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1.
目的 在原核表达系统中表达HPV16 L1蛋白,纯化后在体外自组装成VLPs并鉴定。方法 优化GenBank中HPV16 L1基因序列并截短C末端25个氨基酸,构建至原核表达载体pET-28a上,获得重组表达载体pET28a-16L1△C25。采用镍亲和层析法纯化超声上清,于体外解组装-重组装HPV16 VLPs,采用动态光散射和透射电镜进行形貌分析,纯化后于第0、2和4周免疫小鼠,假病毒中和试验检测HPV16 VLPs免疫后血清中和抗体。结果 双酶切和测序结果表明成功构建pET28a-16L1△C25重组质粒,诱导表达后,经SDS-PAGE和Western blotting分析显示表达的L1蛋白大部分以可溶性形式存在,纯化后的蛋白样品于体外重新组装,动态光散射和透射电镜能够观察到形态与天然病毒颗粒相似的VLPs,第6周小鼠血清中和抗体滴度Log10平均值达到4.43。结论 利用原核表达系统成功表达了截短型HPV16 L1蛋白,并于体外组装成结构完整的VLPs,且具有较好的免疫原性,为低成本HPV预防性疫苗的研发奠定基础。 相似文献
2.
E1 protein of human papillomavirus type 1a is sufficient for initiation of viral DNA replication. 总被引:6,自引:0,他引:6
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V Gopalakrishnan S A Khan 《Proceedings of the National Academy of Sciences of the United States of America》1994,91(20):9597-9601
Previous studies on transient replication of papillomaviruses have shown an absolute requirement for the viral E1 and E2 proteins in DNA replication. Here we demonstrate that for human papillomavirus type 1a (HPV-1a) DNA, the E1 protein alone is sufficient for in vivo replication of plasmids containing the viral origin of replication. Replication was origin-specific and required the presence of a DNA sequence containing a putative E1 binding site, but the E2 binding sites were dispensable. In the presence of the E1 protein, E2 stimulated replication of plasmids containing the E1 and E2 binding sites, but no stimulation was observed when the origin plasmids lacked E2 binding sites. Conversely, in the presence of E1 alone, the E2 binding sites did not affect replication. Plasmids containing the replication origins of HPV-6b, HPV-18, and bovine papillomavirus type 1 (BPV-1) also replicated efficiently in the presence of the HPV-1a E1 and E2 proteins. However, plasmids containing the origins of HPV-6b and HPV-18 failed to replicate in the presence of HPV-1a E1 alone, whereas a plasmid containing the BPV-1 origin replicated to lower levels than the HPV-1a origin-containing plasmid. These results suggest that replication from papillomaviral origins in the presence of E1 alone is presumably dependent on the strength of E1-origin interactions. Additionally, E1-dependent replication is stimulated by the E2 protein in the presence of E2 binding sites. 相似文献
3.
4.
Ishimitsu T Honda T Ohno E Furukata S Sudo Y Nakano N Takahashi T Ono H Matsuoka H 《International heart journal》2010,51(5):359-364
Most previous studies have examined the effects of antihypertensive drugs in hypertensive animals for only a few months, and little information has been provided as to the protective effects of lifetime antihypertensive medication against cardiovascular organ injury. In this study, spontaneously hypertensive rats (SHR) were treated for 1 year with an angiotensin-II receptor antagonist (ARB) and the development of hypertensive organ injury was evaluated. Male 15-week-old SHR (n = 9) were given 25 mg/L candesartan (CS) in their drinking water for 1 year. Twelve SHR and 9 normotensive Wistar-Kyoto rats (WKY) were given normal tap water. Tail-cuff blood pressure was almost normalized by CS throughout 1 year (at 12-months: WKY 132 ± 3, SHR 229 ± 3, CS 137 ± 4 mmHg). After 1 year, cardiac ventricular weight (SHR +33%, CS -2% versus WKY) and aortic thickness (SHR +34%, CS +4% versus WKY) in the CS-treated SHR rats were not different than those of WKY. Echocardiographic midwall fractional shortening (SHR -18%, CS -1% versus WKY) and left ventricular hydroxyproline content (SHR +47%, CS +11% versus WKY) were also improved by CS to the WKY level. With respect to kidney function, GFR (SHR -24%, CS +9% versus WKY) was preserved, proteinuria (SHR +312%, CS +12% versus WKY) was reduced, and the histological glomerular injury rate (SHR +186%, CS +6% versus WKY) was reduced by CS. These results suggest that long-term antihypertensive therapy with CS can completely prevent hypertensive cardiovascular and renal injuries in SHR. 相似文献
5.
BackgroundFrench maritime pine bark extract (Pycnogenol) revealed diverse anti-inflammatory actions by an inhibition of NF-κB–dependent gene expression. The aim of this study was to determine whether Pycnogenol had a beneficial effect on viral myocarditis in mice.Methods and MaterialsFour-week-old inbred male DBA/2 mice were inoculated intraperitoneally with 10 plaque-forming units (pfu) of the encephalomyocarditis (EMC) virus. Pycnogenol was administered orally at a dose of 1 or 10 mg/kg per day for the histologic study, and 10 or 100 mg/kg for the gene expression study, beginning on the day of viral inoculation.ResultsThe area of myocardial infiltration and necrosis on day 7 was significantly smaller in the hearts of mice treated with Pycnogenol 10 mg/kg (16.2 ± 8.9% and 19.2 ± 9.7%, respectively, n = 10, mean ± SEM) compared with controls (27.6 ± 15.0% and 30.1 ± 15.7%, respectively, n = 10, P < .05). There was a nonsignificant trend for less myocardial infiltration in the Pycnogenol 1 mg/kg group. Myocardial virus concentration on day 7 was 8.4 ± 0.3 × 103 pfu/mg in mice treated with 1 mg/kg of Pycnogenol, and 2.7 ± 0.6 × 104 pfu/mg in control mice, and the difference was statistically significant (P < .05). Gene expressions of tumor necrosis factor, type-I procollagen, stem cell factor, and mast cell tryptase were significantly suppressed in the hearts of mice treated with Pycnogenol 100 mg/kg.ConclusionsThese results suggest that Pycnogenol exerts its beneficial effects on viral myocarditis by decreasing virus replication, and by suppressing expression of pro-inflammatory cytokines, genes related to cardiac remodeling, and mast cell–related genes in the hearts of mice. 相似文献
6.
Binding of the human papillomavirus E1 origin-recognition protein is regulated through complex formation with the E2 enhancer-binding protein. 总被引:9,自引:1,他引:8
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M G Frattini L A Laimins 《Proceedings of the National Academy of Sciences of the United States of America》1994,91(26):12398-12402
The papillomavirus E1 and E2 proteins form heteromeric complexes and individually bind specific sequences within the viral origin of replication. The mechanism by which these proteins are recruited to the origin and the role of the E1/E2 complex in replication remain undefined. To examine the interplay of these replication proteins, we have analyzed the binding of human papillomavirus (HPV) type 31b E1 and E2 proteins to the origin of replication. Binding of E1 to the origin was increased by E2 proteins and required the presence of E2 binding sites. This increase was due to the formation of E1/E2 complexes which preferentially bound E2-responsive sequences, and the magnitude was determined by the relative affinity of the E2 binding sites. While the E1 protein alone bound an A/T-rich sequence at the HPV-31b origin with low affinity, complexes of E1 and E2 bound instead to E2 binding sites with high affinity. The E1/E2 complex bound a similar sequence as E2 homodimers, but only E2 homodimer binding induced a significant increase in hypersensitivity as indicated by DNase I footprinting. In the presence of excess E1, E1 proteins bound both the A/T-rich sequence and E2 binding sites through complex formation with E2. In E2 excess, E1/E2 complexes preferentially formed, and binding was specific for E2 sites. Therefore, changes in the relative amounts of E1 and E2 proteins can dramatically alter the pattern of binding of viral replication factors to the origin. These observations suggest a model whereby modulation of the relative levels of E1 and E2 during the viral life cycle may alter the pattern of origin binding and possibly episomal copy number. 相似文献
7.
Interaction between cyclin-dependent kinases and human papillomavirus replication-initiation protein E1 is required for efficient viral replication 总被引:16,自引:0,他引:16
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Tianlin Ma Nianxiang Zou Biing Yuan Lin Louise T. Chow J. Wade Harper 《Proceedings of the National Academy of Sciences of the United States of America》1999,96(2):382-387
We have identified the human papillomavirus (HPV) DNA replication initiation protein E1 as a tight-binding substrate of cyclin E/cyclin-dependent kinase (Cdk) complexes by using expression cloning. E1, a DNA helicase, collaborates with the HPV E2 protein in ori-dependent replication. E1 formed complexes with cyclin E in insect and mammalian cells, independent of Cdks and E2. Additional cyclins, including A-, B-, and F-type (but not D-type), interacted with the E1/E2 complex, and A- and E-type cyclin kinases were capable of phosphorylating E1 and E2 in vitro. Association with cyclins and efficient phosphorylation of E1 required the presence of a cyclin interaction motif (the RXL motif). E1 lacking the RXL motif displayed defects in E2-dependent HPV ori replication in vivo. Consistent with a role for Cdk-mediated phosphorylation in E1 function, an E1 protein lacking all four candidate Cdk phosphorylation sites still associated with E2 and cyclin E but was impaired in HPV replication in vitro and in vivo. Our data reveal a link between cyclin/Cdk function and activation of HPV DNA replication through targeting of Cdk complexes to the E1 replication-initiation protein and suggest a functional role for E1 phosphorylation by Cdks. The use of cyclin-binding RXL motifs is now emerging as a major mechanism by which cyclins are targeted to key substrates. 相似文献
8.
Lee YJ Kim JH Sohn HJ Lee J Jung SY Chwae YJ Kim K Park S Shin HJ 《Parasite immunology》2011,33(7):382-389
Free-living Naegleria fowleri causes primary amoebic meningoencephalitis (PAM) in humans and animals. To examine the effect of immunization with Nfa1 protein on experimental murine PAM because of N. fowleri, BALB/c mice were intra-peritoneally or intra-nasally immunized with a recombinant Nfa1 protein. We analysed Nfa1-specific antibody and cytokine induction, and the mean survival time of infected mice. Mice immunized intra-peritoneally or intra-nasally with rNfa1 protein developed specific IgG, IgA and IgE antibodies; the IgG response was dominated by IgG1, followed by IgG2b, IgG2a and IgG3. High levels of the Th1 cytokine, IFN-γ, and the regulatory cytokine, IL-10, were also induced. The mean survival time of mice immunized intra-peritoneally with rNfa1 protein was prolonged compared with controls, (25.0 and 15.5 days, respectively). Similarly, the mean survival time of mice immunized intra-nasally with rNfa1 protein was 24.7 days, compared with 15.0 days for controls. 相似文献
9.
Lisziewicz J Trocio J Xu J Whitman L Ryder A Bakare N Lewis MG Wagner W Pistorio A Arya S Lori F 《AIDS (London, England)》2005,19(1):35-43
OBJECTIVE: To reconstitute immune responses capable of eliminating infected cells and suppressing viral load during chronic retroviral infection. DESIGN:: A topical, DNA-based therapeutic immunization (DermaVir) was designed to express most of the regulatory and structural viral genes in dendritic cells. METHODS: DermaVir alone and in combination with antiretroviral drugs was tested in chronically SIV-infected macaques. RESULTS: DermaVir provided virological, immunological and clinical benefit for SIV-infected macaques during chronic infection and AIDS. In combination with antiretroviral drugs, DermaVir augmented SIV-specific T-cell responses and enhanced control of viral load rebound during treatment interruptions. CONCLUSIONS: The results indicate the feasibility of therapeutic immunization even in immune compromised hosts, and suggest that DermaVir can complement antiretroviral drugs to sustain suppression of HIV-1 replication. 相似文献
10.
Donald L. Kaminski MD Yashwant G. Deshpande MS Al Li PhD Frances Dysart MD Mihir Nag PhD 《Digestive diseases and sciences》1995,40(5):1157-1164
The development of experimental cholecystitis produced by lysophosphatidylcholine is associated with reversal of the normal absorptive characteristics of gallbladder mucosa, resulting in the intraluminal accumulation of water, glycoprotein, and protein. The purpose of the present study was to attempt to ascertain if the protein leaks into the lumen because of the cytolytic properties of lysophosphatidylcholine or if it is due to an active secretory process and to characterize the protein produced. Experiments were performed on anesthetized cats undergoing gallbladder perfusion with and without lysophosphatidylcholine. The amount of protein in the perfusate was measured and albumin clearance from blood to gallbladder lumen was calculated with and without the administration of vesicular transport inhibitors. In separate experiments, control and lysophosphatidylcholine (LPC) produced gallbladder perfusates were collected and the protein subjected to SDS-PAGE to ascertain the nature of the protein secreted. Inhibitors of both microtubular and microfilament activity decreased the protein accumulation and clearance produced by lysophosphatidylcholine. Gallbladder white blood cell accumulation and inflammation as evaluated byβ-glucuronidase and prostaglandin E levels were not significantly altered by cytochalasin or colchicine administration. Lysophosphatidylcholine also produced significant increases in perfusate LDH levels. The protein produced was primarily a 66-kDa protein. Transfer of the protein to a nitrocellulose membrane and immunoblotting with anti-albumin antibody demonstrated that the protein was albumin. The results suggest that during the development of cholecystitis, lysophosphatidylcholine produces albumin accumulation in the gallbladder primarily by inducing an active secretory process resulting in gallbladder distension. 相似文献
11.
Induction of antiphospholipid antibodies by immunization with synthetic viral and bacterial peptides. 总被引:3,自引:0,他引:3
E E Gharavi H Chaimovich E Cucurull C M Celli H Tang W A Wilson A E Gharavi 《Lupus》1999,8(6):449-455
We previously induced pathogenic antibodies against anionic phospholipids (PL) in experimental animals by immunization with lipid-free purified human beta2glycoprotein I (beta2GPI). We hypothesized that antiphospholipid antibodies (aPL) are induced by in vivo binding of foreign beta2GPI to self-PL, thus forming an immunogenic complex against which aPL antibodies are produced. If this hypothesis is true, other PL-binding proteins that are products of ubiquitous viral/bacterial agents may also induce aPL. To test this hypothesis, groups of NIH/Swiss mice were immunized with synthetic peptides of viral and bacterial origin that share structural similarity with the putative PL-binding region of beta2GPI. Compared with the control groups, animals immunized with the peptides produced significantly higher levels of aPL and anti-beta2GPI antibodies. These findings demonstrate that some PL-binding viral and bacterial proteins function like beta2GPI in inducing aPL and anti-beta2GPI production, and are consistent with a role for such viral and bacterial proteins in inducing aPL antibody production in humans. 相似文献
12.
The cellular DNA polymerase alpha-primase is required for papillomavirus DNA replication and associates with the viral E1 helicase. 总被引:14,自引:1,他引:14
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P Park W Copeland L Yang T Wang M R Botchan I J Mohr 《Proceedings of the National Academy of Sciences of the United States of America》1994,91(18):8700-8704
13.
Human papillomavirus (HPV) origin-binding protein associates with mitotic spindles to enable viral DNA partitioning 总被引:9,自引:0,他引:9
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Van Tine BA Dao LD Wu SY Sonbuchner TM Lin BY Zou N Chiang CM Broker TR Chow LT 《Proceedings of the National Academy of Sciences of the United States of America》2004,101(12):4030-4035
Human papillomaviruses (HPVs) establish long-term infections in patients. The mechanism for extrachromosomal HPV DNA persistence in cycling cells is unknown. We show that HPV origin-containing plasmids partition as minichromosomes, attributable to an association of the viral origin recognition protein E2 with mitotic spindles. alpha-, beta-, and gamma-tubulins were pulled down with a tagged E2. The N-terminal transacting and C-terminal protein dimerization/DNA binding domains independently associated with the spindles. We suggest that this E2 property enables these viruses to establish persistence. Its implication for HPV oncogenesis is presented. 相似文献
14.
目的表达并纯化重组人乳头瘤病毒16型(HPV16)L1蛋白,建立分泌抗HPV16 L1单克隆抗体(mAb)的杂交瘤细胞株。方法以基因工程表达HPV16L1蛋白。用Ni—NTA技术进行纯化后免疫BALB/c小鼠,取脾细胞与SP2/0骨髓瘤细胞融合,经含次黄嘌呤、氨基蝶呤、胸腺嘧啶(HAT)的选择培养基及有限稀释法进行克隆化,用间接ELISA法筛选阳性杂交瘤细胞,对获得的杂交瘤细胞株染色体进行计数.同时测定细胞培养上清mAb的效价。结果筛选出2株可分泌抗HPV16 L1 mAb的杂交瘤细胞,命名为AE3和AG7,染色体数分别为130条和98条。2株杂交瘤细胞AE3和AG7培养上清的效价分别为1:5120和1:80。结论成功建立了2株可分泌抗HPV 16 L1 mAb的杂交瘤细胞,为进一步研制HPV感染检测试剂盒提供实验基础。 相似文献
15.
The E4 and L1 gene products of human papillomavirus (HPV) types 6 and 11 are detected in variable amounts in condylomata acuminata. To study factors associated with detection of these proteins, biopsy specimens containing HPV-6 or -11 were analyzed for E4 protein, L1 protein, and HPV copy number. Seventeen of 50 women biopsied were pregnant. Nine men were also biopsied. Both the E4 and L1 proteins were found more frequently in lesions from pregnant women than from nonpregnant women or men. Both proteins were more often detected in lesions from women than from men. E4 gene products were more often detected in lesions in which L1 protein was detected and a higher HPV copy number was present. Detection of E4 gene products correlates with the detection of L1 protein in condylomata acuminata caused by HPV-6 or -11. 相似文献
16.
The effect of chronic alcohol administration on blood pressure was investigated in 7-week-old Wistar rats. Tail-cuff blood pressure was significantly higher in rats who received 15% ethanol in drinking water than in control rats. Intracellular free calcium concentration of lymphocytes was increased, while magnesium concentration of erythrocyte, aorta, and skeletal muscle and erythrocyte ouabain-sensitive 22Na efflux rate constant (Kos) were decreased in alcohol-induced hypertensive rats but not in control rats. Extracellular fluid volume was also increased in alcohol-administered rats. Oral magnesium supplementation (1% MgO in rat chow) attenuated the development of alcohol-induced hypertension accompanied by increased magnesium concentration of erythrocyte, aorta, skeletal muscle, and Kos and decreased intraerythrocyte sodium concentration. Norepinephrine half-life time of the heart and spleen was also increased in magnesium-supplemented rats. Blood pressure significantly correlated positively with intracellular calcium concentration and extracellular fluid volume, negatively with magnesium concentration of erythrocyte, aorta, skeletal muscle, and Kos. These results suggest that increased intracellular calcium, which was partly due to magnesium depletion and suppressed sodium pump activity, and expanded body fluid volume had a possible role in the development of alcohol-induced hypertension. It is also suggested that oral magnesium supplementation had a hypotensive effect on alcohol-induced hypertension possibly through decreased intracellular sodium concentration caused by an activation of sodium pump and decreased sympathetic nervous activity. 相似文献
17.
Induction of structural changes in the bovine papillomavirus type 1 origin of replication by the viral E1 and E2 proteins. 总被引:15,自引:0,他引:15
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T G Gillette M Lusky J A Borowiec 《Proceedings of the National Academy of Sciences of the United States of America》1994,91(19):8846-8850
Chemical and enzymatic probing techniques were used to examine the interaction of the bovine papillomavirus type 1 E1 and E2 proteins with the viral origin of replication (ori). E1 was found to generate significant distortions to the structure of ori, as assayed by KMnO4 oxidation of DNA. The primary site of ori distortion was located within and adjacent to the AT-element of the core replicator sequence, although a number of minor structural transitions were also detected. The induction of these structural changes required ATP and appeared to require ATP hydrolysis. E2 was found to decrease the amount of E1 required for ori distortion but did not significantly alter the pattern of structural distortion. In contrast, the presence of E2 resulted in a biphasic mechanism for E1 binding to ori, as assayed by nuclease protection. Under these conditions, E1 bound preferentially to the dyad symmetry region containing the conserved Hpa I site. Higher levels of E1 were required for binding to the adjacent ori AT-rich region. Thus, these data suggest that E2 can order the stepwise binding of E1 to ori. 相似文献
18.
Phospholipase A2 inhibition prevents mucosal damage associated with small intestinal ischaemia in rats. 总被引:1,自引:0,他引:1
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The influence of various inflammatory inhibitors on the damaging effects of ischaemia in the small intestinal mucosa has been investigated. A rat experimental model was used, in which a ligated loop of the distal ileum was subjected to ischaemia and revascularisation and the ensuing mucosal damage assessed by lysosomal enzyme release and intestinal permeability measurements. The mucosal content of malondialdehyde - a lipid peroxidation product - and its activity of myeloperoxidase - a neutrophil granulocyte marker was also determined. In the absence of inhibitor, ischaemia and revascularisation caused increased mucosal permeability to sodium fluorescein, increased N-acetyl-beta-glucosaminidase release from the mucosa into the lumen, increased malondialdehyde content in the mucosa and increased myeloperoxidase activity in the mucosa. All these effects were inhibited by the phospholipase A2 inhibitors, quinacrine and nordihydroguaiaretic acid (NDGA), while the lipoxygenase inhibitor, BW755C, had no influence and the cyclooxygenase inhibitor, indomethacin, potentiated the increases in mucosal permeability and N-acetyl-glucosaminidase release. BN 52021, a specific platelet activating factor antagonist, did not influence the myeloperoxidase activity, but it decreased the formation of malondialdehyde and the increases in mucosal permeability and N-acetyl-beta-glucosaminidase release, although not to the same extent as quinacrine and NDGA. These findings indicate that phospholipase A2 inhibition prevents mucosal damage associated with small intestinal ischaemia and suggest that at least part of the ischaemic damage is mediated by products of phospholipase A2 activity that are not arachidonic acid metabolites. 相似文献
19.
GTPase-activating protein interactions with the viral and cellular Src kinases. 总被引:10,自引:3,他引:10
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B K Brott S Decker J Shafer J B Gibbs R Jove 《Proceedings of the National Academy of Sciences of the United States of America》1991,88(3):755-759
GTPase-activating protein (GAP), which regulates the activities of Ras proteins, is implicated in mitogenic signal transduction by growth-factor receptors and oncoproteins with tyrosine kinase activity. Oncogenic viral Src (p60v-src) encoded in Rous sarcoma virus possesses elevated tyrosine kinase activity compared with its nononcogenic normal homolog, cellular Src (p60c-src). To examine molecular interactions between GAP and the two Src kinases, immunoprecipitates of Src or GAP prepared from cell lystates were resolved by gel electrophoresis and analyzed by an immunoblot procedure with antibodies to GAP or Src used as probes. Results suggest that p60c-src is associated with a complex containing GAP in immunoprecipitates from lysates of normal rat and chicken cells. However, GAP is not phosphorylated in p60c-src immunoprecipitates subjected to in vitro kinase reactions. By contrast, GAP undergoes tyrosyl phosphorylation in vitro when immunoprecipitates of p60v-src prepared from transformed cell lysates are incubated with ATP. Our findings suggest that p60v-src and p60c-src associate with complexes containing GAP and provide a biochemical link between both kinases and GAP/Ras signal transduction pathways. These results are consistent with the hypothesis that GAP has a role in mediating normal functions of p60c-src as well as oncogenic activities of p60v-src. 相似文献
20.
Human immunodeficiency virus proteinase dimer as component of the viral polyprotein prevents particle assembly and viral infectivity. 总被引:20,自引:4,他引:20
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H G Kr?usslich 《Proceedings of the National Academy of Sciences of the United States of America》1991,88(8):3213-3217
Enzymatically active retroviral proteinases are dimers of identical polypeptide chains with a fold similar to that of other aspartic proteinases. Each polypeptide chain, encoded on one of the viral polyproteins, is less than half the size of cellular aspartic proteinases and contains only one of the two active-site aspartate residues. A plasmid was constructed to generate a genetically linked dimer of the proteinase (PR) of human immunodeficiency virus (HIV) type 1, composed of two copies of the PR sequence linked by a structurally flexible hinge region. The expression product was stable and active against HIV polyprotein substrates. Mutational analysis revealed that the linked dimer, and not multimers thereof, contained the proteolytic activity. Expression of the linked dimer as a component of a HIV polyprotein by in vitro translation gave rapid autocatalytic processing, whereas the wild-type polyprotein was stable on prolonged incubation. Transfection of HIV subviral or proviral constructs, containing the linked dimer of HIV PR, gave premature processing of the viral polyproteins, thus preventing particle formation and infectivity. Premature processing also led to increased cell toxicity. 相似文献