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1.
The human immunodeficiency virus type 1 (HIV-1) protease (PR) has recently been shown to be inhibited by its propeptide p6* in vitro. As p6* itself is a PR substrate, the primary goal of this study was to determine the importance of p6* cleavage for HIV-1 maturation and infectivity. For that purpose, short peptide variants mimicking proposed cleavage sites within and flanking p6* were designed and analyzed for qualitative and quantitative hydrolysis in vitro. Proviral clones comprising the selected cleavage site mutations were established and analyzed for Gag and Pol processing, virus maturation, and infectivity in cultured cells. Amino-terminal cleavage site mutation caused aberrant processing of nucleocapsid proteins and delayed replication kinetics. Blocking the internal cleavage site resulted in the utilization of a flanking site at a significantly decreased hydrolysis rate in vitro, which however did not affect Gag-Pol processing and viral replication. Although mutations blocking cleavage at the p6* carboxyl terminus yielded noninfectious virions exhibiting severe Gag processing defects, mutations retarding hydrolysis of this cleavage site neither seemed to impact viral infectivity and propagation in cultured cells nor seemed to interfere with overall maturation of released viruses. Interestingly, these mutants were shown to be clearly disadvantaged when challenged with wild-type virus in a dual competition assay. In sum, we conclude that p6* cleavage is absolutely essential to allow complete activation of the PR and subsequent processing of the viral precursors. 相似文献
2.
The aqueous and methanol extracts of thirty-one herbs traditionally used as anti-fever remedies in China were screened for their in vitro inhibition on human immunodeficiency virus type-1 protease (HIV-1 PR). The activity of recombinant HIV-1 protease was determined by sequence-specific cleavage at the Tyr-Pro bond of the fluorogenic substrate (Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Lys(DABCYL)- Arg) or by HPLC anaylsis of the cleavage products after incubation of the enzyme with a synthetic peptide substrate (Acetyl-Ser-Gln-Asn-Tyr-Pro-Val-Val-amide). Among the herbal extracts examined, the aqueous extracts of Prunella vulgaris and Scutellaria baicalensis and the methanol extracts of Woodwardia unigemmata, Paeonica suffruticosa and Spatholobus suberectus elicited significant inhibition (>90%) at a concentration of 200 microg/ml. 相似文献
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Incorporation of functional human immunodeficiency virus type 1 integrase into virions independent of the Gag-Pol precursor protein. 总被引:2,自引:2,他引:0
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Retroviral integrase (IN) is expressed and incorporated into virions as part of the Gag-Pol polyprotein precursor. IN catalyzes integration of the proviral DNA into host cell chromosomes during the early stages of the virus life cycle, and as a component of Gag-Pol, it is involved in virion morphogenesis during late stages. It is unknown whether the scheme, conserved among retroviruses, for expressing and incorporating IN as a component of the Gag-Pol precursor protein is necessary for its function in the infected cell after viral entry. We have developed human immunodeficiency virus (HIV) virion-associated accessory proteins (Vpr and Vpx) as vehicles to deliver both foreign and viral proteins into the virus particle by their expression in trans as heterologous fusion proteins (X. Wu, et al., J. Virol. 69:3389-3398, 1995; X. Wu, et al., J. Virol. 70:3378-3384, 1996; X. Wu, et al., EMBO J. 16:5113-5122, 1977). To analyze IN function independent of its expression as a part of Gag-Pol, we expressed and incorporated IN into HIV type 1 (HIV-1) virions in trans as a fusion partner of Vpr (Vpr-IN). Our results demonstrate that the Vpr-IN fusion protein is efficiently incorporated into virions and then processed by the viral protease to liberate the IN protein. Virus derived from IN-minus provirus is noninfectious. However, this defect is overcome by trans complementation with the Vpr-IN fusion protein. Moreover, complemented virions are able to replicate through a complete cycle of infection, including formation of the provirus (integration). These results show, for the first time, that full IN function can be provided in trans, independent of its expression and incorporation into virions as a component of Gag-Pol. This finding also indicates that the IN domain of Gag-Pol is not required for the formation of infectious virions when IN is provided in trans. The ability to incorporate functional IN into retroviral particles in trans will provide unique opportunities to explore the function of this critical enzyme in a biologically relevant context, i.e., in infected cells as part of the nucleoprotein/preintegration complex. 相似文献
5.
Z Y Zhang I M Reardon J O Hui K L O'Connell R A Poorman A G Tomasselli R L Heinrikson 《Biochemistry》1991,30(36):8717-8721
We report here for the first time that Zn2+ is an effective inhibitor of renin and the protease from HIV-1, two aspartyl proteinases of considerable physiological importance. Inhibition of renin is noncompetitive and is accompanied by binding of 1 mol of Zn2+/mol of enzyme. Depending on the substrate, inhibition of the HIV protease by Zn2+ can be either competitive or noncompetitive, but in neither case is loss of activity due to disruption of the protease dimer. Inhibition of both enzymes is first order with respect to Zn2+ and is rapidly reversed by addition of EDTA. Ki values are strongly pH dependent and optimal in the range of 20 microM at or above pH 7. All of the data in hand suggest that the inhibitory effect of Zn2+ is a consequence of its binding at, or near, the active-site carboxyl groups of these aspartyl proteinases. This inhibition of the viral enzyme may help to explain some of the beneficial effects seen in AIDS patients who have received Zn2+ therapy. 相似文献
6.
Human immunodeficiency virus (HIV) type 1 transframe protein can restore activity to a dimerization-deficient HIV protease variant 总被引:1,自引:0,他引:1
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The protease (PR) from human immunodeficiency virus (HIV) is essential for viral replication: this aspartyl protease, active only as a dimer, is responsible for cleavage of the viral polyprotein precursors (Gag and Gag-Pol), to release the functional mature proteins. In this work, we have studied the structure-function relationships of the HIV PR by combining a genetic test to detect proteolytic activity in Escherichia coli and a bacterial two-hybrid assay to analyze PR dimerization. We showed that a drug-resistant PR variant isolated from a patient receiving highly active antiretroviral therapy is impaired in its dimerization capability and, as a consequence, is proteolytically inactive. We further showed that the polypeptide regions adjacent to the PR coding sequence in the Gag-Pol polyprotein precursor, and in particular, the transframe polypeptide (TF), located at the N terminus of PR, can facilitate the dimerization of this variant PR and restore its enzymatic activity. We propose that the TF protein could help to compensate for folding and/or dimerization defects in PR arising from certain mutations within the PR coding sequence and might therefore function to buffer genetic variations in PR. 相似文献
7.
Triton X-100-extracted human skin fibroblasts were exposed to human immunodeficiency virus type 1 protease and analysed by 2D-gel electrophoresis and immunofluorescence microscopy. Vimentin, two of the tropomyosin isoforms, a protein with Mr ∼ 90,000 and a protein with Mr ∼ 200,000 were found to be degraded. Structurally, this was accompanied by the disintegration of the vimentin filament network and the disappearance of the microfilament network. In contrast to our in vivo observations (Höner et al., 1991), prominent stress fibers and chromatin structure seemed to be rather resistant to the action of this protease. 相似文献
8.
José L. Nieva Félix M. Goñi Arturo Muga Shlomo Nir Francisca Pereira 《International journal of peptide research and therapeutics》1997,4(4-6):365-369
Summary The human immunodeficiency virus type-1 (HIV-1) fusion peptide, corresponding to a sequence of 23 amino acid residues at the
N-terminus of the spike transmembrane subunit gp41, has the capacity to destabilize negatively charged and neutral large unilamellar
vesicles, representing, respectively, the acidic and the neutral fraction of the plasma membrane lipids of viral target cells.
As revealed by infrared spectroscopy, the peptide associated with the vesicles may exist in different conformations. In negatively
charged membranes the structure is mainly an α-helix, while in Ca2+-neutralized negatively charged membranes the conformation switches to a predominantly extended conformation. In membranes
composed of zwitterionic phospholipids and cholesterol, the peptide also adopts a predominant extended structure. The α-helical
structure permeabilizes negatively charged vesicles but does not induce membrane fusion. The peptide in β-type conformation,
on the other hand, permeabilizes neutral membranes and triggers fusion. As seen by31P NMR, the latter structure also exhibits the capacity to alter the lamellar organization of the membrane. 相似文献
9.
José L. Nieva Félix M. Goñi Arturo Muga Shlomo Nir Francisca Pereira 《Letters in Peptide Science》1997,4(4-6):365-369
The human immunodeficiency virus type-1 (HIV-1) fusionpeptide, corresponding to a sequence of 23 amino acidresidues at the N-terminus of the spike transmembranesubunit gp41, has the capacity to destabilizenegatively charged and neutral large unilamellarvesicles, representing, respectively, the acidic andthe neutral fraction of the plasma membrane lipids ofviral target cells. As revealed by infraredspectroscopy, the peptide associated with the vesiclesmay exist in different conformations. In negativelycharged membranes the structure is mainly an-helix, while in Ca2+-neutralizednegatively charged membranes the conformation switchesto a predominantly extended conformation. In membranescomposed of zwitterionic phospholipids andcholesterol, the peptide also adopts a predominantextended structure. The -helical structurepermeabilizes negatively charged vesicles but does notinduce membrane fusion. The peptide in -typeconformation, on the other hand, permeabilizes neutralmembranes and triggers fusion. As seen by31P NMR, the latter structure also exhibits thecapacity to alter the lamellar organization of the membrane. 相似文献
10.
D-penicillamine inhibits transactivation of human immunodeficiency virus type-1 (HIV-1) LTR by transactivator protein 总被引:4,自引:0,他引:4
D-Penicillamine, an amino acid analogue of cysteine, has been shown to inhibit the transactivation of HIV-1 LTR by the transactivator protein, tat protein. The transactivation was studied in Jurkat cells co-transfected with plasmids containing HIV-LTR sequences fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and HIV tat gene. The expression of CAT activity was a measure of transactivation of LTR by the tat protein. Incubation of transfected Jurkat cells with D-penicillamine led to inhibition of CAT activity. This inhibition was found to be concentration-dependent; more than 90% inhibition of chloramphenicol acetylation was seen in extracts prepared from cultures incubated with 40 micrograms/ml of D-penicillamine. Earlier experiments have shown that D-penicillamine at 40 micrograms/ml can completely inhibit HIV-1 (HTLV-III B) replication in H9 cells [(1986) Drug Res. 36, 184-186]. These results suggest that inhibition of transactivation may be the molecular mechanism involved in the inhibition of HIV-1 replication by D-penicillamine. 相似文献
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The integration activity of human immunodeficiency virus type-1 (HIV-1) integrase was characterized in vitro by using pre-processed oligonucleotide substrates. The highest level of integration activity was found at pH 6.5 to 7.0, while the endonucleolytic activity was highest at pH 7.4 to 8.0. Although the endonucleolytic and integration reactions are consecutive in retroviral integration, our result indicates that the optimal conditions of the two reactions are quite different. In addition, it is suggested that the endonucleolytic and integration steps can be separated by control of the cellular physiological state in retroviral therapy. Strong integration was detected in the presence of 0.5-10 mM Mn2+ ion, but weak integration at around 10 mM Mg2+ ion. This observation explains that the Mn2+ ion is preferred to the Mg2+ ion as a cofactor in the integration reaction. Although there was no sequence-specificity in the integration site of the target DNA, integration was found to frequently occur at particular regions of the target DNA. Furthermore, the mutant integrases such as Asp116, Ser147, and Glu152, which had been reported previously, were shown to lose integration activity completely, indicating that these residues are critically involved in catalytic action. 相似文献
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15.
Recognition of the high affinity binding site in rev-response element RNA by the human immunodeficiency virus type-1 rev protein.
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The Human Immunodeficiency Virus type-1 rev protein binds with high affinity to a bubble structure located within the rev-response element (RRE) RNA in stemloop II. After this initial interaction, additional rev molecules bind to the RRE RNA in an ordered assembly process which requires a functional bubble structure, since mutations in the bubble sequence that reduce rev affinity block multiple complex formation. We have used synthetic chemistry to characterize the interaction between rev protein and its high affinity binding site. A minimal synthetic duplex RNA (RBC6) carrying the bubble and 12 flanking base pairs is able to bind rev with 1 to 1 stoichiometry and with high affinity. When the bubble structure is inserted into synthetic RNA molecules carrying longer stretches of flanking double-stranded RNA, rev forms additional complexes resembling the multimers observed with the RRE RNA. The ability of rev to bind to RBC6 analogues containing functional group modifications on base and sugar moieties of nucleoside residues was also examined. The results provide strong evidence that the bubble structure contains specific configurations of non-Watson--Crick G:G and G:A base pairs and suggest that high affinity recognition of RRE RNA by rev requires hydrogen bonding to functional groups in the major groove of a distorted RNA structure. 相似文献
16.
Selection and stabilization of the RNA aptamers against the human immunodeficiency virus type-1 nucleocapsid protein 总被引:5,自引:0,他引:5
Kim SJ Kim MY Lee JH You JC Jeong S 《Biochemical and biophysical research communications》2002,291(4):925-931
The nucleocapsid (NC) protein of the human immunodeficiency virus-1 (HIV-1) plays an important role in the encapsidation of viral RNA and assembly of viral particle. Since the NC protein is resistant for mutation, it might be an excellent target for the anti-viral therapy. RNA aptamers that bind to the mature form of the NC protein were isolated from a RNA library. Surface plasmon resonance measurement and gel shift assay showed that the RNA aptamers specifically bind to the NC protein with high affinity and compete for the psi RNA binding to the NC protein. Mapping of the RNA aptamer showed at least two sites for the protein binding, suggesting a multiple and cooperative binding by the NC to RNA. In addition, the circular form of RNA avidly binds to the NC protein as a linear counter does. Stabilized RNA aptamer is expected to act as an inhibitor for the viral packaging. 相似文献
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Virus life cycles depend on cellular factors. Therefore, targeting cellular in combination with viral enzymes could be an effective control in virus replication. In contrast to viral proteins, cellular proteins are not prone to mutations; therefore, viral escape is not expected from drugs inhibiting cellular factors. Hydroxyurea inhibits the cellular enzyme ribonucleotide reductase, thus reducing DNA synthesis. Furthermore, this drug potentiates the activity of nucleoside analogues, inhibits the escape of A-analogue resistant mutants, and increases the phosphorylation of T-analogues. Besides its antiviral activity, hydroxyurea effects the immune system by decreasing immune activation, inhibiting the expansion of CD8 cells and the depletion of CD4 cells. Hydroxyurea has been used in medicine for 40 years, is well tolerated, and it is the least expensive available anti-HIV-1 drug. These characteristics make hydroxyurea a primary candidate for use in combination therapies for the treatment of HIV-1 infection. 相似文献
19.
Cleavage of p15 protein in vitro by human immunodeficiency virus type 1 protease is RNA dependent. 总被引:7,自引:5,他引:2
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The human immunodeficiency virus (HIV) gag polyprotein is processed by the viral protease to yield the structural proteins of the virus. One of these structural proteins, p15, and its protease cleavage products, p7 and p6, are believed to be responsible for the viral RNA binding which is prerequisite for assembly of infectious virions. To better understand potential interactions between viral RNA, p15, and the HIV protease, we have synthesized p15 in an in vitro system and studied its processing by the viral protease. Using this system, we demonstrate that p15 synthesized in vitro is properly cleaved by the HIV protease in an RNA-dependent reaction. Mutation of cysteine residues in either zinc-binding domain of the p7 portion of p15 does not alter the RNA-dependent cleavage, but mutation of three basic residues located between the zinc-binding domains blocks HIV protease susceptibility. The results support a previously unrecognized role for the interaction of RNA and nucleocapsid-containing gag precursors that may have important consequences for virus assembly. 相似文献
20.
Processing of deoxyuridine mismatches and abasic sites by human immunodeficiency virus type-1 integrase. 总被引:2,自引:0,他引:2
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We have examined the activities of HIV-1 integrase on substrates containing mismatches, composed of deoxyuridine at different positions in either the processed or nonprocessed strand of viral DNA, within and near the conserved CA dinucleotide of the U5 end of the HIV-1 LTR. Substitution in the processed strand of either the C or A of the CA dinucleotide or of the G 5' to the CA reduced strand transfer six-, three- and seven-fold respectively. 3'-processing was also reduced by substitution at the GC but not at the A. Substitution in the nonprocessed strand of the G nucleotide at the processing site abolished strand transfer while substitution of the T had no effect. DNA binding of HIV-1 integrase was not affected by deoxyuridine substitutions. Deoxyuridine substitution outside the trinucleotide remained compatible with enzyme activity. Enzymatically generated abasic sites were created at each mismatch to determine the effect of a missing base on integrase activity. Consistent with the deoxyuridine mismatch observations, 3'-processing and strand transfer were abolished when the abasic site was substituted for either of the nucleotides of the GCA trinucleotide. Integrase was, however, able to tolerate mismatches within this trinucleotide during the disintegration reaction. Taken together, these results suggest that base-mismatched or base-deleted substrates, which can be created by the proofreading-deficient HIV-1 RT, can be tolerated by HIV-1 integrase when located outside of the GCA trinucleotide at the U5 end of the LTR. 相似文献