Association of the sindbis virus RNA methyltransferase activity with the nonstructural protein nsP1

SVLM21 is a mutant of Sindbis virus, which in contrast to SVSTD, is able to replicate in Aedes albopictus mosquito cells deprived of methionine. We have obtained evidence that the basis of this low methionine-resistance (LMR) phenotype is the generation of an altered RNA methyltransferase with an increased affinity for S-adenosylmethionine (ado met). We now report that following the substitution of the nucleotide sequence, 126-504, from SVLM21 cDNA for the corresponding sequence of the Toto 1101 plasmid (infectious Sindbis viral RNA can be transcribed from this plasmid) we were able to generate recombinant Sindbis virus (SVMS-65a) with the LMR phenotype. (SVTOTO virus derived from Toto 1101, like SVSTD, lacks the LMR phenotype.) As was the case with SVLM21, SVMS-65a not only possessed the LMR phenotype but also showed an increased sensitivity to Neplanocin A, a potent inhibitor of S-adenosylhomocysteine (ado hcy) hydrolase. Sequencing of the nucleotide 126-504 region from SVLM21 revealed two mutations; these mutations occurred in adjacent codons and lead to two predicted amino acid changes in the SV nsPl protein; at residue 87, from Arg to Leu, and at residue 88 from Ser to Cys. Since the nucleotide sequence 126-504 lies entirely within the gene for nsP1, we conclude that the RNA methyltransferase activity generated by SV is associated with nsP1. We suggest that residues 87 and 88 in nsP1, where the amino acid changes in SVLM21 nsP1 have occurred, are at or near the binding site for ado met; we also suggest that these changes in nsP1 are responsible for the increased affinity of the SVLM21 RNA methyltransferase for ado met and thereby for the LMR phenotype. Alternatively, it is possible that the binding site for ado met is elsewhere on nsP1 or even on another protein, and that the changes at residues 87 and 88 lead to an alteration of the binding site.     

Phosphorylation of Sindbis virus nsP3 in vivo and in vitro

nsP3 is one of four viral nonstructural proteins required for RNA replication of Sindbis virus. In this report, post-translational modifications of nsP3 which occur in both vertebrate and mosquito cell cultures have been examined. In pulse-chase experiments, analyzed by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, nsP3 was initially observed as a single species (termed nsP3a, approximately 76 kDa) which was gradually converted to slower mobility forms ranging from 78 kDa (termed nsP3b) to 106 kDa (termed nsP3c). The slower mobility forms, but not nsP3a or the other nonstructural proteins, could be labeled in vivo with [32P]orthophosphate. Treatment of nsP3 immunoprecipitates with calf intestinal alkaline phosphatase converted the slower mobility forms to nsP3a. Phosphoamino acid analysis of nsP3b and nsP3c demonstrated that both contained phosphoserine and phosphothreonine but not phosphotyrosine, nsP34, a polyprotein produced by readthrough of the in-frame opal codon preceding nsP4, was also phosphorylated on serine and threonine residues. nsP3 phosphorylation did not require ongoing RNA synthesis since phosphorylated forms were also observed in the absence of Sindbis-specific RNA synthesis. Furthermore, when immunoprecipitates of nsP3 were incubated with [gamma-32P]ATP in the presence of Mg2+ or Mn2+, a kinase activity which was able to phosphorylate nsP3 on serine and threonine residues in vitro was detected. This kinase activity was inhibited by heparin, was activated by spermidine, and could utilize GTP and ATP as the phosphate donor. These latter properties are similar to those of cellular casein kinase II. Although it is possible that this nsP3-associated kinase is of cellular origin, autophosphorylation of nsP3 has not been excluded.     

Cleavage between nsP1 and nsP2 initiates the processing pathway of Sindbis virus nonstructural polyprotein P123

The cleavage between nsP1 and nsP2 and that between nsP2 and nsP3 in the Sindbis virus nonstructural polyproteins was studied with respect to order of processing and enzyme-substrate relationships, using site-specific mutants in which the cleavage sites had been altered. The penultimate Gly in nsP1 or nsP2 or both was substituted by Ala, Val, or Glu, and processing was studied in vitro. Substitution with Ala resulted in partial cleavage whereas substitution with Val or Glu totally abolished cleavage at the mutagenized site. Abolishment of cleavage at the nsP2/nsP3 site did not affect processing at the nsP1/nsP2 site in the precursor polyprotein P123, and nsP1 and P23 were produced. When cleavage at the nsP1/nsP2 site was abolished, however, processing at the nsP2/nsP3 site was also prevented and P123 accumulated. To investigate why cleavage at the nsP1/nsP2 site should be required for cleavage at the nsP2/nsP3 site, the mutagenized polypeptides were used as enzymes in trans-cleavage experiments. We found that P123 can cleave the nsP1/nsP2 site but not the nsP2/nsP3 site, whereas P23 can cleave the nsP2/nsP3 site very efficiently. Thus, cleavage at the nsP1/nsP2 site by P123 is required to produce an enzyme capable of cleaving the nsP2/nsP3 site. Release of nsP4 from P1234 appears to be independent of the other cleavages and occurs primarily immediately after translation. These mutations were also transferred into a full-length cDNA clone of Sindbis virus and virus was recovered. Mutants defective in the cleavage of the nsP2/nsP3 site were temperature sensitive, growing at a slightly reduced rate compared to wild-type virus at 30 degrees but growing poorly at 40 degrees. Mutants defective in the cleavage of both the nsP1/nsP2 site and the nsP2/nsP3 site were viable but grew poorly compared with wild-type at any temperature.     

Characterization of purified Sindbis virus nsP4 RNA-dependent RNA polymerase activity in vitro

The Sindbis virus RNA-dependent RNA polymerase (nsP4) is responsible for the replication of the viral RNA genome. In infected cells, nsP4 is localized in a replication complex along with the other viral non-structural proteins. nsP4 has been difficult to homogenously purify from infected cells due to its interactions with the other replication proteins and the fact that its N-terminal residue, a tyrosine, causes the protein to be rapidly turned over in cells. We report the successful expression and purification of Sindbis nsP4 in a bacterial system, in which nsP4 is expressed as an N-terminal SUMO fusion protein. After purification the SUMO tag is removed, resulting in the isolation of full-length nsP4 possessing the authentic N-terminal tyrosine. This purified enzyme is able to produce minus-strand RNA de novo from plus-strand templates, as well as terminally add adenosine residues to the 3′ end of an RNA substrate. In the presence of the partially processed viral replicase polyprotein, P123, purified nsP4 is able to synthesize discrete template length minus-strand RNA products. Mutations in the 3′ CSE or poly(A) tail of viral template RNA prevent RNA synthesis by the replicase complex containing purified nsP4, consistent with previously reported template requirements for minus-strand RNA synthesis. Optimal reaction conditions were determined by investigating the effects of time, pH, and the concentrations of nsP4, P123 and magnesium on the synthesis of RNA.     

Identification of the active site residues in the nsP2 proteinase of Sindbis virus.

The nonstructural polyproteins of Sindbis virus are processed by a virus-encoded proteinase which is located in the C-terminal domain of nsP2. Here we have performed a mutagenic analysis to identify the active site residues of this proteinase. Substitution of other amino acids for either Cys-481 or His-558 completely abolished proteolytic processing of Sindbis virus polyproteins in vitro. Substitutions within this domain for a second cysteine conserved among alphaviruses, for four other conserved histidines, or for a conserved serine did not affect the activity of the enzyme. These results suggest that nsP2 is a papain-like proteinase whose catalytic dyad is composed of Cys-481 and His-558. Since an asparagine residue has been implicated in the active site of papain, we changed the four conserved asparagine residues in the C-terminal half of nsP2 and found that all could be substituted without total loss of activity. Among papain-like proteinases, the residue following the catalytic histidine is alanine or glycine in the plant and animal enzymes, and the presence of Trp-559 in alphaviruses is unusual. A mutant enzyme containing Ala-559 was completely inactive, implying that Trp-559 is essential for a functional proteinase. All of these mutations were introduced into a full-length clone of Sindbis virus from which infectious RNA could be transcribed in vitro, and the effects of these changes on viability were tested. In all cases it was found that mutations which abolished proteolytic activity were lethal, whether or not these mutations were in the catalytic residues, indicating that proteolysis of the nonstructural polyprotein is essential for Sindbis replication.     

Mutations that confer resistance to mycophenolic acid and ribavirin on Sindbis virus map to the nonstructural protein nsP1.

SVMPA, a mutant of Sindbis virus derived by serial passage on Aedes albopictus mosquito cells maintained after infection in the presence of mycophenolic acid (MPA), is resistant not only to MPA but also to ribavirin. Both of these compounds inhibit the synthesis of GMP and thereby reduce the level of GTP. We had suggested earlier that SVMPA had become resistant to MPA because it coded for an altered RNA guanylyltransferase enzyme with an increased affinity for GTP, enabling it to replicate in cells with reduced levels of GTP. We now report that the MPA-resistant phenotype of SVMPA has been mapped to the coding region for the nonstructural viral protein, nsP1. By replacing the nucleotide sequence between 88 and 1404 of the infectious clone of Sindbis virus (i.e., the Toto 1101 plasmid) with the corresponding sequence from SVMPA cDNA, we were able to generate recombinant Sindbis virus expressing the drug-resistant phenoptype. SVMPA has three base substitutions in the region between nucleotides 88 and 1404 which lead to predicted amino acid changes in the Sindbis virus nsP1 protein: the replacement of Gln at residue 21 by Lys, Ser at residue 23 by Asn, and Val at residue 302 by Met. These results, taken together with previous data from our laboratory associating the RNA methyltransferase with nsP1, (1) are consistent with the idea that an alteration of the RNA guanylyltransferase is responsible for the MPA-resistant phenotype and (2) support the idea that an important function of nsP1 relates to the modification of the 5' terminus of the Sindbis virus mRNAs.     

Microassay for Sindbis virus and interferon activity.

A simple and rapid microplaque assay for Sindbis virus was developed which uses microtiter plates and overlay medium consisting of methylcellulose and specific antibody to Sindbis virus. Discrete plaque formation was consistently observed on baby hamster kidney (BHK-15) cells within 24 h. The assay was reproducible, quantitative, and had about the same sensitivity as the agar overlay technique on chicken embryo cells in 35-mm petri dishes. The microplaque assay could be accurately applied to measuring interferon activity, particularly at low interferon levels. Overall, the microassay methods described here for assay of Sindbis virus yields and interferon activity retain the accuracy of chicken cell macroplaque assays while offering greater simplicity, rapidity, and economy of materials. This assay is also potentially applicable for use with other togaviruses.     

Expression of staphylococcal enterotoxin C1 in Escherichia coli.

The structural gene encoding staphylococcal enterotoxin C1 was cloned into Escherichia coli and localized on a 1.5-kilobase HindIII-ClaI DNA fragment by subcloning. The toxin was partially purified from E. coli clones and shown to be immunologically identical to enterotoxin C1 from Staphylococcus aureus. The cloned toxin also had the same molecular weight (26,000) and charge heterogeneity as staphylococcus-derived enterotoxin. Toxins from both sources were equally biologically active.     

Expression of Japanese encephalitis virus antigens in Escherichia coli

The expression of Japanese encephalitis virus (JE) cDNA in Escherichia coli has been used to study the functional organization of the viral genome. JE protein coding sequences were expressed in E. coli by subcloning random fragments of cloned cDNA (P.C. McAda, P.W. Mason, C.S. Schmaljohn, J.M. Dalrymple, T.L. Mason, and M.J. Fournier, 1987, Virology 158, 348-360) into the bacteriophage lambda gt11 expression vector. Over 120 lambda gt11 recombinants expressing viral protein sequences as beta-galactosidase fusion proteins were identified immunologically with monoclonal antibodies (MAbs) and polyclonal hyperimmune mouse ascites fluid (HMAF). This expression and immunological detection strategy has been used to (1) map viral protein coding sequences to the JE genome; (2) demonstrate that contiguous viral protein coding regions can be expressed as single polypeptides in E. coli, providing functional confirmation for a long viral open reading frame; (3) localize important antigenic domains within the envelope protein E; and (4) identify in JE-infected cells a form of the glycosylated nonstructural protein NS1 that contains a hydrophobic C-terminal extension encoded by portions of the "ns2a" region of the JE genome.     

The nsP3 macro domain is important for Sindbis virus replication in neurons and neurovirulence in mice

Sindbis virus (SINV), the prototype alphavirus, contains a macro domain in the highly conserved N-terminal region of nonstructural protein 3 (nsP3). However, the biological role of the macro domain is unclear. Mutations of amino acids 10 and 24 from asparagine to alanine in the ADP-ribose binding region of the macro domain impaired SINV replication and viral RNA synthesis particularly in neurons, but did not alter binding of poly(ADP-ribose). Mutation at position 10 had the greatest effect and caused nsP3 instability in neurons, decreased SINV-induced death of mature, but not immature neurons, and attenuated virulence in 2 week-old, but not 5 day-old mice. A compensatory mutation at amino acid 31 in the macro domain of nsP3, as well as reversion of mutated amino acid 10, occurred during replication of double mutant SINV in vitro and in vivo. The nsP3 macro domain is important for SINV replication and age-dependent susceptibility to encephalomyelitis.     

Mitogenic activity of Sindbis virus and its isolated glycoproteins.

Purified preparations of Sindbis virus, a member of the togavirus family, are mitogenic for lymphocytes from a number of different mouse strains. Cell separation techniques, as well as studies using lymphocytes from the congenitally athymic BALB/c nu/nu mouse, showed that Sindbis virus is a T-cell-independent B-cell mitogen. Additionally, the envelope glycoproteins of Sindbis virus, isolated by Triton X-100 extraction and butanol precipitation, stimulated lymphocytes to incorporate five times as much [3H]thymidine into their DNA as did the Sindbis virion. These results are similar to those previously reported for vesicular stomatitis virus and herpes simplex virus types I and II and for the purified glycoproteins of vesicular stomatitis virus and influenza virus.     

EB病毒融合蛋白ZtaN-p23在大肠杆菌中的表达、纯化及活性鉴定

目的:克隆EB病毒立即早期蛋白ZtaN和衣壳蛋白p23,构建原核表达载体,并在大肠杆菌中表达融合蛋白,为后续利用蛋白进行疾病诊断奠定基础。方法:采用逆转录聚合酶链反应(RT-PCR)技术从B95-8细胞中分别扩增出目的基因BZLF1N和BLRF2,利用融合PCR法将两个基因进行连接,构建重组质粒pGEX-4T-1-BZLF1N-BLRF2,并转化大肠杆菌BL21(DE3)。加入IPTG诱导表达融合蛋白ZtaN-p23,通过SDS-PAGE和Western blot确定蛋白表达量及活性鉴定。结果:重组质粒pGEX-4T-1-BZLF1N-BLRF2经双酶切鉴定和序列分析正确,证实已成功构建原核表达载体;SDS-PAGE显示诱导的蛋白Mr约46 000,与预期值一致;对融合蛋白表达条件进行优化后,发现其主要以包涵体的形式存在于细胞中;亲和层析结果显示纯化后的ZtaN-p23纯度>95%;Westernblot显示ZtaN-p23具有良好的生物活性。结论:成功构建原核表达载体pGEX-4T-1-BZLF1N-BLRF2,并在大肠杆菌中进行了表达纯化,融合蛋白显示出良好的活性。     

Expression and biochemical characterization of nsP2 cysteine protease of Chikungunya virus

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes epidemic fever, rash and polyarthralgia in Africa and Asia. Although it is known since the 1950s, new epidemiological and clinical features reported during the recent outbreak in the Indian Ocean can be regarded as the emergence of a new disease. Numerous severe forms of the infection have been described that put emphasis on the lack of efficient antiviral therapy. Among the virus-encoded enzymes, nsP2 constitutes an attractive target for the development of antiviral drugs. It is a multifunctional protein of approximately 90 kDa with a helicase motif in the N-terminal portion of the protein while the papain-like protease activity resides in the C-terminal portion. The nsP2 proteinase is an essential enzyme whose proteolytic activity is critical for virus replication. In this work, a recombinant CHIKV nsP2pro and a C-terminally truncated variant were expressed in Escherichia coli and purified by metal-chelate chromatography. The enzymatic properties of the proteinase were then determined using specific synthetic fluorogenic substrates. This study constitutes the first characterization of a recombinant CHIKV nsP2 cysteine protease, which may be useful for future drug screening.     

Expression of the plum pox virus coat protein region in Escherichia coli

A cDNA complementary to the 3 end of plum pox virus (PPV) RNA was sequenced. The sequence was investigated for the presumable coat protein cistron by computer-aided translation. A fragment containing the stop codon of the polyprotein gene and a putative virus-specific protease cleavage site was subcloned into an E. coli expression vector. It is shown by immunological analysis that the coat protein cistron is located within the subcloned region.     

Expression of the Moloney murine leukemia virus and human immunodeficiency virus integration proteins in Escherichia coli

We have constructed expression plasmids containing the genes for the MuLV and the HIV integration proteins. When introduced into Escherichia coli, these plasmids cause the production of proteins of the expected molecular weight (43K for MuLV, 31K for HIV). The wild-type MuLV coding region induces the synthesis of large amounts of integration protein; to obtain large amounts of the full-length HIV integration protein in E. coli, it was necessary to modify the coding region to disrupt a sequence that promotes efficient internal translational initiation in E. coli. It is possible to disrupt this sequence without altering the encoded amino acids; the modified plasmid makes large amounts of the full-length protein and small amounts of the internally initiated protein. Both the MuLV and HIV integration proteins require SDS to be solubilized in E. coli extracts; however, following solubilization with SDS and transfer to a nitrocellulose filter, both integration proteins bind DNA.     

博尔纳病毒核蛋白的原核表达、纯化及鉴定

目的 构建、表达、并纯化重组博尔纳病毒核蛋白(Borna disease vires,BDV),并鉴定该蛋白.方法 通过PCR反应从博尔纳病毒cDNA中扩增博尔纳病毒核蛋白P40基因,构建博尔纳病毒核蛋白P40基因重组质粒pET-14b-BD-VP40,转化大肠杆菌,IPTG诱导融合蛋白的表达,His-tag亲和层析纯化该蛋白,SDS-PAGE分析其表达量、表达形式和纯度;Western-blot法鉴定该蛋白.结果 成功构建蓖组质粒pET-14b-BDVP40,酶切鉴定、核酸序列分析正确,亲和层析纯化后纯度可达90%,Western-blot表明重组蛋白能与博尔纳病毒核蛋白单克隆抗体特异性结合.结论 在大肠杆菌中成功表达了可溶性的pET-14b-BDVP40融合蛋白,为进一步研究博尔纳病毒核蛋白作用机制及开发相应血清学检测试剂盒提供基础.     

Loss of lectin-like activity in aberrant type 1 fimbriae of Escherichia coli.

Growth of a streptomycin-resistant strain of Escherichia coli (VL-2) in the presence of 30 microgram of streptomycin per ml resulted in the production by these bacteria of structurally altered, nonfunctional type 1 fimbriae. This strain, when grown in this subinhibitory concentration of streptomycin, became incapable of producing mannose-sensitive hemagglutination (<1% of that of the control). Adhering ability to epithelial cells and human leukocytes was also diminished (42 and 7% of that of the control, respectively). Although these streptomycin-treated bacteria were as heavily fimbriated as untreated bacteria, their fimbriae were significantly longer. Furthermore, in contrast to the fimbriae of the untreated bacteria, those isolated from the drug-treated bacteria were found to lack mannose binding activity as measured by hemagglutination. It appears, therefore, that streptomycin can cause even resistant bacteria to produce an aberrant fimbrial protein, possibly by causing misreading of messenger RNA. These studies indicate that the use of sublethal doses of certain antibiotics whose mode of action is well known may shed light on the genetic and chemical modulation of bacterial factors involved in mucosal colonization.     

Expression of Mycoplasma pneumoniae antigens in Escherichia coli.

A genomic library of Mycoplasma pneumoniae was generated by using bacteriophage lambda EMBL3 as the vector. Screening of the library for the expression of M. pneumoniae protein antigens with adsorbed anti-M. pneumoniae serum revealed strong reactivity from a third of those clones which contained mycoplasma DNA inserts. Three of the most highly reactive clones were analyzed in detail and found to synthesize discrete mycoplasma proteins. Two carried overlapping fragments of mycoplasma DNA which encoded a protein that was readily detected in Escherichia coli after infection with recombinant bacteriophage. The third clone contained a novel mycoplasma DNA fragment which directed the synthesis of two additional mycoplasma proteins. Further screening of the library with antiserum raised against the major M. pneumoniae adhesin protein P1 (165 kilodaltons [kDa]) yielded one clone which produced an immunologically reactive protein of 140 kDa. Adsorption of anti-P1 serum by this clone selected a population of antibodies that were reactive with M. pneumoniae adhesin P1 (165 kDa). These results demonstrate that immunologically active M. pneumoniae proteins are synthesized in E. coli.     

Expression of the S-1 catalytic subunit of pertussis toxin in Escherichia coli.

The S-1 subunit of pertussis toxin was expressed as a fusion protein in a strain of Escherichia coli deficient in protein degradation. The fusion protein reacted with anti-pertussis toxin antibody, and, like authentic pertussis toxin, it ADP-ribosylated a 41,000-molecular-weight membrane protein from human erythrocytes.