The p7 protein of hepatitis C virus forms an ion channel that is blocked by the antiviral drug,Amantadine

Hepatitis C virus (HCV) cannot be grown in vitro, making biochemical identification of new drug targets especially important. HCV p7 is a small hydrophobic protein of unknown function, yet necessary for particle infectivity in related viruses [Harada, T. et al., (2000) J. Virol. 74, 9498-9506]. We show that p7 can be cross-linked in vivo as hexamers. Escherichia coli expressed p7 fusion proteins also form hexamers in vitro. These and HIS-tagged p7 function as calcium ion channels in black lipid membranes. This activity is abrogated by Amantadine, a compound that inhibits ion channels of influenza [Hay, A.J. et al. (1985) EMBO J. 4, 3021-3024; Duff, K.C. and Ashley, R.H. (1992) Virology 190, 485-489] and has recently been shown to be active in combination with current HCV therapies.     

Characterization of the zinc binding activity of the rubella virus nonstructural protease

The rubella virus (RUB) nonstructural (NS) protein (NSP) ORF encodes a protease that cleaves the NSP precursor (240 kDa) at a single site to produce two products. A cleavage site mutation was introduced into a RUB infectious cDNA clone and found to be lethal, demonstrating that cleavage of the NSP precursor is necessary for RUB replication. Based on computer alignments, the RUB NS protease was predicted to be a papain-like cysteine protease (PCP) with the residues Cys1152 and His1273 as the catalytic dyad; however, the RUB NS protease was recently found to require divalent cations such as Zn, Co, and Cd for activity (X. Liu, S. L. Ropp, R. J. Jackson, and T. K. Frey, J. Virol. 72:4463-4466, 1998). To analyze the function of metal cation binding in protease activity, Zn binding studies were performed using the minimal NS protease domain within the NSP ORF. When expressed as a maltose binding protein (MBP) fusion protein by bacteria, the NS protease exhibited activity both in the bacteria and in vitro following purification when denatured and refolded in the presence of Zn. Atomic absorption analysis detected 1.6 mol of Zn bound per mol of protein refolded in this manner. Expression of individual domains within the protease as MBP fusions and analysis by a Zn(65) binding assay revealed two Zn binding domains: one located at a predicted metal binding motif beginning at Cys1175 and the other one close to the cleavage site. Mutagenesis studies showed that Cys1175 and Cys1178 in the first domain and Cys1227 and His1273, the His in the predicted catalytic site, in the second domain are essential for zinc binding. All of these residues are also necessary for the protease activity, as were several other Cys residues not involved in Zn binding. Far-UV circular dichroism (CD) analysis of the MBP-NS protease fusion protein showed that the protease domain contained a large amount of alpha-helical structure, which is consistent with the results of secondary-structural prediction. Both far-UV-CD and fluorescence studies suggested that Zn did not exert a major effect on the overall structure of the fusion protein. Finally, protease inhibitor assays found that the protease activity can be blocked by both metal ion chelators and the metalloprotease inhibitor captopril. In conjunction with the finding that the previously predicted catalytic site, His1273, is essential for zinc binding, this suggests that the RUB NS protease is actually a novel virus metalloprotease rather than a PCP.     

Purification and refolding of recombinant human IGF II from silkworms infected with recombinant Bombyx mori nuclear polyhedrosis virus.

We have reported that insulin-like growth factor II (IGF II) was produced as a fusion protein in Bombyx mori (silkworm) larval bodies infected with recombinant B. mori nuclear polyhedrosis virus [J. Gen. Virol., 68, 2599-2606 (1987)]. In this study, the purification of IGF II from the infected silkworms is reported. The fusion protein was extracted with 6.0 M guanidine-HCl from the infected larval bodies homogenized in water. The use of organic solvents to remove the impurities, such as lipid derived from the larval bodies, was a very effective method of purification. IGF II was released from the partially purified fusion protein by treatment with CNBr, purified by HPLC, and refolded by air-oxidization. Refolded IGF II had an identical primary structure including disulfide bonds and showed identical thymidine uptake stimulation activity with human IGF II. Furthermore, protein disulfide-isomerase was shown to be able to refold scrambled IGF II rapidly.     

The role of zinc in Bacillus subtilis cytidine deaminase

Cytidine deaminase (CDA) from Bacillus subtilis is a zinc-containing enzyme responsible for the hydrolytic deamination of cytidine to uridine and 2'-deoxycytidine to 2'-deoxyuridine. Titration of the cysteinyl groups of the enzyme with p-hydroxymercuriphenyl sulfonate (PMPS) resulted in release of one zinc ion per subunit. Addition of EDTA to chelate the zinc and dithiothreitol (DTT) to remove PMPS, followed by removal of the low molecular weight compounds by gel filtration, resulted in an apoenzyme with no enzymatic activity. The apoenzyme was almost fully reactivated by addition of zinc chloride, indicating that the zinc ion played a central role in catalysis, in keeping with what has been observed with Escherichia coli CDA [Betts, L., Xiang, S., Short, S. A., Wolfenden, R., and Carter, C. W. J. (1994) J. Mol. Biol. 235, 635-656]. Addition of Cd(2+) or Co(2+) caused partial reactivation of the apoenzyme. Zinc reconstitution of the apoenzyme was strictly dependent on the presence of reducing agents, suggesting that the zinc-ligating cysteines, when unligated, participated in disulfide bond formation. An enzymatically active isoform of the tetrameric CDA protein, containing an extension of 13 amino acids at the C-terminus of each subunit, was used in conjunction with the wild-type CDA in subunit-subunit dissociation studies to show that the zinc ion does not assist in the thermodynamic refolding of the protein. After treatment with PMPS and EDTA, the enzyme existed as unfolded unassociated subunits. Immediately following DTT addition to remove PMPS, the subunits refolded into a tetrameric structure, independent of the presence of zinc.     

Transformation by the oncogenic latent membrane protein correlates with its rapid turnover, membrane localization, and cytoskeletal association.

The latent membrane protein (LMP) of Epstein-Barr virus (EBV) has a short half-life (V. R. Baichwal and B. Sugden, J. Virol, 61:866-875, 1987; K.P. Mann and D. Thorley-Lawson, J. Virol, 61:2100-2108, 1987), is localized in patches in the membrane (D. Liebowitz, D. Wang, and E, Kieff, J. Virol, 58:233-237, 1986), and associates with the cytoskeleton in EBV-immortalized B lymphocytes (D. Liebowitz, R. Kopan, E. Fuchs, J. Sample, and E. Kieff, Mol. Cell. Biol. 7:2299-2308, 1987; K. P. Mann and D. Thorley-Lawson, J. Virol. 61:2100-2108, 1987). Deletion mutants of LMP that are either positive or negative in the induction both of anchorage-independent growth of BALB/c 3T3 cells (V. R. Baichwal and B. Sugden, Oncogene 4:67-74, 1989) and of cytotoxicity in a variety of cells (W. Hammerschmidt, B. Sugden, and V. R. Baichwal, J. Virol. 63:2469-2475, 1989) have been studied to identify the biochemical properties of this protein that correlate with its effects on cell growth. Mutant LMP proteins that are metabolically stable, do not associate with the cytoskeleton, and exhibit a diffuse plasma membrane localization also do not induce anchorage-independent growth in rodent cells or cytotoxicity in B lymphoblastoid cells. In contrast, a mutant of LMP that is functionally identical to the wild-type protein has a half-life, membrane localization, and cytoskeletal association similar or identical to those of LMP. These results are consistent with the hypothesis that LMP's rapid turnover, association with the cytoskeleton, and patching in the membrane are required for it to affect cell growth.     

huGM-CSF(9-127)-IL-6(29-184)融合蛋白的复性及纯化研究

利用Q Sepharose H.P.离子交换柱层析在8mol/L尿素变性条件下对huGM-CSF(9-127)-IL-6(29-184）融合蛋白进行初步纯化,然后再利用Sephacryl S-200分子筛柱层析复性及纯化后获得目的蛋白,其纯度达到95％以上。该纯化方案成功地解决了稀释复性或透析复性产物在进行Q Sepharose H.P.离子交换柱层析时目的蛋白不稳定而沉积于柱上的问题,获得了较好的复性效果,复性率达到80％以上。使用该纯化方案,1天内便可基本完成重组蛋白的复性及纯化过程,而且也便于扩大。     

Cell fusion activity of hepatitis C virus envelope proteins

To examine the cell fusion activity of hepatitis C virus (HCV) envelope proteins (E1 and E2), we have established a sensitive cell fusion assay based on the activation of a reporter gene as described previously (O. Nussbaum, C. C. Broder, and E. A. Berger, J. Virol. 68:5411-5422, 1994). The chimeric HCV E1 and E2 proteins, each consisting of the ectodomain of the E1 and E2 envelope protein and the transmembrane and cytoplasmic domains of the vesicular stomatitis virus G glycoprotein, were expressed on the cell surface. Cells expressing the chimeric envelope proteins and T7 RNA polymerase were cocultured with the various target cell lines transfected with a reporter plasmid encoding the luciferase gene under the control of the T7 promoter. After cocultivation, the cell fusion activity was determined by the expression of luciferase in the cocultured cells. The induction of cell fusion requires both the chimeric E1 and E2 proteins and occurs in a low-pH-dependent manner. Although it has been shown that HCV E2 protein binds human CD81 (P. Pileri, Y. Uematsu, S. Campagnoli, G. Galli, F. Falugi, R. Petracca, A. J. Weiner, M. Houghton, D. Rosa, G. Grandi, and S. Abrignani, Science 282:938-941, 1998), the expression of human CD81 alone is not sufficient to confer susceptibility to cell fusion in the mouse cell line. Treatment of the target cells with pronase, heparinase, or heparitinase reduced the cell fusion activity induced by the chimeric envelope proteins. These results suggest (i) that both HCV E1 and E2 proteins are responsible for fusion with the endosomal membrane after endocytosis and (ii) that certain protein molecules other than human CD81 and some glycosaminoglycans on the cell surface are also involved in the cell fusion induced by HCV.     

Expression and characterization of recombinant soluble porcine CD3 ectodomain molecules: mapping the epitope of an anti-porcine CD3 monoclonal antibody 898H2-6-15

The porcine CD3 specific monoclonal antibody 898H2-6-15 has been used in allo- and xeno-transplantation studies as a porcine CD3 marker and as an effective T cell depletion reagent when conjugated to the diphtheria toxin mutant, CRM9. A recombinant anti-porcine CD3 immuntoxin was recently developed using single-chain variable fragments (scFv) derived from 898H2-6-15. In this study, using published sequence data, we have expressed the porcine CD3 ectodomain molecules in E. coli through inclusion body isolation and in vitro refolding approach. The expressed and refolded porcine CD3 ectodomain molecules include CD3ε, CD3γ, CD3δ, CD3εγ heterodimer, CD3εδ heterodimer, CD3εγ single-chain fusion protein and CD3εδ single-chain fusion protein. These refolded porcine CD3 ectodomain molecules were purified with a strong anion exchange resin Poros 50HQ. ELISA analysis demonstrated that only the porcine CD3εγ ectodomain single-chain fusion protein can bind to the porcine CD3 specific monoclonal antibody 898H2-6-15. The availability of this porcine CD3εγ ectodomain single-chain fusion protein will allow screening for affinity matured variants of scFv derived from 898H2-6-15 to improve the recombinant anti-porcine CD3 immunotoxin. Porcine CD3εγ ectodomain single-chain fusion protein will also be a very useful reagent to study the soluble phase interaction between porcine CD3εγ and porcine CD3 antibodies such as 898H2-6-15.     

Cloning of the vaccinia virus ribonucleotide reductase small subunit gene. Characterization of the gene product expressed in Escherichia coli.

During its infectious cycle, vaccinia virus expresses a virus-encoded ribonucleotide reductase which is distinct from the host cellular enzyme (Slabaugh, M.B., and Mathews, C.K. (1984) J. Virol. 52, 501-506; Slabaugh, M.B., Johnson, T.L., and Mathews, C.K. (1984) J. Virol. 52, 507-514). We have cloned the gene for the small subunit of vaccinia virus ribonucleotide reductase (designated VVR2) into Escherichia coli and expressed the protein using a T7 RNA polymerase plasmid expression system. After isopropyl beta-D-thiogalactopyranoside induction, accumulation of a 37-kDa peptide was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and this peptide reacted with polyclonal antiserum raised against a TrpE-VVR2 fusion protein. The 37-kDa protein was purified to homogeneity, and gel filtration of the purified protein revealed that the recombinant protein existed as a dimer in solution. Purified recombinant VVR2 protein was shown to complement the activity of purified recombinant ribonucleotide reductase large subunit, with a specific activity that was similar to native VVR2 from a virus-infected cell extract. A CD spectrum of the recombinant viral protein showed that like the mouse protein, the vaccinia virus protein has 50% alpha-helical structure. Like other iron-containing ribonucleotide reductase small subunits, recombinant VVR2 protein contained a stable organic free radical that was detectable by EPR spectroscopy. The EPR spectrum of purified recombinant VVR2 was identical to that of vaccinia virus-infected mammalian cells. Both the hyperfine splitting character and microwave saturation behavior of VVR2 were similar to those of mouse R2 and distinct from E. coli R2. By using amino acid analysis to determine the concentration of VVR2, we determined that approximately 0.6 radicals were present per R2 dimer. Our results indicate that vaccinia virus small subunit is similar to mammalian ribonucleotide reductases.     

重组PLZF蛋白锌指结构域的表达、提纯和活性分析

PLZF(promyelocytic leukaemia zinc finger protein)是一种重要的转录抑制因子,它由位于N端的BTB结构域和C端的锌指结构域构成。鉴于目前对于锌指结构域的立体结构还不是十分清楚,对其进行了高效表达和提纯。为了表达PLZF蛋白的锌指结构域,在其编码序列的5'端加上起始密码ATG后插入到表达载体PET-11a的多克隆位点。构建好的表达质粒转化到BL21 (DE3)大肠杆菌内并用IPTG诱导表达,发现重组蛋白主要以不溶性的包涵体形式在胞内表达。用含有SDS变性剂的缓冲液溶解包涵体后,采用凝胶过滤方法将重组蛋白纯化到纯度达96%以上。对纯化后的蛋白质用反透析的方法进行复性,然后用DNA结合实验进行活性分析,发现复性后的蛋白质具有特异的DNA结合活性,这为进一步研究PLZF蛋白锌指结构域的立体结构打下了重要基础。     

Purification and characterization of the gene 1.2 protein of bacteriophage T7

Gene 1.2 of bacteriophage T7, located near the primary origin of DNA replication at position 15.37 on the T7 chromosome, encodes a 10,059-dalton protein that is essential for growth on Escherichia coli optA1 strains (Saito, H., and Richardson, C. C. (1981) J. Virol. 37, 343-351). In the absence of the T7 1.2 and E. coli optA gene products, the degradation of E. coli DNA proceeds normally, and T7 DNA synthesis is initiated at the primary origin. However, T7 DNA synthesis ceases prematurely and the newly synthesized DNA is degraded; no viable phage particles are released. The gene 1.2 protein has been purified to apparent homogeneity from cells in which the cloned 1.2 gene is overexpressed. Purification of the [35S] methionine-labeled protein was followed by monitoring the radioactivity of the protein and by gel electrophoresis. The purified protein has been identified as the product of gene 1.2 on the basis of molecular weight and partial amino acid sequence. We have found that extracts of E. coli optA1 cells infected with T7 gene 1.2 mutants are defective in packaging exogenous T7 DNA when such extracts are prepared late in infection. Purified gene 1.2 protein restores packaging activity to these defective extracts, thus providing a biological assay for gene 1.2 protein. No specific enzymatic activity has been found associated with the purified gene 1.2 protein.     

Guanidine-resistant mutants of poliovirus have distinct mutations in peptide 2C.

In previous work in our laboratory, 12 guanidine-resistant (gr) mutants of poliovirus were selected from 12 separate stocks of plaque-purified guanidine-sensitive (gs) viruses (K. Anderson-Sillman, S. Bartal, and D. R. Tershak, J. Virol. 50:922-928, 1984). Peptide mapping of protein 2C and evaluation of virus growth at different temperatures enabled us to subdivide these mutants into several distinct groups (D. R. Tershak, Can. J. Microbiol. 31:1166-1168, 1985; Anderson-Sillman et al., J. Virol.). Studies by Pincus et al. indicate that two nucleotide changes in codon 179 of protein 2C leads to an Asn-to-Gly or Asn-to-Ala change and that these missense modifications account for guanidine resistance (S. E. Pincus, H. Rohl, and E. Wimmer, Virology 157:83-88, 1987; S. E. Pincus and E. Wimmer, J. Virol. 60:793-796, 1986). In the present report, we confirm their findings but also show that a single amino acid change of Phe to Tyr in residue 164 of protein 2C or a Met-to-Leu replacement in amino acid 187 coupled with mutations in codons 233 or 295 and 309 could confer guanidine resistance.     

Mutational analysis of heptad repeats in the membrane-proximal region of Newcastle disease virus HN protein

For most paramyxoviruses, syncytium formation requires the expression of both surface glycoproteins (HN and F) in the same cell, and evidence suggests that fusion involves a specific interaction between the HN and F proteins (X. Hu et al., J. Virol. 66:1528-1534, 1992). The stalk region of the Newcastle disease virus (NDV) HN protein has been implicated in both fusion promotion and virus specificity of that activity. The NDV F protein contains two heptad repeat motifs which have been shown by site-directed mutagenesis to be critical for fusion (R. Buckland et al., J. Gen. Virol. 73:1703-1707, 1992; T. Sergel-Germano et al., J. Virol. 68:7654-7658, 1994; J. Reitter et al., J. Virol. 69:5995-6004, 1995). Heptad repeat motifs mediate protein-protein interactions by enabling the formation of coiled coils. Upon analysis of the stalk region of the NDV HN protein, we identified two heptad repeats. Secondary structure analysis of these repeats suggested the potential for these regions to form alpha helices. To investigate the importance of this sequence motif for fusion promotion, we mutated the hydrophobic a-position amino acids of each heptad repeat to alanine or methionine. In addition, hydrophobic amino acids in other positions were also changed to alanine. Every mutant protein retained levels of attachment activity that was greater than or equal to the wild-type protein activity and bound to conformation-specific monoclonal as well as polyclonal antisera. Neuraminidase activity was variably affected. Every mutation, however, showed a dramatic decrease in fusion promotion activity. The phenotypes of these mutant proteins indicate that individual amino acids within the heptad repeat region of the stalk domain of the HN protein are important for the fusion promotion activity of the protein. These data are consistent with the idea that the HN protein associates with the F protein via specific interactions between the heptad repeat regions of both proteins.     

Spike protein oligomerization control of Semliki Forest virus fusion.

We have recently shown, using cleavage-deficient mutants of the p62-E1 membrane protein complex of Semliki Forest virus that p62 cleavage to E2 is necessary for the activation of the fusion function of the complex at pH 5.8 (a pH optimal for virus fusion) (M. Lobigs and H. Garoff, J. Virol. 64:1233-1240, 1990). In this study, we show that the mutant precursor complexes can be induced to activate membrane fusion when treated with more acidic buffers (pH 5.0 and 4.5), which also appear to dissociate most of the p62-E1 complexes and change the conformation of the E1 subunit (the supposed fusion protein of Semliki Forest virus into a form which is resistant to trypsin digestion. These data suggest that p62 cleavage is not essential for membrane fusion per se but that the crucial event activating this process seems to be the apparent dissociation of the heterodimer, which in turn is facilitated by the spike precursor cleavage.     

A single amino acid change in the E2 glycoprotein of Sindbis virus confers neurovirulence by altering an early step of virus replication.

Amino acid changes in the envelope glycoproteins of Sindbis virus have been linked to neurovirulence; however, the molecular mechanisms by which these amino acid changes alter neurovirulence are not known. Recombinant-virus studies have mapped an important determinant of neurovirulence in adult mice to a single amino acid change, glutamine to histidine, at position 55 of the E2 glycoprotein (P. C. Tucker, E. G. Strauss, R. J. Kuhn, J. H. Strauss, and D. E. Griffin, J. Virol. 67:4605-4610, 1993). To investigate how histidine confers neurovirulence, we examined the various stages of the virus life cycle in neural (N18) and nonneural (BHK) cells. In BHK cells, recombinant viruses 633 (E255Q) and TE (E255H) replicated similarly. In contrast, in N18 neuroblastoma cells, TE established infection more efficiently, replicated faster, and achieved higher rates of virus release than did 633. Viral structural protein synthesis was similar in 633- and TE-infected BHK cells, while in N18 cells, structural protein synthesis was detected only in TE-infected cells at 6 h and remained higher for at least 16 h postinfection. Viral RNA synthesis was initiated more rapidly and was up to fivefold greater in TE- versus 633-infected N18 cells. Taken together with other data demonstrating minimal effects on virus binding and entry (P. C. Tucker, S. H. Lee, N. Bui, D. Martinie, and D. E. Griffin, J. Virol. 71:6106-6112, 1997), these data suggest that E2 position 55 plays an important role at early stages of infection of neural cells, thereby facilitating neurovirulence.     

An adenovirus mRNA which encodes a 14,700-Mr protein that maps to the last open reading frame of region E3 is expressed during infection.

The E3 regions of adenovirus types 2 and 5, respectively, are known to synthesize proteins of 19,000 Mr (19K) and 11.6K, but information regarding the identity and characterization of other potential E3 proteins encoded by the six remaining open reading frames (ORFs) is lacking. In this study, we show that the last ORF of region E3, which encodes a 14.7K protein, is expressed in adenovirus-infected cells. This information was largely derived from analysis of an E3 deletion mutant (H2dl801) in which an extensive deletion (1,939 base pairs) was found to eliminate all ORFs except for two proteins of 12.5K and 14.7K. The 14.7K protein was translated from RNA isolated from H2dl801-infected cells that had been hybridization selected to E3 DNA; hybridization-selected RNA from wild-type adenovirus type 5-infected cells translated both the 19K and the 14.7K proteins. Moreover, an antiserum directed against a bacterial 14.7K fusion protein (A. E. Tollefson and W. S. M. Wold, J. Virol. 62:33-39, 1988) immunoprecipitated the 14.7K translation product synthesized by wild-type and mutant H2dl801 adenovirus mRNAs.     

Efficient recovery of the functional IP10-scFv fusion protein from inclusion bodies with an on-column refolding system

A functional IP10-scFv fusion protein retaining the antibody specificity for acidic isoferritin and chemokine function was produced at high level in Esherichia coli (E. coli). IP10-scFv gene from the recombinant plasmid pc3IP104c9 was subcloned into pET28a fused to N-terminal His-tag sequence in frame and overexpressed in E. coli BL21(DE3). With an on-column refolding procedure based on Ni-chelating chromatography, the active fusion protein was recovered efficiently from inclusion bodies with a refolding yield of approximate 45% confirmed by spectrophotometer. The activity of refolded IP10-scFv was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting and enzyme-linked immunosorbent assay. The results showed the fusion protein retains the specific binding activity to AIF with an affinity constant of 4.48x10(-8) M as well as the chemokine function of IP-10. The overall yield of IP10-scFv with bioactivity in E. coli flask culture was more than 40 mg/L.     

Identification of a specific region in the e1 fusion protein involved in zinc inhibition of semliki forest virus fusion

The enveloped alphaviruses infect cells via a low-pH-triggered membrane fusion reaction mediated by the viral transmembrane protein E1. During fusion, E1 inserts into the target membrane and refolds to a hairpin-like postfusion conformation in which domain III (DIII) and the juxtamembrane stem pack against a central core trimer. Although zinc has previously been shown to cause a striking block in alphavirus fusion with liposome target membranes, the mechanism of zinc's effect on the E1 fusion protein is not understood. Here we developed a cell culture system to study zinc inhibition of fusion and infection of the alphavirus Semliki Forest virus (SFV). Inclusion of 2 mM ZnCl(2) in the pH 5.75 fusion buffer caused a decrease of ～5 logs in SFV fusion at the plasma membrane. Fusion was also inhibited by nickel, a chemically related transition metal. Selection for SFV zinc resistance identified a key histidine residue, H333 on E1 DIII, while other conserved E1 histidine residues were not involved. An H333N mutation conferred resistance to both zinc and nickel, with properties in keeping with the known pH-dependent chelation of these metals by histidine. Biochemical studies demonstrated that zinc strongly inhibits formation of the postfusion E1 trimer in wild-type SFV but not in an H333 mutant. Together our results suggest that zinc acts by blocking the fold-back of DIII via its interaction with H333.     

Identification and characterization of the herpes simplex virus type 1 virion protein encoded by the UL35 open reading frame.

The UL35 open reading frame (ORF) of herpes simplex virus type 1 (HSV-1) has been predicted from DNA sequence analysis to encode a small polypeptide with a molecular weight of 12,095. We have investigated the protein product of the UL35 ORF by using a trpE-UL35 gene fusion to produce a corresponding fusion protein in Escherichia coli. The TrpE-UL35 chimeric protein was subsequently isolated and used as a source of immunogen for the production of rabbit polyclonal antiserum directed against the UL35 gene product. The TrpE-UL35 antiserum was found to recognize a 12-kDa protein which was specifically present in HSV-1-infected cells. By utilizing the TrpE-UL35 antiserum, the kinetics of synthesis of the UL35 gene product was examined, and these studies indicate that UL35 is expressed as a gamma 2 (true late) gene. The 12-kDa protein recognized by the TrpE-UL35 antiserum was associated with purified HSV-1 virions and type A and B capsids, suggesting that the UL35 ORF may encode the 12-kDa capsid protein variably designated p12, NC7, or VP26. To confirm this assignment, immunoprecipitation and immunoblotting studies were performed to demonstrate that the TrpE-UL35 antiserum reacts with the same polypeptide as an antiserum directed against the purified p12 capsid protein (anti-NC7) (G.H. Cohen, M. Ponce de Leon, H. Diggelmann, W.C. Lawrence, S.K. Vernon, and R.J. Eisenberg, J. Virol. 34:521-531, 1980). Furthermore, the anti-NC7 serum was also found to react with the TrpE-UL35 chimeric protein isolated from E. coli, providing additional evidence that the UL35 gene encodes p12. On the basis of these studies, we conclude that UL35 represents a true late gene which encodes the 12-kDa capsid protein of HSV-1.     

人B淋巴细胞刺激因子C端肽的免疫增强作用

用套式PCR从人胎脑cDNA文库中克隆了B淋巴细胞刺激因子C端肽 (C terminalpeptideofBlymphocytestimulator,C BLyS)的cDNA。在大肠杆菌BL2 1CodonPlus (DE3)RIL中以包含体形式表达了C BLyS。对包含体的复性条件进行了摸索 ,建立了C BLyS的透析复性与纯化方法。经复性和纯化的C BLyS可结合其受体B细胞成熟抗原 (Bcellmaturationantigen ,BCMA) 人IgG1Fc融合蛋白 ,刺激体外培养的小鼠脾脏细胞增殖 ,并且可明显增强小鼠对溶菌酶的免疫应答水平。