Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P30536 (PBS)
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In the HIV Seroprevalence Survey among Childbearing Women (SCBW), antibodies to human immunodeficiency virus type 1 are detected using enzyme immunoassays (EIA) and Western blot (WB) methods modified to accommodate samples of blood dried on special collection paper. Dried blood spot (DBS) eluates positive by EIA are tested by one of two WB methods, the miniblot technique using equipment from Immunetics Corporation and the PBS Integra assay (pageblot) from Genetic Systems. In this report we compared the performance of the two WB methods. The identity and position of the viral proteins on the WB were identified using monoclonal antibodies and monospecific antisera. The blots differed substantially in their composition and concentration of viral glycoproteins. Performance of the WB assays with DBS elution buffers from different EIA kits was equivalent except for samples eluted in the Abbott buffer, which reduced detection of antibodies to the p31, p51, p55, and p66 viral proteins. Case classification of DBS, positive sera, dilution curve samples, and seroconversion panels was equivalent by both tests in the presence of all elution buffers. Proficiency evaluation panels sent to SCBW participating laboratories over a 3-year period were used to note the differences between the two WB methods in detection of antibodies to the viral glycoproteins.
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PMID:Factors influencing HIV-1 banding patterns in miniaturized western blot testing of dried blood spot specimens. 140 56

The interactions between the Reverse Transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) and the natural tRNA(Lys3) primer for initiation of viral DNA synthesis were examined. We constructed a set of HIV-1 RNA templates in which the wild-type primer binding site (PBS(Lys3)) is replaced by sequences complementary to tRNA(lle), tRNA(Lys1,2), tRNA(Phe), tRNA(Pro) or tRNA(Trp) and tested the ability of RT enzymes of different retroviral species to initiate cDNA synthesis from self versus non-self tRNA primers. We demonstrate that initiation of HIV-1 reverse transcription is a specific process that is most efficient with the self tRNA(Lys3) primer. Interestingly, the property of HIV-1 RT to discriminate against non-self tRNA primers is lost upon extension of the tRNA by only two deoxyribonucleotides. Furthermore, selective tRNA priming by HIV-1 RT was not observed with viral RNA-tRNA(Lys3) duplexes isolated from HIV-1 virion particles, suggesting that the majority of tRNA(Lys3) primers annealed to viral RNA in particles is extended by a variable number of deoxyribonucleotides. This result indicates that reverse transcription is initiated relatively early in nascently assembled virions.
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PMID:HIV-1 reverse transcriptase discriminates against non-self tRNA primers. 895 74

Three experimental vaccines against feline immunodeficiency virus (FIV), all based on viral antigens presented via immune stimulating complexes (iscoms), were tested for their capacity to induce protection in cats from FIV infection. The respective vaccines consisted of FIV propagated in Crandell feline kidney (CrFK) cells (FIV-iscoms); FIV-iscoms spiked with recombinant vaccinia virus expressed FIV envelope glycoprotein incorporated into iscoms (FIV-iscoms + vGR657x15-iscoms) and vGR657x15-iscoms spiked with recombinant FIV Gag protein incorporated into iscoms (vGR657x15-iscoms + FIV-Gag-iscoms). Simian immunodeficiency virus envelope glycoprotein incorporated into iscoms, iscoms prepared with uninfected CrFK cells, and PBS served as controls. All cats vaccinated with vGR657x15-iscoms combined with FIV-iscoms or FIV-Gag-iscoms developed Env-specific plasma antibody responses. These antibodies neutralised FIV infection in CrFK cells, but failed to neutralise FIV infection in primary feline thymocytes. FIV-iscoms induced poor Env-specific responses and only one out of six cats developed antibodies that neutralised FIV in the CrFK cell based assay. Four weeks after challenge all cats proved to be infected, showing that none of the vaccine preparations provided protection. In contrast, 2 weeks after infection, virus infected peripheral blood mononuclear cells were only observed in cats vaccinated with FIV-iscoms + vGR657x15-iscoms or CrFK-iscoms and to a lesser extent in cats vaccinated with FIV-iscoms and vGR657x15-iscoms + FIV-Gag-iscoms, but not in cats vaccinated with SIV-iscoms or PBS. The differences found in cell associated virus loads amongst the respective groups are discussed in the light of antibody mediated enhancement of infectivity and protective effects provided by Gag-specific T cell responses.
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PMID:Feline immunodeficiency virus subunit vaccines that induce virus neutralising antibodies but no protection against challenge infection. 960 28

The spread of sexually transmitted infections caused by herpes simplex virus type 2 (HSV-2) has continued unabated despite educational efforts generated in response to the human immunodeficiency virus (HIV) epidemic. Given the absence of effective vaccines, this indicates the need to develop prophylactic measures such as topical antiviral agents. Chemical modification of bovine beta-lactoglobulin (beta-LG), the major protein of whey, by hydroxyphthalic anhydride (3HP) led to the generation of a potent HIV-1 inhibitor designated 3HP-beta-LG. This agent was shown to also have antiviral activity against HSV-2 and HSV-1 in vitro. Recent studies indicate that 3HP-beta-LG binds to HSV-1 virions, which, at least in part, involves the viral glycoprotein gE. Here we show that 3HP-beta-LG inhibits HSV-2 infection in the mouse model of genital HSV-2 infection. Simultaneous exposure to HSV-2 and 3HP-beta-LG caused a significant decrease in the proportion of infected animals (27% virus shedding, 5% lesion development and 0% fatality for 3HP-beta-LG as compared to 80% shedding, 60% lesion development and 53% fatality in mice treated with PBS). The proportion of animals with HSV-2 infection after treatment with beta-LG was similar to that in the PBS-treated group. Pretreatment with 3HP-beta-LG formulated in a gel, which prolongs the presence of the agent in the vagina, also significantly reduced the proportion of HSV-2-infected mice (5% virus shedding, 5% lesion development and 0% fatality for 3HP-beta-LG as compared to 70% shedding, 60% lesion development and 40% fatality in vehicle-treated mice). These differences were significant (P < or = 0.0005, 0.002 and 0.008 for shedding, lesion development and fatality, respectively). Virus titres in the minority of mice that developed infection were similar to those in untreated mice. HSV-2 infection was not inhibited by treatment of an ongoing infection, indicating that 3HP-beta-LG interferes with the initial infection. These data suggest that 3HP-beta-LG may be an efficacious agent for preventing vaginal transmission of genital herpesvirus infections.
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PMID:3-Hydroxyphthaloyl beta-lactoglobulin. IV Antiviral activity in the mouse model of genital herpesvirus infection. 987 14

Reverse transcription of the RNA genome of retroviruses has to proceed through some highly structured regions of the template. The RNA genome of the human immunodeficiency virus type 1 (HIV-1) contains two hairpin structures within the repeat (R) region at the 5' end of the viral RNA (Fig. 1Fig. 1Template RNA structure of the HIV-1 R region and the position of reverse transcription pause sites. The HIV-1 R region (nucleotides +1/97) encodes two stable RNA structures, the TAR and polyA hairpins [5]. The latter hairpin contains the AAUAAA hexamer motif (marked by a box) that is involved in polyadenylation. The lower panel shows the predicted structures of the wild-type and two mutant forms of the polyA hairpin that were used in this study. Nucleotide substitutions are boxed, deletions are indicated by black triangle. The thermodynamic stability (free energy or DeltaG, in kcal/mol) was calculated according to the Zucker algorithm [71]. The TAR hairpin has a DeltaG of -24.8 kcal/mol. Minus-strand DNA synthesis on these templates was initiated by a DNA primer annealed to the downstream PBS. The position of reverse transcription pause sites observed in this study are summarized. All numbers refer to nucleotide positions on the wild-type HIV-1 transcript. Filled arrows represent stops observed on the wild-type template, and open arrows mark the pause sites that are specific for the structured A-mutant template. The sizes of the arrows correspond to the relative frequency of pausing. Little pausing was observed on the B-mutant template with the destabilized polyA hairpin.). These structures, the TAR and polyA hairpins, fulfil important functions in the viral life cycle. We analyzed the in vitro elongation properties of the HIV-1 reverse transcriptase (RT) enzyme on the wild-type RNA template and mutants thereof with either a stabilized or a destabilized polyA hairpin. Stable RNA structure was found to interfere with efficient elongation of the RT enzyme, as judged by the appearance of pause cDNA products. A direct relation was measured between the stability of template RNA structure and the extent of RT pausing. However, the position of structure-induced pause sites is rather diverse, with significant stops at a position approximately 6 nt ahead of the basepaired stem of the TAR and polyA hairpins. This suggests that the RT enzyme is stalled when its most forward domain contacts the RNA duplex. Addition of the viral nucleocapsid protein (NC) to the in vitro assay was found to overcome such structure-induced RT stops. These results indicate that the RT polymerase has problems penetrating regions of the template with stable RNA structure. This effect was more pronounced at high Mg2+ concentrations, which is known to stabilize RNA secondary structure. Such a structure-induced defect was not apparent in reverse transcription assays performed in virus-infected cells, which is either caused by the NC protein or other components of the virion particle. Thus, retroviruses can use relatively stable RNA structures to control different steps in the viral life cycle without interfering with the process of reverse transcription.
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PMID:The effect of template RNA structure on elongation by HIV-1 reverse transcriptase. 1009 59

The formation of an infectious retrovirus particle requires several RNA-RNA interaction events. In particular, the genomic RNA molecules form a dimeric structure, and a cellular tRNA molecule is annealed to an 18-base complementary region (the primer binding site, or PBS) on the genomic RNA, where it will serve as primer for reverse transcription. tRNAs normally possess a highly stable secondary and tertiary structure; it seems unlikely that annealing of a tRNA molecule to the PBS, which involves unwinding of this structure, could occur efficiently at physiological temperatures without the assistance of a cofactor. Many prior studies have shown that the viral nucleocapsid (NC) protein can act as a nucleic acid chaperone (i.e., facilitate annealing events between nucleic acids), and the assays used to demonstrate this activity include its ability to catalyze dimerization of transcripts representing retroviral genomes and the annealing of tRNA to the PBS in vitro. However, mature NC is not required for these events in vivo, since protease-deficient viral mutants, in which NC is not cleaved from the parental Gag polyprotein, are known to contain dimeric RNAs with tRNA annealed to the PBS. In the present experiments, we have tested recombinant human immunodeficiency virus type 1 Gag polyprotein for nucleic acid chaperone activity. The protein was positive by all of our assays, including the ability to stimulate dimerization and to anneal tRNA to the PBS in vitro. In quantitative experiments, its activity was approximately equivalent on a molar basis to that of NC. Based on these results, we suggest that the Gag polyprotein (presumably by its NC domain) catalyzes the annealing of tRNA to the PBS during (or before) retrovirus assembly in vivo.
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PMID:The human immunodeficiency virus type 1 Gag polyprotein has nucleic acid chaperone activity: possible role in dimerization of genomic RNA and placement of tRNA on the primer binding site. 1019 21

Retroviral reverse transcription is initiated from a cellular tRNA molecule and all known exogenous isolates of murine leukemia virus utilise a tRNA(Pro)molecule. While several studies suggest flexibility in murine leukemia virus primer utilisation, studies on human immunodeficiency virus and avian retro-viruses have revealed evidence of molecular adapt-ation towards the specific tRNA isoacceptor used as replication primer. In this study, murine leukemia virus tRNA utilisation is investigated by in vivo screening of a retroviral vector combinatorial library with randomised primer binding sites. While most of the selected primer binding sites are complementary to the 3'-end of tRNA((Pro)), we also retrieved PBS sequences matching four other tRNA molecules and demonstrate that Akv murine leukemia virus vectors may efficiently replicate using tRNA(Arg(CCU)), tRNA(Phe(GAA))and a hitherto unknown human tRNA(Ser(CGA)).
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PMID:Selection of functional tRNA primers and primer binding site sequences from a retroviral combinatorial library: identification of new functional tRNA primers in murine leukemia virus replication. 1063 32

Reverse transcription by human immunodeficiency virus (HIV) reverse transcriptase (RT) entails several distinct, early steps in the synthesis of double-stranded DNA from viral RNA templates. These steps include tRNA(lys3) priming of RNA-dependent DNA polymerization and template switching for synthesis of near full-length (-) DNA. However, HIV RT lacks the fluent processivity of other viral reverse transcriptases or cellular polymerases. Previous studies in our laboratory showed that RT reactions primed with tRNA(lys3) had higher efficiencies of template switching than those primed by synthetic oligonucleotides. To further study primer extension, pausing, and template switching, we utilized an in vitro reconstituted reverse transcription/template switching reaction consisting of HIV RNA templates, recombinant HIV RT and primer tRNA(lys3). We observed initial pause sites within the first five nucleotides of the primer terminus; these were extended efficiently to full-length (-) strong-stop DNA with prolonged incubation. Other pause sites occurred in the R/U5/PBS template adjacent to homopolymeric sequences and regions thought to be involved in secondary structure. This assay provides a sensitive means of assessing template switching at times between 10 s and 3 h. Copyright 1995 S. Karger AG, Basel
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PMID:Analysis of Primer Extension and the First Template Switch during Human Immunodeficiency Virus Reverse Transcription. 1172 68

The conversion of the single-stranded RNA genome into double-stranded DNA by virus-coded reverse transcriptase (RT) is an essential step of the retrovirus life cycle. In human immunodeficiency virus type 1 (HIV-1), RT uses the cellular tRNA(Lys)3 to initiate the (-) strand DNA synthesis. Placement of the primer tRNA(Lys)3 involves binding of its 3'-terminal 18 nt to a complementary region of genomic RNA termed PBS. However, the PBS sequence is not the unique determinant of primer usage and additional contacts are important. This placement is believed to be achieved in vivo by the nucleocapsid domain of Gag or by the mature protein NCp. Up to now, structural information essentially arose from heat-annealed primer-template complexes (Isel et al., J Mol Biol, 1995, 247:236-250; Isel et al., EMBO J, 1999, 18:1038-1048). Here, we investigated the formation of the primer-template complex mediated by NCp and compared structural and functional properties of heat- and NCp-annealed complexes. We showed that both heat- and NCp-mediated procedures allow comparable high yields of annealing. Then, we investigated structural features of both kinds of complexes by enzymatic probing, and we compared their relative efficiency in (-) strong stop DNA synthesis. We did not find any significant differences between these complexes, suggesting that information derived from the heat-annealed complex can be transposed to the NCp-mediated complex and most likely to complexes formed in vivo.
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PMID:Structural and functional properties of the HIV-1 RNA-tRNA(Lys)3 primer complex annealed by the nucleocapsid protein: comparison with the heat-annealed complex. 1187 59

The prevalence of human immunodeficiency virus antibodies in mothers and their neonates was determined through unlinked anonymous HIV testing at delivery. Two hundred and forty-six apparently healthy volunteer anonymous parturients at the University of Benin Teaching Hospital, Nigeria, and their neonates were recruited for the study. Blood samples were collected from the mothers and cord blood from their neonates at delivery. The blood samples were coded and assayed for HIV antibodies using ImmunoComb HIV 1 and 2 bispot test and ImmunoComb II HIV-1 and 2 CombFirm, both from PBS Orgenics, France. The results for maternal and neonatal blood sample pairs were matched. The maternal seroprevalence for HIV antibodies was 2.4% (6/246) while only two infants had HIV antibodies. The mother-to-child transmission of HIV antibodies was 33.3%. The study highlights the increasing HIV infection among pregnant women in this hospital. The risk of vertical transmission is therefore high. Universal antenatal HIV testing with an opt-out system is suggested. The need for our maternity centres and special care baby units to establish management protocols and anti-retroviral therapy for HIV infected women and their neonates is recommended.
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PMID:Maternal and neonatal seroprevalence of human immunodeficiency virus antibodies in Benin City, Nigeria. 1551 70


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