Interaction of human plasma fibronectin with viral proteins of human immunodeficiency virus

Abstract Fibronectin (FN) is present in soluble and matrix forms in various body fluids and tissues, and has been shown to bind to several pathogens, including viruses. The interaction of FN with viral proteins of human immunodeficiency virus (HIV-1) was investigated by immunofluorescence technique using a cell line chronically infected with HIV-1 (H9-V). The results of this study showed that FN binds to HIV-1 infected cells. especially at FN concentration of 5 μg/ml. In addition, FN-pentapeptide has shown the ability to bind to HIV-1 infected cells. On the other hand, preincubation with antibodies against FN abolished the binding of FN to HIV-1 infected cells. Finally, FN has shown to bind to HIV-1 glycoproteins, including gp41 and pg120. In contrast, no binding to HIV-1 core proteins, including p15 and p24, was noted. We suggest that FN, in binding HIV-1 particles, may reduce viremia and thus may be involved in the clearance of viral proteins from the cells.     

Maternal-fetal transmission of human immunodeficiency virus

The World Health Organization estimates that by year 2000, 10 million children will be infected with human immunodeficiency virus type 1 (HIV-1) at birth and will subsequently develop AIDS. Perinatally acquired infections account for the majority of all HIV-1 cases in children, with an estimated mother-to-infant transmission rate of more than 30%. It is not clear why more than half of the children born to HIV-1-infected mothers are uninfected. Maternal transmission of HIV-1 occurs at three levels: prepartum, intrapartum, and postpartum. Several maternal parameters including advanced clinical stages of the mother, low CD4+ lymphocyte counts, maternal immune response to HIV-1, recent infection, high level of circulating HIV-1, and maternal disease progression have been implicated in an increased risk of mother-to-infant transmission of HIV-1. Viral factors influencing mother-to-infant transmission are not known. Furthermore, several other factors such as acute infection during pregnancy, presence of other sexually transmitted diseases (STD) or other chronic infections, vaginal bleeding, disruption of placental integrity, premature rupture of membrane (PROM), and preterm PROM have been associated with mother-to-infant transmission of HIV-1. In addition, tobacco and cigarette smoking during pregnancy have been shown to triple the rate of maternal transmission of HIV-1. The AIDS Clinical Trial Group (ACTG) suggested that zidovudine (ZDV) can reduce the rate of mother-to-infant transmission of HIV-1 if administered to HIV-1-infected pregnant women with CD4 counts greater than 200. Moreover, this study failed to take into consideration several factors that may influence maternal transmission of HIV-1. However, the molecular mechanisms involved in mother-to-infant transmission of HIV-1 are not understood, which makes it more difficult to define strategies for effective treatment and prevention of HIV-1 infection in children. Several groups are engaged in the understanding of the molecular and biological properties of HIV-1 influencing mother-to-infant transmission. Results from my and several other laboratories suggest that the minor genotypes, subtypes, or variants of HIV-1 found in a genetically heterogeneous virus population of infected mothers are transmitted to their infants. The minor HIV-1 genotype predominates initially as a homogeneous population in the infant and then becomes diverse as the infant matures. Furthermore, transmission of a major or multiple HIV-1 genotypes from mother to infant has been reported. Taken together, these results strongly suggest that there are differences among the molecular and biological properties of the maternal variants that are transmitted to the infants and the maternal variants that are not transmitted to the infants. The understanding of the molecular and biological properties of the transmitted viruses will enable researchers to target a particular subtype in the mothers that is transmitted to the infants.     

人类免疫缺陷病毒与细胞凋亡

人类免疫缺陷病毒1型(HIV-1)感染可使被感染者体内CD4细胞数量减少,最终导致艾滋病。关于HIV-1如何杀死免疫细胞的精确机制仍是1个争论的问题。现已知道,细胞凋亡为HIV-1诱导细胞死亡的一个重要机制。HIV可直接诱导细胞凋亡,也可以通过活化作用,同源被感染的细胞的介导,以及CD^8 T细胞诱导细胞凋亡。且细胞因子在HIV诱导细胞凋亡的过程中发挥着重要的作用。本综述主要从以上几个方面总结HIV-1诱导细胞凋亡的机制。     

The effect of human immunodeficiency virus-1 protease inhibitors on the toxicity of a variety of cells

Protease inhibitors in combination with other antiretroviral drugs have been shown to be efficacious in treating human immunodeficiency virus-1 (HIV-1) infection. The side effects of such a treatment usually involve perturbations of fat metabolism and insulin responsiveness. This has led to a number of studies on the adverse effects of these drugs in vitro. The concentrations of various protease inhibitors used in many of these studies were >20 microM. Although some investigators did address the toxicity of protease inhibitors, no overall effort was made to examine this effect during differentiation of fat or muscle. In this study, we assessed the toxicity of HIV-1 protease inhibitors over a range of concentrations (i.e., 0 to 100 microM) in nondifferentiating (e.g., human fibroblasts, 3T3-L1 preadipocytes, and L6 myoblasts) and differentiated cells (e.g., L6 myotubes). The most toxic protease inhibitor in all cell types was Saquinavir (sqv), whereas the least toxic protease inhibitor was Indinavir (idv). Ritonavir (rtv) and Amprenavir (apv) were more toxic than idv but not quite as toxic as sqv. In 3T3-L1 preadipocytes, treatment with sqv, rtv, and apv resulted in toxicity, whereas idv was not toxic even at the highest concentration used. Indinavir was not toxic to L6 myoblasts or L6 myotubes; however, sqv, rtv, and apv caused toxicity in L6 myoblasts. Saquinavir decreased L6 myotube viability in a dose-dependent manner. Human immunodeficiency virus-1 protease inhibitors were shown to be toxic in a variety of cell types. These effects on human fibroblasts and muscle cells have not been reported previously.     

Stimulation of human flap endonuclease 1 by human immunodeficiency virus type 1 integrase: possible role for flap endonuclease 1 in 5'-end processing of human immunodeficiency virus type 1 integration intermediates

Human immunodeficiency virus type 1 (HIV-1) DNA integration intermediates consist of viral and host DNA segments separated by a 5-nucleotide gap adjacent to a 5'-AC unpaired dinucleotide. These short-flap (pre-repair) integration intermediates are structurally similar to DNA loci undergoing long-patch base excision repair in mammalian cells. The cellular proteins flap endonuclease 1 (FEN-1), proliferating cell nuclear antigen, replication factor C, DNA ligase I and DNA polymerase delta are required for the repair of this type of DNA lesion. The role of FEN-1 in the base excision repair pathway is to cleave 5'-unpaired flaps in forked structures so that DNA ligase can seal the single-stranded breaks that remain following gap repair. The rate of excision by FEN-1 of 5'-flaps from short- and long-flap oligonucleotide substrates that mimic pre- and post-repair HIV-1 integration intermediates, respectively, and the effect of HIV-1 integrase on these reactions were examined in the present study. Cleavage of 5'-flaps by FEN-1 in pre-repair HIV-1 integration intermediates was relatively inefficient and was further decreased 3-fold by HIV-1 integrase. The rate of removal of 5'-flaps by FEN-1 from post-repair HIV-1 integration intermediates containing relatively long (7-nucleotide) unpaired 5'-tails and short (1-nucleotide) gaps was increased 3-fold relative to that seen with pre-repair substrates and was further stimulated 5- to 10-fold by HIV-1 integrase. Overall, post-repair structures were cleaved 18 times more effectively in the presence of HIV-1 integrase than pre-repair structures. The site of cleavage was 1 or 2 nucleotides 3' of the branch point and was unaffected by HIV-1 integrase. Integrase alone had no detectable activity in removing 5'-flaps from either pre- or post-repair substrates.     

Selection of peptides interfering with a ribosomal frameshift in the human immunodeficiency virus type 1

The human immunodeficiency virus of type 1 (HIV-1) uses a programmed -1 ribosomal frameshift to produce the precursor of its enzymes, and changes in frameshift efficiency reduce replicative fitness of the virus. We used a fluorescent two-reporter system to screen for peptides that reduce HIV-1 frameshift in bacteria, knowing that the frameshift can be reproduced in Escherichia coli. Expression of one reporter, the green fluorescent protein (GFP), requires the HIV-1 frameshift, whereas the second reporter, the red fluorescent protein (RFP), is used to assess normal translation. A peptide library biased for RNA binding was inserted into the sequence of the protein thioredoxin and expressed in reporter-containing bacteria, which were then screened by fluorescence-activated cell sorting (FACS). We identified peptide sequences that reduce frameshift efficiency by over 50% without altering normal translation. The identified sequences are also active against different frameshift stimulatory signals, suggesting that they bind a target important for frameshifting in general, probably the ribosome. Successful transfer of active sequences to a different scaffold in a eukaryotic test system demonstrates that the anti-frameshift activity of the peptides is neither due to scaffold-dependent conformation nor effects of the scaffold protein itself on frameshifting. The method we describe identifies peptides that will provide useful tools to further study the mechanism of frameshift and may permit the development of lead compounds of therapeutic interest.     

Covalent bonded Gag multimers in human immunodeficiency virus type-1 particles

The oligomerization of HIV-1 Gag and Gag-Pol proteins, which are assembled at the plasma membrane, leads to viral budding. The budding generally places the viral components under non-reducing conditions. Here the effects of non-reducing conditions on Gag structures and viral RNA protection were examined. Using different reducing conditions and SDS-PAGE, it was shown that oligomerized Gag possesses intermolecular covalent bonds under non-reducing conditions. In addition, it was demonstrated that the mature viral core contains a large amount of covalent bonded Gag multimers, as does the immature core. Viral genomic RNA becomes sensitive to ribonuclease in reducing conditions. These results suggest that, under non-reducing conditions, covalent bonded Gag multimers are formed within the viral particles and play a role in protection of the viral genome.     

Shared antigenic epitopes of the major core proteins of human and simian immunodeficiency virus isolates.

Antigenic epitopes on the major core (gag) protein of isolates of simian and human immunodeficiency virus (SIV and HIV) were compared using a panel of eleven mouse monoclonal antibodies (Mabs) that recognized nine distinct gag epitopes. Viral isolates used for comparison were HIV-1IIIb, HIV-2ROD, and SIV isolates from macaque (SIVmac), sooty mangabey (SIVsm-UCD), African green monkey (SIVagm), and stump-tailed macaque (SIVstm-UCD). The relatedness of the various HIV and SIV isolates, as determined by Mabs to core protein epitopes, paralleled that ascertained by genetic sequencing.     

A novel short peptide is a specific inhibitor of the human immunodeficiency virus type 1 integrase

The retroviral encoded protein integrase (IN) is required for the insertion of the human immunodeficiency virus type 1 (HIV-1) proviral DNA into the host genome. In spite of the crucial role played by IN in the retroviral life cycle, which makes this enzyme an attractive target for the development of new anti-AIDS agents, very few inhibitors have been described and none seems to have a potential use in anti-HIV therapy. To obtain potent and specific IN inhibitors, we used the two-hybrid system to isolate short peptides. Using HIV-1 IN as a bait and a yeast genomic library as the source of inhibitory peptides (prey), we isolated a 33-mer peptide (I33) that bound tightly to the enzyme. I33 inhibited both in vitro IN activities, i.e. 3' end processing and strand transfer. Further analysis led us to select a shorter peptide, EBR28, corresponding to the N-terminal region of I33. Truncated variants showed that EBR28 interacted with the catalytic domain of IN interfering with the binding of the DNA substrate. Alanine single substitution of each EBR28 residue (alanine scanning) allowed the identification of essential amino acids involved in the inhibition. The EBR28 NMR structure shows that this peptide adopts an alpha-helical conformation with amphipathic properties. Additionally, EBR28 showed a significant antiviral effect when assayed on HIV-1 infected human cells. Thus, this potentially important short lead peptide may not only be helpful to design new anti-HIV agents, but also could prove very useful in further studies of the structural and functional characteristics of HIV-1 IN.     

Dynamic tradeoffs in the raft-mediated entry of human immunodeficiency virus type 1 into cells

To initiate an infection human immunodeficiency virus type 1 (HIV-1) particles must first bind to receptors on the surface of their host cells, a process that eventually leads to fusion of viral and cellular membranes and release of the viral genome into the cytoplasm. Understanding the molecular mechanisms of these processes may enable the development of new anti-HIV strategies. Disagreement currently prevails on the role in virus entry of microdomains within the cellular plasma membrane known as lipid rafts. Experiments have suggested that lipid rafts, in their interactions with cellular receptors and viral particles, either promote or have minimal effect on viral entry. Here we develop a dynamic model for HIV-1 entry that enables us to identify and quantitatively assess tradeoffs that can arise from the clustering of receptors in rafts. Specifically, receptor clustering can be detrimental to the initiation of viral infection by reducing the probability that a virus particle finds its primary receptor, CD4. However, receptor clustering can also enable a virus particle, once bound, to rapidly form multivalent interactions with receptors and co-receptors that are required for virus-cell membrane fusion. We show how the resolution of such tradeoffs hinges on the level and spatial distribution of receptors and co-receptors on the cell surface, and we discuss implications of these effects for the design of therapeutics that inhibit HIV-1 entry.     

Activation of a cryptic splice donor in human immunodeficiency virus type-1

The human immunodeficiency virus type-1 regulatory protein Rev is absolutely required for the production of viral structural proteins. Splice sites have been seen to function ascis-acting repressor sequendes (CRS) and inhibit expression of the Rev-dependent RNAs. In order to analyze the role of a splice donor in Rev dependence, the wild-type 5 splice donor of HIV-1 was mutated in the context of othergag sequences. Following transient transfection, RNA expression by RT-PCR was analyzed. The unspliced RNA produced by the mutant construct still required Rev for the cytoplasmic accumulation of the RNA. Despite deletion of the wild-type 5 splice donor and thetat splice acceptor was used. A cryptic splice donor was identified by PCR and subsequent cloning of the spliced RNA. The cryptic site is 5/9 to the consensus sequence and located immediately downstream of the initiation codon (ATG) for Gag. Analysis of the RNA product containing the cryptic splice donor revealed that the Rev was required for the cytoplasmic accumulation of unspliced RNA, while spliced RNA was Rev independent. Transfection of a wild-type construct also demonstrated usage of the cryptic splice donor. These results indicate that a cryptic splice donor can be activated when the wild-type splice donor is inactivated and that the cryptic splice donor may retain Rev regulation. The findings also suggest the potential for cryptic splice sites to serve as CRS in the determining the Rev dependence of viral RNAs.     

Extrachromosomal human immunodeficiency virus type-1 DNA can initiate a spreading infection of HL-60 cells

In this report, we describe a human immunodeficiency virus type-1 (HIV-1)-infected promyelocytic cell line, OM, derived from HL-60 cells. Although the OM cell line was biologically cloned twice, the pattern of HIV-1 expression during culture appeared analogous to a classical acute spreading infection and was inhibited by both azidothymidine and recombinant soluble CD4 treatment. The number of OM cells actually expressing HIV-1 at the beginning of culture was 0%, reached a peak of nearly 100% at 6 weeks, and then fell to less than 10% HIV-1+ cells by 10 weeks. Clonal analysis of the surviving cells verified that stable HIV-1+ OM cells resulted from the spreading infection. Southern analysis confirmed the transmission of HIV-1 through these OM cultures and the occurrence of stable clones which resulted. The initial percentage of OM cells actually harboring the HIV-1 genome was less than 0.1%, indicating nonfaithful transmission of an unintegrated HIV-1 genome during clonal expansion. These results demonstrate that extrachromosomal HIV-1 DNA can contribute to the spread of HIV-1 infection and give rise to cells which have stably integrated HIV-1 provirus.     

Inoculation of New World primates with the human immunodeficiency virus

The susceptibility of common marmosets and cotton-top tamarins to infection by HIV-2 in vivo was tested. One year and 19 months, respectively, post-inoculation, sera taken from three of four animals from each species are reactive for HIV-2 antibodies and HIV-specific nucleotide sequences were demonstrated in short-term cultures of PBL from two cotton-top tamarins. The animals remain in good health.     

Evaluation of protective efficacy of recombinant subunit vaccines against simian immunodeficiency virus infection of macaques.

Simian immunodeficiency virus (SIV) was used as a model to study the protective efficacy of an immunization regimen currently being evaluated as candidate vaccines against HIV in human subjects. Four Macaca fascicularis were first immunized with recombinant vaccinia virus expressing the envelope glycoprotein gp160 of SIVmne and then boosted with subunit gp160. Both cell-mediated and humoral immune responses against SIV, including neutralizing antibodies, were elicited. The macaques were shown to be protected from a homologous virus infection as determined by serology, lymphocyte cocultivation, polymerase chain reactions and in vivo transmission analyses. Four unimmunized control animals were readily infected. However, viremia in infected control animals could decrease substantially following the initial phase of infection so that persistent infection might not be readily detectable.