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1.
DNA amplification for direct detection of HIV-1 in DNA of peripheral blood mononuclear cells 总被引：140，自引：0，他引：140
C Y Ou S Kwok S W Mitchell D H Mack J J Sninsky J W Krebs P Feorino D Warfield G Schochetman 《Science (New York, N.Y.)》1988,239(4837):295-297
By means of a selective DNA amplification technique called polymerase chain reaction, proviral sequences of the human immunodeficiency virus (HIV-1) were identified directly in DNA isolated from peripheral blood mononuclear cells (PBMCs) of persons seropositive but not in DNA isolated from PBMCs of persons seronegative for the virus. Primer pairs from multiple regions of the HIV-1 genome were used to achieve maximum sensitivity of provirus detection. HIV-1 sequences were detected in 100% of DNA specimens from seropositive, homosexual men from whom the virus was isolated by coculture, but in none of the DNA specimens from a control group of seronegative, virus culture-negative persons. However, HIV-1 sequences were detected in 64% of DNA specimens from seropositive, virus culture-negative homosexual men. This method of DNA amplification made it possible to obtain results within 3 days, whereas virus isolation takes up to 3 to 4 weeks. The method may therefore be used to complement or replace virus isolation as a routine means of determining HIV-1 infection. 相似文献
2.
V Manzari A Gismondi G Barillari S Morrone A Modesti L Albonici L De Marchis V Fazio A Gradilone M Zani 《Science (New York, N.Y.)》1987,238(4833):1581-1583
A new human retrovirus was isolated from a continuous cell line derived from a patient with CD4+ Tac- cutaneous T cell lymphoma/leukemia. This virus is related to but distinct from human T cell leukemia/lymphoma virus types I and II (HTLV-I and HTLV-II) and human immunodeficiency virus (HIV-1). With the use of a fragment of provirus cloned from one patient with T cell leukemia, closely related sequences were found in DNA of the cell line and of tumor cells from seven other patients with the same disease; these sequences were only distantly related to HTLV-I. The phenotype of the cells and the clinical course of the disease were clearly distinguishable from leukemia associated with HTLV-I. All patients and the wife of one patient showed a weak serological cross-reactivity with both HTLV-I and HIV-1 antigens. None of the patients proved to be at any apparent risk for HIV-1 infection. The name proposed for this virus is HTLV-V, and the date indicate that it may be a primary etiological factor in the major group of cutaneous T cell lymphomas/leukemias, including the sporadic lymphomas known as mycoses fungoides. 相似文献
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4.
设计含有与ERG6基因两侧序列同源的长引物,以质粒pBlue-Kan为模板进行PCR扩增,构建含有Cre/lox P系统的酿酒酵母ERG6基因敲除组件.将基因敲除组件转化至酿酒酵母(Saccharomyce cerevisiae)AD1-8,通过同源重组的方式使目的基因缺失,获得为lox P-Kan-lox P序列组件所替换而产生Kanr的阳性克隆子.然后再将质粒pHis-Cre转入阳性克隆子表达Cre重组酶敲除筛选标记,成功获得酿酒酵母ERG6基因缺失的突变株,并命名为AD1-8-δ. 相似文献
5.
To better understand the basis for human immunodeficiency virus type 1 (HIV-1) persistence and latency, the form in which viral DNA exists in the peripheral T lymphocyte reservoir of infected individuals was investigated. In asymptomatic individuals, HIV-1 was harbored predominantly as full-length, unintegrated complementary DNA. These extrachromosomal DNA forms retained the ability to integrate upon T cell activation in vitro. In patients with acquired immunodeficiency syndrome (AIDS), there was an increase in integrated relative to extrachromosomal DNA forms. By analysis of DNA from patient lymphocyte subpopulations depleted of human lymphocyte antigen-Dr receptor-positive cells, quiescent T cells were identified as the source of extrachromosomal HIV-1 DNA. Thus quiescent T lymphocytes may be a major and inducible HIV-1 reservoir in infected individuals. 相似文献
6.
Activation of the HIV-1 LTR by T cell mitogens and the trans-activator protein of HTLV-I 总被引：102，自引：0，他引：102
M Siekevitz S F Josephs M Dukovich N Peffer F Wong-Staal W C Greene 《Science (New York, N.Y.)》1987,238(4833):1575-1578
To investigate the mechanism by which immune activation augments replication of the human immunodeficiency virus type 1 (HIV-1) in infected T cells, four different classes of T cell mitogens were evaluated for their effects on the HIV-1 long terminal repeat (LTR). Phytohemagglutinin (PHA), a mitogenic lectin; phorbol 12-myristic 13-acetate, a tumor promoter; ionomycin, a calcium ionophore; and tat-1, the trans-activator protein from the human T cell leukemia/lymphoma virus type I (HTLV-I) each stimulated the HIV-1 LTR. Studies of deleted forms of the LTR supported a central role in these responses for the HIV-1 enhancer, which alone was sufficient for mitogen inducibility, but also suggested that other 5' positive and negative regulatory elements contribute to the overall magnitude of the response. Synergistic activation of the HIV-1 LTR (up to several thousandfold) was observed with combinations of these mitogens and the HIV-1--derived tat-III protein. Cyclosporin A, an immunosuppressive agent, inhibited PHA-mediated activation of the HIV-1 LTR but was without effect in the presence of other mitogens. Thus, HIV-1 gene expression and replication appear to be regulated, via the HIV-1 LTR, by the same mitogenic signals that induce T cell activation. 相似文献
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Nef protein of HIV-1 is a transcriptional repressor of HIV-1 LTR 总被引：73，自引：0，他引：73
9.
Recombinase-mediated gene activation and site-specific integration in mammalian cells 总被引：50，自引：0，他引：50
A binary system for gene activation and site-specific integration, based on the conditional recombination of transfected sequences mediated by the FLP recombinase from yeast, was implemented in mammalian cells. In several cell lines, FLP rapidly and precisely recombined copies of its specific target sequence to activate an otherwise silent beta-galactosidase reporter gene. Clones of marked cells were generated by excisional recombination within a chromosomally integrated copy of the silent reporter. By the reverse reaction, integration of transfected DNA was targeted to a specific chromosomal site. The results suggest that FLP could be used to mosaically activate or inactivate transgenes for analysis of vertebrate development, and to efficiently integrate transfected DNA at predetermined chromosomal locations. 相似文献
10.
Antibody-enhanced infection by HIV-1 via Fc receptor-mediated entry 总被引：49，自引：0，他引：49
Monocytes and macrophages, which may play a central role in the pathogenesis of infection with human immunodeficiency virus type 1 (HIV-1), express the CD4 molecule and Fc receptors (FcR) for immunoglobulin G (IgG). To explore the possibility that FcR mediate HIV-1 infection of monocytes, studies were conducted with the human monocytic cell line U937. These cells were exposed to HIV-1 complexed with various concentrations of serum from HIV-1 antibody-positive individuals and monitored for HIV-1 replication. Serum samples from antibody-negative normal individuals did not affect virus yields. High concentrations of antibody-positive sera showed virus-neutralizing activity; however, cells infected with HIV-1 in the presence of antibody-positive sera at subneutralizing concentrations significantly enhanced virus replication. This infection enhancement was blocked by heat-aggregated gamma-globulin. Moreover, the IgG fraction from an HIV-1 antibody-positive serum enhanced HIV-1 infection at the same serum dilution equivalents. In contrast, IgG-F(ab')2 did not enhance HIV-1 infection but showed neutralizing activity with HIV-1. These results are compatible with the concept of FcR-mediated infection enhancement and suggest that this immunological response to HIV-1, instead of protecting the host, potentially facilitates the infection. 相似文献
11.
Deleted HTLV-I provirus in blood and cutaneous lesions of patients with mycosis fungoides. 总被引：12，自引：0，他引：12
W W Hall C R Liu O Schneewind H Takahashi M H Kaplan G R?upe A Vahlne 《Science (New York, N.Y.)》1991,253(5017):317-320
Mycosis fungoides, a rare form of cutaneous T cell leukemia/lymphoma, is suspected of having a viral etiology on the basis of certain similarities to adult T cell leukemia, which is associated with human T cell leukemia/lymphoma virus type I (HTLV-I) infection. Cell lines were established from peripheral blood mononuclear cells (PBMC) of an HTLV-I-seronegative patient with mycosis fungoides. DNA hybridization analysis revealed the presence of HTLV-I-related sequences with unusual restriction endonuclease sites. Sequence analysis of subcloned fragments demonstrated the presence of a monoclonally integrated provirus with a 5.5-kilobase deletion involving large regions of gag and env and all of pol. Additional evidence for the presence of deleted proviruses was found by polymerase chain reaction (PCR) amplification of DNA from cutaneous lesions of five other HTLV-I-seronegative patients. The findings suggest that HTLV-I infection may be involved in the etiology of at least certain cases of mycosis fungoides. 相似文献
12.
Human immunodeficiency virus type 1 (HIV-1) gene expression is activated by Tat, a virally encoded protein. Tat trans-activation requires viral (trans-activation--responsive; TAR) RNA sequences located in the R region of the long terminal repeat (LTR). Existing evidence suggests that Tat probably cooperates with cellular factors that bind to TAR RNA in the overall trans-activation process. A HeLa complementary DNA was isolated and characterized that encodes a TAR RNA-binding protein (TRBP). TRBP activated the HIV-1 LTR and was synergistic with Tat function. 相似文献
13.
[目的]建立PIWIL1基因启动子调控的特异性表达Cre重组酶的转基因猪,为研究piRNA及其相关基因的功能和机制奠定基础。[方法]通过PCR扩增,从猪的全基因组上扩增出PIWIL1基因的启动子。切除猪源Cre重组酶的表达载体pET28a-MHC-Cre-BGHpo1yA-FRT2neor的启动子MHC,将其与PIWIL1基因启动子连接,构建成特异性表达Cre重组酶的pPIWIL1-Cre载体。将该载体转染小型猪胎儿成纤维细胞,通过G418筛选,获得阳性克隆进行核移植。[结果]获得2头PIWIL1基因启动子调控的特异表达Cre酶的转基因猪。[结论]成功构建了PIWIL1基因启动子调控的特异表达Cre重组酶的转基因猪,为研究piRNA及其相关基因的功能和机制提供了工具猪。 相似文献
14.
Characterization of a noncytopathic HIV-2 strain with unusual effects on CD4 expression 总被引：22，自引：0，他引：22
L A Evans J Moreau K Odehouri H Legg A Barboza C Cheng-Mayer J A Levy 《Science (New York, N.Y.)》1988,240(4858):1522-1525
A new isolate of the human immunodeficiency virus type 2, designated HIV-2UC1, was recovered from an Ivory Coast patient with normal lymphocyte numbers who died with neurologic symptoms. Like some HIV-1 isolates, HIV-2UC1 grows rapidly to high titers in human peripheral blood lymphocytes and macrophages and has a differential ability to productively infect established human cell lines of lymphocytic and monocytic origin. Moreover, infection with this isolate also appears to involve the CD4 antigen. However, unlike other HIV isolates, HIV-2UC1 does not cause cytopathic effects in susceptible T cells nor does it lead to loss of CD4 antigen expression on the cell surface. These results indicate that HIV-2 may be found in individuals with neurologic symptoms and that the biological characteristics of this heterogeneous subgroup can differ from those typical of HIV-1. 相似文献
15.
Saphire EO Parren PW Pantophlet R Zwick MB Morris GM Rudd PM Dwek RA Stanfield RL Burton DR Wilson IA 《Science (New York, N.Y.)》2001,293(5532):1155-1159
We present the crystal structure at 2.7 angstrom resolution of the human antibody IgG1 b12. Antibody b12 recognizes the CD4-binding site of human immunodeficiency virus-1 (HIV-1) gp120 and is one of only two known antibodies against gp120 capable of broad and potent neutralization of primary HIV-1 isolates. A key feature of the antibody-combining site is the protruding, finger-like long CDR H3 that can penetrate the recessed CD4-binding site of gp120. A docking model of b12 and gp120 reveals severe structural constraints that explain the extraordinary challenge in eliciting effective neutralizing antibodies similar to b12. The structure, together with mutagenesis studies, provides a rationale for the extensive cross-reactivity of b12 and a valuable framework for the design of HIV-1 vaccines capable of eliciting b12-like activity. 相似文献
16.
Contribution of human alpha-defensin 1, 2, and 3 to the anti-HIV-1 activity of CD8 antiviral factor 总被引：1，自引：0，他引：1
Zhang L Yu W He T Yu J Caffrey RE Dalmasso EA Fu S Pham T Mei J Ho JJ Zhang W Lopez P Ho DD 《Science (New York, N.Y.)》2002,298(5595):995-1000
It has been known since 1986 that CD8 T lymphocytes from certain HIV-1-infected individuals who are immunologically stable secrete a soluble factor, termed CAF, that suppresses HIV-1 replication. However, the identity of CAF remained elusive despite an extensive search. By means of a protein-chip technology, we identified a cluster of proteins that were secreted when CD8 T cells from long-term nonprogressors with HIV-1 infection were stimulated. These proteins were identified as alpha-defensin 1, 2, and 3 on the basis of specific antibody recognition and amino acid sequencing. CAF activity was eliminated or neutralized by an antibody specific for human alpha-defensins. Synthetic and purified preparations of alpha-defensins also inhibited the replication of HIV-1 isolates in vitro. Taken together, our results indicate that alpha-defensin 1, 2, and 3 collectively account for much of the anti-HIV-1 activity of CAF that is not attributable to beta-chemokines. 相似文献
17.
RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination 总被引：154，自引：0，他引：154
The vast repertoire of immunoglobulins and T cell receptors is generated, in part, by V(D)J recombination, a series of genomic rearrangements that occur specifically in developing lymphocytes. The recombination activating gene, RAG-1, which is a gene expressed exclusively in maturing lymphoid cells, was previously isolated. RAG-1 inefficiently induced V(D)J recombinase activity when transfected into fibroblasts, but cotransfection with an adjacent gene, RAG-2, has resulted in at least a 1000-fold increase in the frequency of recombination. The 2.1-kilobase RAG-2 complementary DNA encodes a putative protein of 527 amino acids whose sequence is unrelated to that of RAG-1. Like RAG-1, RAG-2 is conserved between species that carry out V(D)J recombination, and its expression pattern correlates precisely with that of V(D)J recombinase activity. In addition to being located just 8 kilobases apart, these convergently transcribed genes are unusual in that most, if not all, of their coding and 3' untranslated sequences are contained in single exons. RAG-1 and RAG-2 might activate the expression of the V(D)J recombinase but, more likely, they directly participate in the recombination reaction. 相似文献
18.
The accuracy of reverse transcriptase from HIV-1 总被引：72，自引：0，他引：72
19.
S H Weiss J J Goedert S Gartner M Popovic D Waters P Markham F di Marzo Veronese M H Gail W E Barkley J Gibbons 《Science (New York, N.Y.)》1988,239(4835):68-71
In a prospective cohort study of 265 laboratory and affiliated workers, one individual with no recognized risk factors for human immunodeficiency virus type 1 (HIV-1) infection was HIV-1 seropositive at the time of entry into the study. Molecular analyses of two HIV-1 isolates derived in two independent laboratories from a blood sample from this worker showed that the isolates were indistinguishable from a genotypic form of HIV-1 present in the H9/HTLV-IIIB cell line. Exposure to this strain of virus most probably occurred during work with concentrated virus or culture fluids from virus-producing cell lines under standard Biosafety Level 3 containment. Although no specific incident leading to this infection has been identified, undetected skin contact with virus culture supernatant might have occurred. This worker was the only one found to be positive among the subgroup of 99 workers who shared a work environment involving exposure to concentrated virus. The incidence rate of 0.48 per 100 person-years exposure indicates that prolonged laboratory exposure to concentrated virus is associated with some risk of HIV-1 infection, which is comparable to the risk for health care workers experiencing a needle stick exposure. While none of the ten workers with parenteral exposure to HIV-1 in this cohort became infected, a worker in another laboratory did seroconvert following an injury with a potentially contaminated needle. Strict Biosafety Level 3 containment and practices should be followed when working with concentrated HIV-1 preparations, and further refinement of the procedures may be necessary. 相似文献
20.
Type-restricted neutralization of molecular clones of human immunodeficiency virus 总被引：23，自引：0，他引：23
D J Looney A G Fisher S D Putney J R Rusche R R Redfield D S Burke R C Gallo F Wong-Staal 《Science (New York, N.Y.)》1988,241(4863):357-359
In a study of the immunologic significance of the genetic diversity present within single isolates of human immunodeficiency virus type 1 (HIV-1), the neutralization of viruses derived from molecular clones of the HIV-1 strain HTLV-IIIB by an extensive panel of sera was compared. Sera from HIV-1-infected patients and from goats immunized with polyacrylamide gel-purified HIV-1 envelope glycoprotein (gp120), native gp120, or gp120-derived recombinant peptides, showed marked heterogeneity in neutralizing activity against these closely related viruses. The change of a single amino acid residue in gp120 may account for such "clonal restriction" of neutralizing activity. 相似文献