with all of the needed properties. Light microscopy can follow a cell and its descendants after they have been labeled by either the infection of embryonic cells with a recombinant retrovirus or the microinjection of individual precursor cells with enzymes or fluorescent dyes. However, light microscopy cannot image events deeper than a few hundred micrometers within an embryo due to light scattering and aberrations in the objective lenses and other optics. Magnetic resonance imaging (MRI) does not suffer from these limitations, routinely being used to image in 3 dimensions through specimens as large as adult humans. However, it is relatively slow and, as implemented to date, it cannot routinely achieve cellular resolution. Here, we present our attempts to meet the technical challenges posed by in vivo MRI microscopy. As an example of both the progress and the future challenges, we present images of cells within the developing frog embryo over a several day time course.     

Temporary seronegativity in a human immunodeficiency virus type 1-infected man

In addition to financial constraints there are significant social, educational, moral and religious barriers to the prevention and treatment of reproductive tract infections in rural India. A pilot project aimed at achieving progress in this field is reported below.     

Common themes of antibody maturation to simian immunodeficiency virus, simian-human immunodeficiency virus, and human immunodeficiency virus type 1 infections

Characterization of virus-specific immune responses to human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) is important to understanding the early virus-host interactions that may determine the course of virus infection and disease. Using a comprehensive panel of serological assays, we have previously demonstrated a complex and lengthy maturation of virus-specific antibody responses elicited by attenuated strains of SIV that was closely associated with the development of protective immunity. In the present study, we expand these analyses to address several questions regarding the nature of the virus-specific antibody responses to pathogenic SIV, SIV/HIV-1 (SHIV), and HIV-1 infections. The results demonstrate for the first time a common theme of antibody maturation to SIV, SHIV, and HIV-1 infections that is characterized by ongoing changes in antibody titer, conformational dependence, and antibody avidity during the first 6 to 10 months following virus infection. We demonstrate that this gradual evolution of virus-specific antibody responses is independent of the levels of virus replication and the pathogenicity of the infection viral strain. While the serological assays used in these studies were useful in discriminating between protective and nonprotective antibody responses during evaluation of vaccine efficacy with attenuated SIV, these same assays do not distinguish the clinical outcome of infection in pathogenic SIV, SHIV, or HIV-1 infections. These results likely reflect differences in the immune mechanisms involved in mediating protection from virus challenge compared to those that control an established viral infection, and they suggest that additional characteristics of both humoral and cellular responses evolve during this early immune maturation.     

Conservation of an intact vif gene of human immunodeficiency virus type 1 during maternal-fetal transmission

The human immunodeficiency virus type 1 (HIV-1) vif gene is conserved among most lentiviruses, suggesting that vif is important for natural infection. To determine whether an intact vif gene is positively selected during mother-to-infant transmission, we analyzed vif sequences from five infected mother-infant pairs following perinatal transmission. The coding potential of the vif open reading frame directly derived from uncultured peripheral blood mononuclear cell DNA was maintained in most of the 78,912 bp sequenced. We found that 123 of the 137 clones analyzed showed an 89.8% frequency of intact vif open reading frames. There was a low degree of heterogeneity of vif genes within mothers, within infants, and between epidemiologically linked mother-infant pairs. The distances between vif sequences were greater in epidemiologically unlinked individuals than in epidemiologically linked mother-infant pairs. Furthermore, the epidemiologically linked mother-infant pair vif sequences displayed similar patterns that were not seen in vif sequences from epidemiologically unlinked individuals. The functional domains, including the two cysteines at positions 114 and 133, a serine phosphorylation site at position 144, and the C-terminal basic amino acids essential for vif protein function, were highly conserved in most of the sequences. Phylogenetic analyses of 137 mother-infant pair vif sequences and 187 other available vif sequences from HIV-1 databases revealed distinct clusters for vif sequences from each mother-infant pair and for other vif sequences. Taken together, these findings suggest that vif plays an important role in HIV-1 infection and replication in mothers and their perinatally infected infants.