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1.
African monkeys are resistant to HIV-1 infection due to intrinsic restriction mechanisms found in their cells. However, although they can be infected by monkey-adapted modified HIV-1 particles that are designed to overcome known restriction factors, virus numbers drop to undetectable levels in immunocompetent animals. These results indicate the possibility of the presence of yet unidentified factor(s) that restrict HIV-1 in old-world monkey (OWM) cells after integration of the viral genome into the host cell chromosome. In the light of these findings, we hypothesized that OWMs might have evolved resistance mechanism(s) against HIV-1 by switching specific gene(s) on in response to the synthesis of viral proteins in infected cells. In an attempt to mimic post-infection status, we expressed HIV-1 Tat gene in African green monkey cells and compared the whole proteome with normal cells and identified secretory leukocyte protease inhibitor (SLPI), a protein with known extracellular anti-HIV-1 activity, as an over-expressed protein in the presence of HIV-1 Tat protein by 2D-PAGE and mass spectrometry analysis. We also showed that overexpression of SLPI in the presence of HIV-1 Tat was specific to monkey cells. Our results also suggest that SLPI had a previously undiscovered intracellular anti-HIV activity in addition to its extracellular activity. 相似文献
2.
Human immunodeficiency virus type-1 (HIV-1) Pr55 Gag virus-like particles (VLP) represent an interesting HIV vaccine component since they stimulate strong humoral and cellular immune responses. We demonstrated that VLP expressed by recombinant baculoviruses activate human PBMC to release pro-inflammatory (lL-6, TNF-α), anti-inflammatory (IL-10) and Th1-polarizing (IFN-γ) cytokines as well as GM-CSF and MIP-1α in a dose-and time-dependent manner. Herein, residual baculoviruses within the VLP preparations showed no or minor effects. Monocytes could be identified as a main target for VLP to induce cytokine production. Furthermore, VLP-induced monocyte activation was shown by upregulation of molecules involved in antigen presentation (MHC II, CD80, CD86) and cell adhesion (CD54). Exposure of VLP to serum inactivates its capacity to stimulate cytokine production. In summary, these investigations establish VLP as strong activators of PBMC and monocytes therein, potently enhancing their functionality and potency to promote an efficient immune response. This capacity makes VLP an interesting component of combination vaccines. 相似文献
3.
Human immunodeficiency virus (HIV) Gag proteins are assembled into virus particles and then cleaved by the virion-associated HIV protease. Concomitant with Gag processing, doughnut-like HIV particles (the immature form) are converted to particles containing condensed cores (the mature form). Here we describe the in vitro processing of immature HIV Gag virus-like particles (VLP) by exogenously added HIV protease. Following delipidization, sequential processing of immature VLP showed that the matrix (MA)/capsid (CA) junction was cleaved faster than the CA/nucleocapsid (NC) junction, an altered order of processing when compared with authentic processing. When the in vitro processed VLP were analyzed on density gradients, most of the MA, CA-p15 intermediate, and NC were detected as a highly multimeric form, equivalent to the unprocessed VLP. In contrast, CA was found as a monomer dissociated from the multimeric CA-p15 following cleavage of the CA/NC junction. Electron microscopy revealed that the in vitro processing was accompanied by conversion of the doughnut-like particles to particles containing condensed cores and spherical outer shells. The cores, however, lacked core shells, which are normally observed for authentic HIV, suggesting that the in vitro processing of immature VLP failed to produce core shells. 相似文献
4.
A method is described to visualise directly human immunodeficiency virus type-1 (HIV-1) particles. HIV-1 containing samples were adsorbed onto a plastic surface and doubly labeled with antibodies specific for viral proteins and sensitive nucleic acids dyes. Laser scanning confocal microscopy detected co-localization of viral proteins and nucleic acids, thus allowing specific identification of HIV. Using this technique, we have quantified eight different HIV-1 sub-types and three HIV-1 groups in tissue culture supernatants from infected peripheral blood mononuclear cells (PBMCs). Confocal counts correlated well with electron microscopy (EM) counts and HIV-1 RNA loads as determined by quantitative PCR. Confocal microscopy may prove to be a simple alternative to electron microscopy for virus identification and quantitation. 相似文献
5.
Expression of human immunodeficiency virus Gag protein and the N-terminal matrix (MA) domain in Escherichia coli yielded spherical structures in the cytoplasm. When human N-myristoyltransferase was coexpressed, both Gag and MA were fully myristoylated and spherical structures were relocated in close proximity to the cytoplasmic membrane. However, neither myristoylated Gag nor MA exhibited tight binding to E. coli membrane, suggesting that myristoylation in E. coli did not confer membrane affinity on Gag despite the relocation. Our data also suggest that the morphogenetic pathway of Gag particles in prokaryotic cells differs from that in eukaryotic cells despite biochemical similarities of in the form of Gag expressed. 相似文献
6.
Infection of cells with human immunodeficiency virus type-1 (HIV-1) results in the production of both infectious and non-infectious virions. At present, several assays are available for the quantitation of virus particles based on the presence of either viral capsid protein or nucleic acid. However, the ability to detect the total number of virus particles, both infectious and non-infectious, has been an elusive goal that would advance the study of virus assembly and egress. A rapid optical detection scheme for real-time label-free quantitation of HIV-1 virus particles was developed. Virions produced in cell cultures transfected transiently were evaluated with a nanospectroscopic assay. Quantitation with the optical detection scheme correlated with routine conventional assays. Nanospectroscopy can be used for the detection of both infectious and non-infectious, wild type and mutant strains of HIV-1 in solution at concentrations as low as 7×10(10)particles/ml, requiring volumes as small as 2 μl per assay, and in significantly less time than standard techniques. This assay provides a rapid, reliable system for quantifying virus particles in solution and could be applied to the study of viral particle production in cell culture. 相似文献
7.
Expression of the murine leukaemia virus (MLV) major Gag antigen p65(Gag) using the baculovirus expression system leads to efficient assembly and release of virus-like particles (VLP) representative of immature MLV. Expression of p180(Gag-Pol), facilitated normally in mammalian cells by readthrough of the p65(Gag) termination codon, also occurs efficiently in insect cells to provide a source of the MLV protease and a pattern of p65(Gag) processing similar to that observed in mammalian cells. VLP release from p180(Gag-Pol)-expressing cells however remains essentially immature with disproportionate levels of the uncleaved p65(Gag) precursor when compared to the intracellular Gag profile. Changing the p65(Gag) termination codon altered the level of p65(Gag) and p180(Gag-Pol) within expressing cells but did not alter the pattern of released VLP, which remained immature. Coexpression of p65(Gag) with a fixed readthrough p180(Gag-Pol) also led to only immature VLP release despite high intracellular protease levels. Our data suggest a mechanism that preferentially selects uncleaved p65(Gag) for the assembly of MLV in this heterologous expression system and implies that, in addition to their relative levels, active sorting of the correct p65(Gag) and p180(Gag-Pol) ratios may occur in producer cells. 相似文献
8.
Group C rotavirus (GpC RV) is a causative agent of acute gastroenteritis in children and adults. We expressed the three major capsid proteins VP2, VP6 and VP7 of human GpC RV in baculovirus and demonstrated the self-assembly of VP2/6/7 or VP6/7 virus-like particles (VLPs) in insect cells. We examined a number of parameters, including the kinetics of protein synthesis in different cell lines and media, to optimize the most favorable conditions for the synthesis of recombinant viral proteins and the production of VLPs in Sf9 cells. Hyperimmune serum to VP2/6/7 and VP6/7 VLPs recognized individual recombinant proteins of human GpC RV by Western blot analysis. This serum also showed specific reactivities with the corresponding GpC VLPs but not GpA RV by using immune electron microscopy (IEM) and enzyme immunoassay (EIA). The ability to produce an unlimited amount of GpC RV antigen and the availability of high quality antibody will allow us to develop sensitive and specific diagnostic assays to better determine the epidemiology and disease burden of GpC RV in humans. 相似文献
9.
Background HIV protease (PR) is a virus-encoded aspartic protease that is essential for viral replication and infectivity. The fully
active and mature dimeric protease is released from the Gag-Pol polyprotein as a result of precursor autoprocessing. 相似文献
10.
Human immunodeficiency virus type-1 (HIV-1) affects mostly men and women in their reproductive years. For those who have access to highly active antiretroviral therapy (HAART), the course of HIV-1 infection has shifted from a lethal to a chronic disease. As a result of this, many patients with HIV-1 consider having offspring, as do other patients of reproductive age with chronic illnesses. This article summarizes the current knowledge on the presence of HIV in the male and female genital tract, the effects of HIV-1 infection and HAART on male and female fertility and the results of various assisted reproduction techniques (ART) in HIV-1-infected men and women who wish to have offspring. 相似文献
11.
Current therapy for human immunodeficiency virus (HIV-1) infection relies primarily on the administration of anti-retroviral
nucleoside analogues, either alone or in combination with HIV-protease inhibitors. Although these drugs have a clinical benefit,
continuous therapy with the drugs leads to drug-resistant strains of the virus. Recently, significant progress has been made
towards the development of natural and synthetic agents that can directly inhibit HIV-1 replication or its essential enzymes.
We previously reported on the pharmacological cyclin-dependent kinase inhibitor (PCI) r-roscovitine as a potential inhibitor of HIV-1 replication. PCIs are among the most promising novel antiviral agents to emerge
over the past few years. Potent activity on viral replication combined with proliferation inhibition without the emergence
of resistant viruses, which are normally observed in HAART patients; make PCIs ideal candidates for HIV-1 inhibition. To this
end we evaluated twenty four cdk inhibitors for their effect on HIV-1 replication in vitro. Screening of these compounds identified alsterpaullone as the most potent inhibitor of HIV-1 with activity at 150 nM. We
found that alsterpaullone effectively inhibits cdk2 activity in HIV-1 infected cells with a low IC 50 compared to control uninfected cells. The effects of alsterpaullone were associated with suppression of cdk2 and cyclin expression.
Combining both alsterpaullone and r-roscovitine (cyc202) in treatment exhibited even stronger inhibitory activities in HIV-1 infected PBMCs. 相似文献
12.
CD40 ligand is a costimulatory molecule which acts a potent immunomodulator. We found the mice inoculated with human CD40 ligand expression plasmid (pMEhCD40L) combined with human immunodeficiency virus type-1 (HIV-1) DNA vaccine exhibited both humoral and cellular antigen-specific immunological enhancement. The expression of hCD40L induced predominantly antigen-specific immunoglobulin G (IgG) antibody response while it failed to induce mucosal IgA response. Delayed-type hypersensitivity (DTH) and cytotoxic T lymphocyte (CTL) activity were induced in a dose-dependent manner. Examination of the relative levels of the two IgG subclasses showed that co-injection of pMEhCD40L enhanced IgG2a response without suppressing IgG1 response. Similarly, the expression of pMEhCD40L enhanced not only T helper 1 (Th1)- but also Th2-type cytokine production. In conclusion, co-inoculation of pMEhCD40L with DNA vaccine was shown to be a useful way to enhance CTL responses without suppressing the humoral immune response in acquired immune deficiency syndrome (AIDS) patients. 相似文献
14.
Protease inhibitors are widely used in the treatment of human immunodeficiency virus type 1 (HIV-1)-infected individuals and show a drastic effect on the reduction of virus load. We previously reported that doughnut-shaped, protease-defective gp120-containing HIV-1 particles from an L-2 cell clone, carrying a provirus with mutations at the pol (protease), env (gp41) and nef genes, rapidly and more effectively induces virus particle-mediated syncytia formation of uninfected T-cells, than a parental wild-type laboratory strain of HIV-1 (LAI). In this study, we examined the possibility of whether enhanced syncytia formation is mediated by morphologically similar doughnut-shaped particles obtained after treatment of LAI-infected cells with the protease inhibitors L-689, 502, DMP-323, RO-31-8959, and KNI-272. Utilizing such protease inhibitor-induced particles and a clone of MOLT-4 cells, we could not detect any enhancement of syncytia formation, over that seen with wild-type LAI particles. This result should alleviate concerns of patients on highly active antiretroviral therapy (HAART), that protease inhibitors might accelerate progression of the disease through enhanced production of defective, 'immature'-appearing particles. 相似文献
15.
Regions of the gag-pol gene of human immunodeficiency virus (HIV), the causative agent of AIDS, have been cloned into the polyhedrin gene of the baculovirus Autographa californica nuclear polyhedrosis virus. When these recombinant viruses were used to infect insect cells, the cells produced gag-related proteins which could be immunoprecipitated with serum from AIDS patients. The major proteins produced by Acgag1, which contained the entire gag gene and a small portion of the pol gene, had molecular weights of 55,000 and 40,000 Da. Acgag2, which contained a larger portion of the pol gene in addition to the gag coding sequences, produced a major protein of 24,000 Da and only minor amounts of the 55,000- and 40,000-Da proteins. The implications of these results with respect to proteolytic processing of HIV gag proteins as well as the potential diagnostic use of this system are discussed. 相似文献
16.
Recent findings have brought optimism that development of a successful human immunodeficiency virus type-1 (HIV-1) vaccine lies within reach. Studies of early events in HIV-1 infection have revealed when and where HIV-1 is potentially vulnerable to vaccine-targeted immune responses. With technical advances in human antibody production, clues about how antibodies recognize HIV-1 envelope proteins have uncovered new targets for immunogen design. A recent vaccine regimen has shown modest efficacy against HIV-1 acquisition. However, inducing long-term T and B cell memory and coping with HIV-1 diversity remain high priorities. Mediators of innate immunity may play pivotal roles in blocking infection and shaping immunity; vaccine strategies to capture these activities are under investigation. Challenges remain in integrating basic, preclinical and clinical research to improve predictions of types of immunity associated with vaccine efficacy, to apply these insights to immunogen design, and to accelerate evaluation of vaccine efficacy in persons at-risk for infection. 相似文献
17.
Studies of HIV-1 capsid formation in a cell-free system revealed that capsid assembly occurs via an ordered series of assembly intermediates and requires host machinery. Here we use this system to examine 12 mutations in HIV-1 Gag that others studied previously in intact cells. With respect to capsid formation, these mutations generally produced the same phenotype in the cell-free system as in cells, indicating the cell-free system's high degree of fidelity. Analysis of assembly intermediates reveals that a mutation in the distal region of CA (322 LDeltaS) and truncations proximal to the second cys-his box in NC block multimerization of Gag at early stages in the cell-free capsid assembly pathway. In contrast, mutations in the region of amino acids 56-68 (located in the proximal portion of MA) inhibit assembly at a later point in the pathway. Other mutations, including truncations distal to the first cys-his box in NC and mutations in the distal half of MA (88HDeltaG, 85YDeltaG, Delta104-115, and Delta115-129), do not affect formation of immature capsids in the cell-free system. These data provide new information on the role of different domains in Gag during the early events of capsid assembly. 相似文献
18.
The incorporation of human immunodeficiency virus type 1 (HIV-1) Gag-beta-galactosidase (Gag-beta-gal; GBG) fusion proteins into HIV virus-like particles in the presence of HIV Gag proteins was studied. HIV Gag-beta-gal fusion constructs were cotransfected individually into COS7 cells with or without an HIV Gag protein expression plasmid. Release of HIV GBG fusion proteins from the cells were measured by assay of the medium versus intracellular beta-gal activities. Analysis indicates that fusion proteins (constructs HIVGBG, GBG 1919 and 1877) retaining the C-terminal portion of the CA and the adjacent NC domains were efficiently assembled into virus-like particles. Fusion proteins with deleted sequences covering the N-terminal portions of the gag sequences (GBG 831, 1147, 1419, 1447, 1511, 1552, 1600, 1630, 1684, 1715, and 1752) were impaired in entry into virus-like particles. The presence of CA major homology region (MHR) in the fusion proteins had no significant effects on inducing fusion protein incorporation when the C-terminal CA sequences in the fusion proteins were truncated (GBG 1841 and 1801). Subcellular fractionation studies indicated that most fusion proteins including the nonmyristylated one were enriched in the crude membrane fraction. Exceptions to this rule were fusion proteins with intact MHR but truncated C-terminal CA sequences, which possessed low levels of membrane association. However, assembly of fusion proteins into HIV Gag particles did not correlate with their subcellular fractionation or immunofluorescence localization patterns. Overall, the studies suggest that the very C-terminal CA and adjacent NC sequences are the primary determinants for incorporation of HIV Gag-beta-gal fusion proteins into virus particles. 相似文献
19.
Recombinant baculoviruses (rBV) expressing Ebola virus VP40 (rBV-VP40) or GP (rBV-GP) proteins were generated. Infection of Sf9 insect cells by rBV-VP40 led to assembly and budding of filamentous particles from the cell surface as shown by electron microscopy. Ebola virus-like particles (VLPs) were produced by coinfection of Sf9 cells with rBV-VP40 and rBV-GP, and incorporation of Ebola GP into VLPs was demonstrated by SDS-PAGE and Western blot analysis. Recombinant baculovirus infection of insect cells yielded high levels of VLPs, which were shown to stimulate cytokine secretion from human dendritic cells similar to VLPs produced in mammalian cells. The immunogenicity of Ebola VLPs produced in insect cells was evaluated by immunization of mice. Analysis of antibody responses showed that most of the GP-specific antibodies were of the IgG2a subtype, while no significant level of IgG1 subtype antibodies specific for GP was induced, indicating the induction of a Th1-biased immune response. Furthermore, sera from Ebola VLP immunized mice were able to block infection by Ebola GP pseudotyped HIV virus in a single round infection assay, indicating that a neutralizing antibody against the Ebola GP protein was induced. These results show that production of Ebola VLPs in insect cells using recombinant baculoviruses represents a promising approach for vaccine development against Ebola virus infection. 相似文献
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