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1.
A sequence-specific ribozyme (M1GS RNA) derived from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the mRNA encoding human cytomegalovirus (HCMV) protease (PR), a viral protein that is responsible for the processing of the viral capsid assembly protein. We showed that the constructed ribozyme cleaved the PR mRNA sequence efficiently in vitro. Moreover, a reduction of about 80% in the expression level of the protease and a reduction of about 100-fold in HCMV growth were observed in cells that expressed the ribozyme stably. In contrast, a reduction of less than 10% in the expression of viral protease and viral growth was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. Further examination of the antiviral effects of the ribozyme-mediated cleavage of PR mRNA indicates that (1) the proteolytic cleavage of the capsid assembly protein is inhibited significantly, and (2) the packaging of the viral genomic DNA into the CMV capsids is blocked. These observations are consistent with the notion that the protease functions to process the capsid assembly protein and is essential for viral capsid assembly. Moreover, our results indicate that the RNase P ribozyme-mediated cleavage specifically reduces the expression of the protease, but not other viral genes examined. Thus, M1GS ribozyme is highly effective in inhibiting HCMV growth by targeting the PR mRNA and may represent a novel class of general gene-targeting agents for the studies and treatment of infections caused by human viruses, including HCMV.  相似文献   

2.
Kim K  Trang P  Umamoto S  Hai R  Liu F 《Nucleic acids research》2004,32(11):3427-3434
By linking a guide sequence to the catalytic RNA subunit of RNase P (M1 RNA), we constructed a functional ribozyme (M1GS RNA) that targets the overlapping mRNA region of two human cytomegalovirus (HCMV) capsid proteins, the capsid scaffolding protein (CSP) and assemblin, which are essential for viral capsid formation. The ribozyme efficiently cleaved the target mRNA sequence in vitro. Moreover, a reduction of >85% in the expression of CSP and assemblin and a reduction of 4000-fold in viral growth were observed in the HCMV-infected cells that expressed the functional ribozyme. In contrast, there was no significant reduction in viral gene expression and growth in virus-infected cells that either did not express the ribozyme or produced a ‘disabled’ ribozyme carrying mutations that abolished its catalytic activity. Characterization of the effects of the ribozyme on the HCMV lytic replication cycle further indicates that the expression of the functional ribozyme specifically inhibits the expression of CSP and assemblin, and consequently blocks viral capsid formation and growth. Our results provide the direct evidence that RNase P ribozymes can be used as an effective gene-targeting agent for antiviral applications, including abolishing HCMV growth by blocking the expression of the virus-encoded capsid proteins.  相似文献   

3.
A ribozyme (M1GS RNA) constructed from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the overlapping region of two human cytomegalovirus (HCMV) mRNAs, which encode for the viral essential protease (PR) and capsid assembly proteins (AP), respectively. The results show a reduction of >80% in the expression levels of PR and AP and an inhibition of approximately 2000-fold of viral growth in cells that stably expressed the ribozyme. In comparison, <10% reduction in the expression of the targets and viral growth was found in cells that either did not express the ribozyme or produced a "disabled" ribozyme carrying mutations that abolished its catalytic activity. Examination of replication of the virus in the ribozyme-expressing cells indicates that packaging of the viral genomic DNA into capsids is blocked, and suggests that the antiviral effects are because the ribozyme specifically inhibits the AP and PR expression and, consequently, abolishes viral capsid formation and growth. Our results show that RNase P ribozymes are highly effective in blocking HCMV growth by targeting the PR and AP mRNAs and demonstrate the feasibility to use these ribozymes in gene therapy for antiviral applications.  相似文献   

4.
丙型肝炎病毒 (Hepatitis C virus,HCV) 是引起慢性肝炎的重要病因之一,严重危害公众健康。目前临床HCV感染常采用干扰素联合病毒唑进行治疗,然而应答率不高且易反复。因此,探索新型抗HCV治疗策略及药物显得尤为迫切。针对HCV核心基因的序列,设计与之互补的引导序列 (Guide Sequence,GS),通过PCR的方法将其共价连接于大肠杆菌核糖核酸酶P(RNase P) 催化性亚基 (M1 RNA) 的3¢末端,成功构建了一种靶向性M1GS核酶 (M1GS-HCV/C141)。经体外切割试验、胞内反义效应及胞内毒性研究,结果表明:M1GS-HCV/C141核酶不仅能够在体外对靶RNA片段产生特异性切割,在HCV感染的Huh7.5.1细胞中,也能显著抑制病毒核心蛋白的表达,进而使HCV RNA的拷贝数减少约1 000倍。因此,文中构建的M1GS-HCV/C141核酶在体外具有显著的抗HCV活性,这为HCV治疗研究提供了一条新的潜在途径。  相似文献   

5.
Developing RNase P ribozymes for gene-targeting and antiviral therapy   总被引:5,自引:0,他引:5  
RNase P, a tRNA processing enzyme, contains both RNA and protein subunits. M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli, recognizes its target RNA substrate mainly on the basis of its structure and cleaves a double stranded RNA helix at the 5' end that resembles the acceptor stem and T-stem structure of its natural tRNA substrate. Accordingly, a guide sequence (GS) can be covalently attached to the M1 RNA to generate a sequence specific ribozyme, M1GS RNA. M1GS ribozyme can target any mRNA sequence of choice that is complementary to its guide sequence. Recent studies have shown that M1GS ribozymes efficiently cleave the mRNAs of herpes simplex virus 1 and human cytomegalovirus, and the BCR-ABL oncogenic mRNA in vitro and effectively reduce the expression of these mRNAs in cultured cells. Moreover, an in vitro selection scheme has been developed to select for M1 GS ribozyme variants with more efficient catalytic activity in cleaving mRNAs. When expressed in cultured cells, these selected ribozymes also show an enhance ability to inhibit viral gene expression and growth. These recent results demonstrate the feasibility of developing the M1GS ribozyme-based technology as a promising gene targeting approach for basic research and clinical therapeutic application.  相似文献   

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Using an in vitro selection procedure, we have previously isolated RNase P ribozyme variants that efficiently cleave an mRNA sequence in vitro. In this study, a variant was used to target the HIV RNA sequence in the tat region. The variant cleaved the tat RNA sequence in vitro about 20 times more efficiently than the wild type ribozyme. Our results provide the first direct evidence that combined mutations at nucleotide 83 and 340 of RNase P catalytic RNA from Escherichia coli (G83 -> U83 and G340 -> A340) increase the overall efficiency of the ribozyme in cleaving an HIV RNA sequence. Moreover, the variant is more effective in reducing HIV-1 p24 expression and intracellular viral RNA level in cells than the wild type ribozyme. A reduction of about 90% in viral RNA level and a reduction of 150 fold in viral growth were observed in cells that expressed the variant, while a reduction of less than 10% was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. Thus, engineered ribozyme variants are effective in inhibiting HIV infection. These results also demonstrate the potential of engineering RNase P ribozymes for anti-HIV application.  相似文献   

8.
The herpes simplex virus type 1 (HSV-1) protease and its substrate, ICP35, are involved in the assembly of viral capsids and required for efficient viral growth. The full-length protease (Pra) consists of 635 amino acid (aa) residues and is autoproteolytically processed at the release (R) site and the maturation (M) site, releasing the catalytic domain No (VP24), Nb (VP21), and a 25-aa peptide. To understand the biological importance of cleavage at these sites, we constructed several mutations in the cloned protease gene. Transfection assays were performed to determine the functional properties of these mutant proteins by their abilities to complement the growth of the protease deletion mutant m100. Our results indicate that (i) expression of full-length protease is not required for viral replication, since a 514-aa protease molecule lacking the M site could support viral growth; and that (ii) elimination of the R site by changing the residue Ala-247 to Ser abolished viral replication. To better understand the functions that are mediated by proteolytic processing at the R site of the protease, we engineered an HSV-1 recombinant virus containing a mutation at this site. Analysis of the mutant A247S virus demonstrated that (i) the mutant protease retained the ability to cleave at the M site and to trans process ICP35 but failed to support viral growth on Vero cells, demonstrating that release of the catalytic domain No from Pra is required for viral replication; and that (ii) only empty capsid structures were observed by electron microscopy in thin sections of A247S-infected Vero cells, indicating that viral DNA was not encapsidated. Our results demonstrate that processing of ICP35 is not sufficient to support viral replication and provide genetic evidence that the HSV-1 protease has nuclear functions other than enzymatic activity.  相似文献   

9.
Engineered RNase P ribozymes are promising gene-targeting agents that can be used in both basic research and clinical applications. We have previously selected ribozyme variants for their activity in cleaving an mRNA substrate from a pool of ribozymes containing randomized sequences. In this study, one of the variants was used to target the mRNA encoding thymidine kinase (TK) of herpes simplex virus 1 (HSV-1). The variant exhibited enhanced cleavage and substrate binding and was at least 30 times more efficient in cleaving TK mRNA in vitro than the ribozyme derived from the wild type sequence. Our results provide the first direct evidence to suggest that a point mutation at nucleotide 95 of RNase P catalytic RNA from Escherichia coli (G(95) --> U(95)) increases the rate of cleavage, whereas another mutation at nucleotide 200 (A(200) --> C(200)) enhances substrate binding of the ribozyme. A reduction of about 99% in TK expression was observed in cells expressing the variant, whereas a 70% reduction was found in cells expressing the ribozyme derived from the wild type sequence. Thus, the RNase P ribozyme variant is highly effective in inhibiting HSV-1 gene expression. Our study demonstrates that ribozyme variants increase their cleavage activity and efficacy in blocking gene expression in cells through enhanced substrate binding and rate of cleavage. These results also provide insights into the mechanism of how RNase P ribozymes efficiently cleave an mRNA substrate and, furthermore, facilitate the development of highly active RNase P ribozymes for gene-targeting applications.  相似文献   

10.
Moloney leukemia virus 10 protein (MOV10) is an interferon (IFN)-inducible RNA helicase implicated in antiviral activity against RNA viruses, yet its role in herpesvirus infection has not been investigated. After corneal inoculation of mice with herpes simplex virus 1 (HSV-1), we observed strong upregulation of both MOV10 mRNA and protein in acutely infected mouse trigeminal ganglia. MOV10 suppressed HSV-1 replication in both neuronal and non-neuronal cells, and this suppression required the N-terminus, but not C-terminal helicase domain of MOV10. MOV10 repressed expression of the viral gene ICP0 in transfected cells, but suppressed HSV-1 replication independently of ICP0. MOV10 increased expression of type I IFN in HSV-1 infected cells with little effect on IFN downstream signaling. Treating the cells with IFN-α or an inhibitor of the IFN receptor eliminated MOV10 suppression of HSV-1 replication. MOV10 enhanced IFN production stimulated by cytoplasmic RNA rather than DNA. IKKε co-immunoprecipitated with MOV10 and was required for MOV10 restriction of HSV-1 replication. Mass spectrometry identified ICP27 as a viral protein interacting with MOV10. Co-immunoprecipitation results suggested that this interaction depended on the RGG box of ICP27 and both termini of MOV10. Overexpressed ICP27, but not its RGG-Box deletion mutant, rendered MOV10 unable to regulate HSV-1 replication and type I IFN production. In summary, MOV10 is induced to restrict HSV-1 lytic infection by promoting the type I IFN response through an IKKε-mediated RNA sensing pathway, and its activity is potentially antagonized by ICP27 in an RGG box dependent manner.  相似文献   

11.
S A Rice  V Lam    D M Knipe 《Journal of virology》1993,67(4):1778-1787
The herpes simplex virus type 1 (HSV-1) alpha protein ICP27 regulates the transition between the delayed-early and late phases of the viral infection. Previous genetic analyses have suggested that the important functional domains of ICP27 map to its carboxyl-terminal half. One striking feature of the primary sequence of ICP27, however, is an extremely acidic region near its amino terminus. To determine whether this region is required for ICP27 function, we deleted the sequences in the ICP27 gene which encode it (codons 12 through 63). In transient expression assays, the deletion mutant was unable to efficiently repress the expression of a cotransfected reporter gene or to efficiently complement the growth of d27-1, an HSV-1 ICP27 null mutant. These results suggested that the acidic region of ICP27 is involved in a regulatory function required for lytic growth. To test this possibility further, we introduced the mutant allele into the HSV-1 genome by marker transfer. Two independently derived isolates of the mutant virus, designated d1-2a and d1-2b, were recovered and analyzed. Both isolates were defective for growth in Vero cells, exhibiting a 100-fold reduction in virus yield compared with the wild-type infection. Vero cells infected with the d1-2 isolates showed a three- to eightfold reduction in viral DNA replication, a moderate reduction in the expression of viral gamma genes, and a delay in the repression of beta genes. The phenotype of the d1-2 isolates differs substantially from the phenotypes of previously isolated ICP27 mutants, which show much more severe defects in viral gene expression. Our results demonstrate that the amino-terminal half of ICP27 participates in its regulatory activities in both infected and transfected cells.  相似文献   

12.
Bunnell SM  Rice SA 《Journal of virology》2000,74(16):7362-7374
ICP27 is an essential herpes simplex virus type 1 (HSV-1) immediate-early protein that regulates viral gene expression by poorly characterized mechanisms. Previous data suggest that its carboxyl (C)-terminal portion is absolutely required for productive viral infection. In this study, we isolated M16R, a second-site revertant of a viral ICP27 C-terminal mutant. M16R harbors an intragenic reversion, as demonstrated by the fact that its cloned ICP27 allele can complement the growth of an HSV-1 ICP27 deletion mutant. DNA sequencing demonstrated that the intragenic reversion is a frameshift alteration in a homopolymeric run of C residues at codons 215 to 217. This results in the predicted expression of a truncated, 289-residue molecule bearing 72 novel C-terminal residues derived from the +1 reading frame. Consistent with this, M16R expresses an ICP27-related molecule of the predicted size in the nuclei of infected cells. Transfection-based viral complementation assays confirmed that the truncated, frameshifted protein can partially substitute for ICP27 in the context of viral infection. Surprisingly, its novel C-terminal residues are required for this activity. To see if the frameshift mutation is all that is required for M16R's viability, we re-engineered the M16R ICP27 allele and inserted it into a new viral background, creating the HSV-1 mutant M16exC. An additional mutant, exCd305, was constructed which possesses the frameshift in the context of an ICP27 gene with the C terminus deleted. We found that both M16exC and exCd305 are nonviable in Vero cells, suggesting that one or more extragenic mutations are also required for the viability of M16R. Consistent with this interpretation, we isolated two viable derivatives of exCd305 which grow productively in Vero cells despite being incapable of encoding the C-terminal portion of ICP27. Studies of viral DNA synthesis in mutant-infected cells indicated that the truncated, frameshifted ICP27 protein can enhance viral DNA replication. In summary, our results demonstrate that the C-terminal portion of ICP27, conserved widely in herpesviruses and previously believed to be absolutely essential, is dispensable for HSV-1 lytic replication in the presence of compensatory genomic mutations.  相似文献   

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We previously reported that a recombinant ICP27-null virus stimulated, but did not prevent, apoptosis in human HEp-2 cells during infection (M. Aubert and J. A. Blaho, J. Virol. 73:2803-2813, 1999). In the present study, we used a panel of 15 recombinant ICP27 mutant viruses to determine which features of herpes simplex virus type 1 (HSV-1) replication are required for the apoptosis-inhibitory activity. Each virus was defined experimentally as either apoptotic, partially apoptotic, or nonapoptotic based on infected HEp-2 cell morphologies, percentages of infected cells with condensed chromatin, and patterns of specific cellular death factor processing. Viruses d27-1, d1-5, d1-2, M11, M15, M16, n504R, n406R, n263R, and n59R are apoptotic or partially apoptotic in HEp-2 cells and severely defective for growth in Vero cells. Viruses d2-3, d3-4, d4-5, d5-6, and d6-7 are nonapoptotic, demonstrating that ICP27 contains a large amino-terminal region, including its RGG box RNA binding domain, which is not essential for apoptosis prevention. Accumulations of viral TK, VP16, and gD but not gC, ICP22, or ICP4 proteins correlated with prevention of apoptosis during the replication of these viruses. Of the nonapoptotic viruses, d4-5 did not produce gC, indicating that accumulation of true late gene products is not necessary for the prevention process. Analyses of viral DNA synthesis in HEp-2 cells indicated that apoptosis prevention by HSV-1 requires that the infection proceeds to the stage in which viral DNA replication takes place. Infections performed in the presence of the drug phosphonoacetic acid confirmed that the process of viral DNA synthesis and the accumulation of true late (gamma(2)) proteins are not required for apoptosis prevention. Based on our results, we conclude that the accumulation of HSV-1 early (beta) and leaky-late (gamma(1)) proteins correlates with the prevention of apoptosis in infected HEp-2 cells.  相似文献   

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为评价抗caspase 3核酶在阻抑细胞凋亡发生中的潜在价值 ,以RNaseP催化亚基M1RNA为模板 ,设计合成 3个特异性针对人caspase 3的核酶pM1 GS716、pM1 GS337和pM1 GS2 35 ,并对它们的体内外切割活性进行探讨 .3 2 P标记的caspase 3基因片段体外转录物作为靶RNA ,体外切割实验表明 ,pM1 GS716和pM1 GS337均有切割活性 ,其中pM1 GS716的切割效率可达到 93% .3个核酶转染HeLa细胞 ,评价其在体内的切割活性 .在TNF α作用下 ,转染pM1 GS716的HeLa细胞内caspase 3mRNA下降了 75 % ,蛋白含量下降了 6 9% ,caspase 3蛋白酶活性下降了 5 2 % .Hoechst 332 5 8染色表明 ,细胞凋亡率较对照明显下降 (分别为 2 1 6± 0 7%和 4 9 4± 0 2 % ,P <0 0 1) .提示体外制备的pM1 GS716具有良好的特异催化切割活性 ,有望通过切割caspase 3而抑制细胞凋亡 .  相似文献   

18.
ICP27 is an essential herpes simplex virus type 1 (HSV-1) alpha protein that is required for the transition from the beta to the gamma phase of infection. To identify functional regions of ICP27, we constructed 16 plasmids that contain nucleotide substitution mutations in the ICP27 gene. The mutations created XhoI restriction sites, altered one or two codons, and were spaced at semiregular intervals throughout the coding region. Three mutations completely inactivated an essential function of ICP27, as demonstrated by the inability of the transfected plasmids to complement the growth of an HSV-1 ICP27 deletion mutant. These mutations, M11, M15, and M16, mapped in the carboxyl-terminal one-third of ICP27 at residues 340 and 341, 465 and 466, and 488, respectively. In cotransfection assays, all three defective-plasmid mutants retained the transrepression function of ICP27 but were defective at transactivation. To define the lytic functions that are mediated by the transactivation activity of ICP27, we engineered HSV-1 recombinants containing the M11, M15, or M16 mutation. All three viral mutants failed to grow in Vero cells and possessed similar phenotypes. The viral mutants replicated their DNA similarly to the wild-type virus but showed several defects in viral gene expression. These were a failure to down-regulate alpha and beta genes at late times after infection and an inability to induce certain gamma-2 genes. Our results demonstrate that the transactivation function of ICP27 (as it is defined in cotransfection assays) mediates an essential gene regulation function during the HSV-1 infection. This activity is not required for ICP27-dependent enhancement of viral DNA replication. Our work supports and extends previous studies which suggest that ICP27 carries out two distinct regulatory activities during the HSV-1 infection.  相似文献   

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