The role of the membrane-spanning domain sequence in glycoprotein-mediated membrane fusion.
(33/1525)
The role of glycoprotein membrane-spanning domains in the process of membrane fusion is poorly understood. It has been demonstrated that replacing all or part of the membrane-spanning domain of a viral fusion protein with sequences that encode signals for glycosylphosphatidylinositol linkage attachment abrogates membrane fusion activity. It has been suggested, however, that the actual amino acid sequence of the membrane-spanning domain is not critical for the activity of viral fusion proteins. We have examined the function of Moloney murine leukemia virus envelope proteins with substitutions in the membrane-spanning domain. Envelope proteins bearing substitutions for proline 617 are processed and incorporated into virus particles normally and bind to the viral receptor. However, they possess greatly reduced or undetectable capacities for the promotion of membrane fusion and infectious virus particle formation. Our results imply a direct role for the residues in the membrane-spanning domain of the murine leukemia virus envelope protein in membrane fusion and its regulation. They also support the thesis that membrane-spanning domains possess a sequence-dependent function in other protein-mediated membrane fusion events. (+info)
Efficient human immunodeficiency virus ( HIV)-1 Gag-Env pseudovirion formation elicited from mammalian cells by a canarypox HIV vaccine candidate.
(34/1525)
Canarypox viruses undergo abortive replication in mammalian cells. Despite this restriction on replication in mammalian cells, significant immune responses have been shown in animals and in humans receiving recombinant canarypox vaccine vectors expressing heterologous immunogens. A recombinant canarypox vaccine candidate (vCP205), which expresses human immunodeficiency virus (HIV)-1 Gag, Env, and protease proteins, is presently under investigation in phase I and phase II human trials in the United States and elsewhere. In this study, the ability of vCP205 to elicit HIV Gag-Env pseudovirion formation in avian and mammalian cells was investigated. Gag-Env pseudovirions were produced from both avian and mammalian cell lines infected by this vaccine vector. A subset of mammalian cells was identified in which pseudovirion production and release was very efficient, surpassing the production from infected avian cells. The production of Gag-Env pseudovirions by canarypox HIV vaccine vectors may have important implications for future HIV vaccine design. (+info)
Inhibition of infection of incoming HIV-1 virus by RNA-cleaving DNA enzyme.
(35/1525)
Nine different DNA enzymes (DzV3-n, n=1-9) targeting the V3 loop region of HIV-1 HXB2 were synthesized. One of those, DzV3-9, efficiently cleaved the target in the conserved sequence in the RNA transcript in vitro. DzV3-9 was stable in the cells and inhibited replication of both NL432 and SF162 strains in U87 cells expressing CD4 and co-receptors. The inhibitory effect of DNAzyme on incoming HIV-1 was also demonstrated with pseudotype virions generated by NL432-based luciferase reporter genes. Thus, an efficient, stable DNAzyme against a functionally important region of HIV-1 was identified, and it may be useful for prevention of HIV-1 infection. (+info)
Role of immune responses against the envelope and the core antigens of simian immunodeficiency virus SIVmne in protection against homologous cloned and uncloned virus challenge in Macaques.
(36/1525)
We previously showed that envelope (gp160)-based vaccines, used in a live recombinant virus priming and subunit protein boosting regimen, protected macaques against intravenous and intrarectal challenges with the homologous simian immunodeficiency virus SIVmne clone E11S. However, the breadth of protection appears to be limited, since the vaccines were only partially effective against intravenous challenge by the uncloned SIVmne. To examine factors that could affect the breadth and the efficacy of this immunization approach, we studied (i) the effect of priming by recombinant vaccinia virus; (ii) the role of surface antigen gp130; and (iii) the role of core antigens (Gag and Pol) in eliciting protective immunity. Results indicate that (i) priming with recombinant vaccinia virus was more effective than subunit antigen in eliciting protective responses; (ii) while both gp130 and gp160 elicited similar levels of SIV-specific antibodies, gp130 was not as effective as gp160 in protection, indicating a possible role for the transmembrane protein in presenting functionally important epitopes; and (iii) although animals immunized with core antigens failed to generate any neutralizing antibody and were infected upon challenge, their virus load was 50- to 100-fold lower than that of the controls, suggesting the importance of cellular immunity or other core-specific immune responses in controlling acute infection. Complete protection against intravenous infection by the pathogenic uncloned SIVmne was achieved by immunization with both the envelope and the core antigens. These results indicate that immune responses to both antigens may contribute to protection and thus argue for the inclusion of multiple antigens in recombinant vaccine designs. (+info)
The molecular clock of HIV-1 unveiled through analysis of a known transmission history.
(37/1525)
Detailed knowledge about the rate and mode of the genetic variation is vital for understanding how HIV-1 induces disease and develops resistance as well as for studies on the molecular epidemiology and origin of the virus. To unveil the molecular clock of HIV-1 we analyzed a unique set of viruses from a known transmission history with separation times between samples of up to 25 years. The env V3 and p17gag regions of the genome were sequenced, and genetic distances were estimated by using the true tree and a nucleotide substitution model based on a general reversible Markov process with a gamma distribution to account for differences in substitution rates among sites. Linear regression analysis showed that separation times were significantly correlated with synonymous as well as nonsynonymous nucleotide distances in both V3 and p17, giving strong support for the existence of a molecular clock. The estimated rate of nucleotide substitution was 6.7 +/- 2.1 x 10(-3) substitutions/site per year in V3 and 2.7 +/- 0.5 x 10(-3) in p17. Importantly, the regression analyses showed that there was a significant genetic distance at zero divergence times. This pretransmission interval exists because the ramifications in the phylogenetic trees do not correspond to time of transmission, but rather to the coalescence time of the most recent common ancestor of the viruses carried by the transmitter and the recipient. Simulation experiments showed that neither the V3 nor the p17 clocks were overdispersed, which indicates that the introduction of nucleotide substitutions can be described adequately by a simple stochastic Poisson process. (+info)
Linkage disequilibrium test implies a large effective population number for HIV in vivo.
(38/1525)
The effective size of the HIV population in vivo, although critically important for the prediction of appearance of drug-resistant variants, is currently unknown. To address this issue, we have developed a simple virus population model, within which the relative importance of stochastic factors and purifying selection for genetic evolution differs over, at least, three broad intervals of the effective population size, with approximate boundaries given by the inverse selection coefficient and the inverse mutation rate per base per cycle. Random drift and selection dominate the smallest (stochastic) and largest (deterministic) population intervals, respectively. In the intermediate (selection-drift) interval, random drift controls weakly diverse populations, whereas strongly diverse populations are controlled by selection. To estimate the effective size of the HIV population in vivo, we tested 200 pro sequences isolated from 11 HIV-infected patients for the presence of a linkage disequilibrium effect which must exist only in small populations. This analysis demonstrated a steady-state virus population of 10(5) infected cells or more, which is either in or at the border of the deterministic regime with respect to evolution of separate bases. (+info)
Retrotransposons and retroviruses: analysis of the envelope gene.
(39/1525)
Retroviruses and long terminal repeat (LTR) retrotransposons share a common structural organization. The main difference between these retroelements is the presence of a functional envelope (env) gene in retroviruses, which is absent or nonfunctional in LTR retrotransposons. Several similarities between these two groups of retroelements have been detected for the reverse transcriptase, gag, and integrase domains. Assuming that each of these domains shares a common ancestral sequence, several hypotheses could account for the emergence of retroviruses from LTR retrotransposons. In this context, the positions of elements such as gypsy and the members of the Ty3 subfamily are not clear, since they are classified as retroviruses but phylogenetically they are assigned to the LTR retrotransposon group. We compared the env gene products of these retroelements and identified two similar motifs in retroviruses and LTR retrotransposons. These two regions do not occur in the same order. If we assume that they are derived from the same ancestral sequence, this could result from independent acquisition of the various domains rather than the single acquisition of the whole env gene. However, we cannot exclude the possibility that the env gene was reorganized after being acquired. Trees based on these regions show that these two groups of elements are clearly distinguished. These trees are similar to those obtained from reverse transcriptase or integrase. In trees based on reverse transcriptase, the retroviruses with complete or partial env genes can be distinguished from the other LTR retrotransposons. (+info)
The appearance of escape variants in vivo does not account for the failure of recombinant envelope vaccines to protect against simian immunodeficiency virus.
(40/1525)
The presence or evolution of immune escape variants has been proposed to account for the failure of recombinant envelope vaccines to protect macaques against challenge with simian immunodeficiency virus (SIVmac). To address this issue, two groups of three cynomolgus macaques were immunized with recombinant SIV Env vaccines using two different vaccine schedules. One group of macaques received four injections of recombinant SIV gp120 in SAF-1 containing threonyl muramyl dipeptide as adjuvant. A second group were primed twice with recombinant vaccinia virus expressing SIV gp160 and then boosted twice with recombinant SIV gp120. Both vaccine schedules elicited neutralizing antibodies to Env. However, on the day of challenge, titres of anti-Env antibodies measured by ELISA were higher in macaques primed with recombinant vaccinia virus. Following intravenous challenge with 10 monkey infectious doses of the SIVmac J5M challenge stock, five of the six immunized macaques and all four naive controls became infected. The virus burdens in PBMC of macaques that were primed with recombinant vaccinia virus were lower than those of naive controls, as determined by virus titration and quantitative DNA PCR. Sequence analysis was performed on SIV env amplified from the blood of immunized and naive infected macaques. No variation of SIV env sequence was observed, even in macaques with a reduced virus load, suggesting that the appearance of immune escape variants does not account for the incomplete protection observed. In addition, this study indicates that the measurement of serum neutralizing antibodies may not provide a useful correlate for protection elicited by recombinant envelope vaccines. (+info)