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.     

非洲猪瘟病毒j5R膜蛋白的电脑预测和实验证实

蛋白多肽二级结构的电脑预测表明,非洲猪瘟病毒（ Ａｆｒｉｃａｎ ｓｗｉｎｅ ｆｅｖｅｒ ｖｉｒｕｓ , Ａ Ｓ Ｆ Ｖ）ｊ５ Ｒ阅读框编码１２ ．９ ｋ Ｄａ 膜蛋白。该蛋白的 Ｃ 末端含有一个潜在抗原决定簇,针对其合成肽的抗体能在 Ａ Ｓ Ｆ Ｖ 感染细胞和病毒颗粒中检测到２３ 或２５ ｋ Ｄａ（ 取决于不同毒株） 特异蛋白。免疫荧光试验显示,ｊ５ Ｒ 蛋白主要位于感染细胞的病毒复制部位。油水两相分离和细胞分级分离试验结果证明ｊ５ Ｒ 蛋白是膜相关蛋白     

African swine fever virus encodes a serine protein kinase which is packaged into virions.

Nucleotide sequencing of the SalI j region of the virulent Malawi (LIL20/1) strain of African swine fever virus (ASFV) identified an open reading frame (ORF), designated j9L, with extensive similarity to the family of protein kinases. This ORF encodes a 35.1-kDa protein of 299 amino acids which shares 24.6% amino acid identity with the human pim-1 proto-oncogene and 21.0% identity with the vaccinia virus B1R-encoded protein kinase. The ASFV ORF contains the motifs characteristic of serine-threonine protein kinases, with the exception of the presumed ATP-binding site, which is poorly conserved. The ORF was expressed to high levels in Escherichia coli, and the recombinant enzyme phosphorylated a calf thymus histone protein on serine residues in vitro. An antibody raised to an amino-terminal peptide of the ASFV protein kinase was reactive with the recombinant protein in Western immunoblot analyses and was used to demonstrate the presence of the protein kinase in ASF virions.     

The UL49.5 gene of pseudorabies virus codes for an O-glycosylated structural protein of the viral envelope.

Sequence analysis of BamHI fragment 1 of the pseudorabies virus (PrV) genome identified a novel PrV gene located upstream of the UL50 gene encoding PrV dUTPase. The deduced protein product displayed homology to the product of the herpes simplex virus type 1 UL49.5 protein. The predicted PrV UL49.5 protein consists of 98 amino acids with a calculated molecular mass of 10,155 Da. It contains putative signal peptide and transmembrane domains but lacks a consensus sequence for N glycosylation. PrV UL49.5 was expressed as a fusion protein with glutathione S-transferase in Escherichia coli, and a rabbit antiserum was generated. In Western blots (immunoblots) of purified virions, the antiserum detected a protein with an apparent molecular mass of 14 kDa. After fractionation of the virions, the 14-kDa protein was detected in the envelope fraction. Localization of the UL49.5 protein in the viral envelope was confirmed by immunoelectron microscopy. The treatment of purified virions with glycosidases led to a reduction of the apparent molecular mass in Western blots by approximately 2 kDa following digestion with neuraminidase and O-glycosidase. Our results demonstrate that the PrV UL49.5 protein is an O-glycosylated structural component of the viral envelope. It represents the 10th PrV glycoprotein described. According to the unified nomenclature for alphaherpesvirus glycoproteins, we propose to designate it glycoprotein N (gN).     

Proteins encoded by open reading frames 3 and 4 of the genome of Lelystad virus (Arteriviridae) are structural proteins of the virion.

Four structural proteins of Lelystad virus (Arteriviridae) were recognized by monoclonal antibodies in a Western immunoblotting experiment with purified virus. In addition to the 18-kDa integral membrane protein M and the 15-kDa nucleocapsid protein N, two new structural proteins with molecular masses of 45 to 50 kDa and 31 to 35 kDa, respectively, were detected. Monoclonal antibodies that recognized proteins of 45 to 50 kDa and 31 to 35 kDa immunoprecipitated similar proteins expressed from open reading frames (ORFs) 3 and 4 in baculovirus recombinants, respectively. Therefore, the 45- to 50-kDa protein is encoded by ORF3 and the 31- to 35-kDa protein is encoded by ORF4. Peptide-N-glycosidase F digestion of purified virus reduced the 45- to 50-kDa and 31- to 35-kDa proteins to core proteins of 29 and 16 kDa, respectively, which indicates N glycosylation of these proteins in the virion. Monoclonal antibodies specific for the 31- to 35-kDa protein neutralized Lelystad virus, which indicates that at least part of this protein is exposed at the virion surface. We propose that the 45- to 50-kDa and 31- to 35-kDa structural proteins of Lelystad virus be named GP3 and GP4, to reflect their glycosylation and the ORFs from which they are expressed. Antibodies specific for GP3 and GP4 were detected by a Western immunoblotting assay in swine serum after an infection with Lelystad virus.     

非洲猪瘟病毒CP204L基因的原核表达与单抗制备

由非洲猪瘟病毒(ASFV)引起的非洲猪瘟(ASF)给我国养猪业带来了不可估量的经济损失,严重阻碍了我国养猪业的发展,研发ASFV快速诊断试剂是目前最重要的内容之一。CP204L基因编码ASFV结构蛋白p30。本研究以克隆ASFV的CP204L基因为基础,通过基因重组技术,加入His标签,将构建的重组质粒命名为pET-28a-CP204L。将重组质粒转化至大肠杆菌BL21(DE3)感受态细胞,37℃经1mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达6h,表达蛋白进行SDS-PAGE鉴定和Western Blot检测。重组蛋白纯化后免疫小鼠制备筛选单克隆抗体,Western Blot和IFA验证单抗的结合特异性。结果表明,重组的pET-28a-CP204L诱导后表达蛋白为30kD,以不可溶性包涵体形式存在;表达蛋白利用His标签进行纯化,获得纯化蛋白2mg,单克隆抗体筛选获得5株IgG亚型的ASFV p30蛋白的单抗,且均具有良好的结合活性。本研究为发展ASFV检测方法提供了基础。     

Identification, subviral localization, and functional characterization of the pseudorabies virus UL17 protein

Homologs of the UL17 gene of the alphaherpesvirus herpes simplex virus 1 (HSV-1) are conserved in all three subfamilies of herpesviruses. However, only the HSV-1 protein has so far been characterized in any detail. To analyze UL17 of pseudorabies virus (PrV) the complete 597-amino-acid protein was expressed in Escherichia coli and used for rabbit immunization. The antiserum recognized a 64-kDa protein in PrV-infected cell lysates and purified virions, identifying PrV UL17 as a structural virion component. In indirect immunofluorescence analyses of PrV-infected cells the protein was predominantly found in the nucleus. In electron microscopic studies after immunogold labeling of negatively stained purified virion preparations, UL17-specific label was detected on single, mostly damaged capsids, whereas complete virions and the majority of capsids were free of label. In ultrathin sections of infected cells, label was primarily found dispersed around scaffold-containing B-capsids, whereas on DNA-filled C-capsids it was located in the center. Empty intranuclear A-capsids were free of label, as were extracellular capsid-less L-particles. Functional characterization of PrV-DeltaUL17F, a deletion mutant lacking codons 23 to 444, demonstrated that cleavage of viral DNA into unit-length genomes was inhibited in the absence of UL17. In electron microscopic analyses of PrV-DeltaUL17F-infected RK13 cells, DNA-containing capsids were not detected, while numerous capsidless L-particles were observed. In summary, our data indicate that the PrV UL17 protein is an internal nucleocapsid protein necessary for DNA cleavage and packaging but suggest that the protein is not a prominent part of the tegument.     

Characterization of a pre-S polypeptide on the surfaces of infectious avian hepadnavirus particles.

DNA from the pre-S region of the duck hepatitis B virus (DHBV) genome was inserted into an open reading frame vector designed to give high-level expression in Escherichia coli. The resulting fusion protein contained the first 8 amino acids of beta-galactosidase, 86 amino acids of the DHBV pre-S region, and 219 amino acids of chloramphenicol acetyltransferase at the C terminus (beta-gal:pre-S:CAT). Rabbit antiserum against purified beta-gal:pre-S:CAT was used to identify pre-S-containing polypeptides in DHBV particles by Western blotting. A dominant species of 36 kilodaltons (kDa) was identified. Antiserum against the major 17-kDa DHBsAg polypeptide also reacted with the 36-kDa protein. This suggests that the DHBV envelope gene polypeptides share the same carboxyl terminus, but differ in the sites from which translation is initiated. N-linked carbohydrate was not detected on either the 17- or 36-kDa envelope proteins. Anti-beta-gal:pre-S:CAT abolished infectivity of the virus in an in vitro assay. Thus, the pre-S region is exposed on the surfaces of infectious virions and may be directly involved in binding of virus to host-cell receptors.     

A Virally Encoded Chaperone Specialized for Folding of the Major Capsid Protein of African Swine Fever Virus

It is generally believed that cellular chaperones facilitate the folding of virus capsid proteins, or that capsid proteins fold spontaneously. Here we show that p73, the major capsid protein of African swine fever virus (ASFV) failed to fold and aggregated when expressed alone in cells. This demonstrated that cellular chaperones were unable to aid the folding of p73 and suggested that ASFV may encode a chaperone. An 80-kDa protein encoded by ASFV, termed the capsid-associated protein (CAP) 80, bound to the newly synthesized capsid protein in infected cells. The 80-kDa protein was released following conformational maturation of p73 and dissociated before capsid assembly. Coexpression of the 80-kDa protein with p73 prevented aggregation and allowed the capsid protein to fold with kinetics identical to those seen in infected cells. CAP80 is, therefore, a virally encoded chaperone that facilitates capsid protein folding by masking domains exposed by the newly synthesized capsid protein, which are susceptible to aggregation, but cannot be accommodated by host chaperones. It is likely that these domains are ultimately buried when newly synthesized capsid proteins are added to the growing capsid shell.     

A ubiquitin conjugating enzyme encoded by African swine fever virus.

The post-translational modification of proteins by covalent attachment of ubiquitin occurs in all eukaryotes by a multi-step process. A family of E2 or ubiquitin conjugating (UBC) enzymes catalyse one step of this process and these have been implicated in several diverse regulatory functions. We report here the sequence of a gene encoded by African swine fever virus (ASFV) which has high homology with UBC enzymes. This ASFV encoded enzyme has UBC activity when expressed in Escherichia coli since it forms thiolester bonds with [125I]ubiquitin in the presence of purified ubiquitin activating enzyme (E1) and ATP, and subsequently transfers [125I]ubiquitin to specific protein substrates. These substrates include histones, ubiquitin and the UBC enzyme itself. The ASFV encoded UBC enzyme is similar in structure and enzyme activity to the yeast ubiquitin conjugating enzymes UBC2 and UBC3. This is the first report of a virus encoding a functionally active UBC enzyme and provides an example of the exploitation of host regulatory mechanisms by viruses.     

Identification and characterization of a filament-associated protein encoded by Amsacta moorei entomopoxvirus.

A novel protein which is expressed at high levels in insect cells infected with Amsacta moorei entomopoxvirus was identified by our laboratory. This viral gene product migrates as a 25/27-kDa doublet when subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. It is expressed at late times of infection and is present in infected cells but is absent in purified extracellular virions and occlusion bodies. The gene encoding this polypeptide was mapped on the viral genome, and cDNA clones were generated and sequenced. The predicted protein was shown to be phosphorylated and contained an unusual 10-unit proline-glutamic acid repeat element. A polyclonal antiserum was produced against a recombinant form of the protein expressed in Escherichia coli, and a monoclonal antibody which reacted with the proline-glutamic acid motif was also identified. Immunofluorescence and immunoelectron microscopy techniques revealed that this protein is associated with large cytoplasmic fibrils which accumulate in the cytoplasm between 96 and 120 h postinfection. We subsequently called this viral polypeptide filament-associated late protein of entomopoxvirus. The fibrils containing this polypeptide are closely associated with occlusion bodies and may play a role in their morphogenesis and maturation.     

African swine fever virus protease, a new viral member of the SUMO-1-specific protease family

African swine fever virus (ASFV) is a complex DNA virus that employs polyprotein processing at Gly-Gly-Xaa sites as a strategy to produce several major core components of the viral particle. The virus gene S273R encodes a 31-kDa protein that contains a "core domain" with the conserved catalytic residues characteristic of SUMO-1-specific proteases and the adenovirus protease. Using a COS cell expression system, it was found that protein pS273R is capable of cleaving the viral polyproteins pp62 and pp220 in a specific way giving rise to the same intermediates and mature products as those produced in ASFV-infected cells. Furthermore, protein pS273R, like adenovirus protease and SUMO-1-specific enzymes, is a cysteine protease, because its activity is abolished by mutation of the predicted catalytic histidine and cysteine residues and is inhibited by sulfhydryl-blocking reagents. Protein pS273R is expressed late after infection and is localized in the cytoplasmic viral factories, where it is found associated with virus precursors and mature virions. In the virions, the protein is present in the core shell, a domain where the products of the viral polyproteins are also located. The identification of the ASFV protease will allow a better understanding of the role of polyprotein processing in virus assembly and may contribute to our knowledge of the emerging family of SUMO-1-specific proteases.     

The 1,629-nucleotide open reading frame located downstream of the Autographa californica nuclear polyhedrosis virus polyhedrin gene encodes a nucleocapsid-associated phosphoprotein.

A 78-kDa protein was produced in bacteria from a clone of the 1,629-nucleotide open reading frame located immediately downstream from the polyhedrin gene of Autographa californica nuclear polyhedrosis virus. The identity of this protein was confirmed by its reactivity with peptide antiserum and amino terminal peptide sequencing after purification from transformed bacteria. The polypeptide was used to produce polyclonal antisera in rabbits. Immunoblot analysis of insect cells infected with the baculovirus indicated that two related proteins with molecular masses of 78 and 83 kDa were synthesized late in infection. Biochemical fractionation studies indicated that both of these proteins were present in purified nucleocapsids from budded and occluded virus preparations. Immunoprecipitation of 32P-labeled proteins and treatment of purified nucleocapsids with alkaline phosphatase demonstrated that the 83-kDa protein was a phosphorylated derivative of the 78-kDa protein. Furthermore, immunoelectron microscopy revealed that the proteins were localized to regions of nucleocapsid assembly within the infected cell and appeared to be associated with the end structures of mature nucleocapsids.     

Pseudorabies virus UL36 tegument protein physically interacts with the UL37 protein

The UL36 open reading frame encoding the tegument protein ICP1/2 represents the largest open reading frame in the genome of herpes simplex virus type 1 (HSV-1). Polypeptides homologous to the HSV-1 UL36 protein are present in all subfamilies of HERPESVIRIDAE: We sequenced the UL36 gene of the alphaherpesvirus pseudorabies virus (PrV) and prepared a monospecific polyclonal rabbit antiserum against a bacterial glutathione S-transferase (GST)-UL36 fusion protein for identification of the protein. The antiserum detected a >300-kDa protein in PrV-infected cells and in purified virions. Interestingly, in coprecipitation analyses using radiolabeled infected-cell extracts, the anti-UL36 serum reproducibly coprecipitated the UL37 tegument protein, and antiserum directed against the UL37 protein coprecipitated the UL36 protein. This physical interaction could be verified using yeast two-hybrid analysis which demonstrated that the UL37 protein interacts with a defined region within the amino-terminal part of the UL36 protein. By use of immunogold labeling, capsids which accumulate in the cytoplasm in the absence of the UL37 protein (B. G. Klupp, H. Granzow, E. Mundt, and T. C. Mettenleiter, J. Virol. 75:8927-8936, 2001) as well as wild-type intracytoplasmic and extracellular virions were decorated by the anti-UL36 antiserum, whereas perinuclear primary enveloped virions were not. We postulate that the physical interaction of the UL36 protein, which presumably constitutes the innermost layer of the tegument (Z. Zhou, D. Chen, J. Jakana, F. J. Rixon, and W. Chiu, J. Virol. 73:3210-3218, 1999), with the UL37 protein is an important early step in tegumentation during virion morphogenesis in the cytoplasm.     

African Swine Fever Virus Structural Protein pE120R Is Essential for Virus Transport from Assembly Sites to Plasma Membrane but Not for Infectivity

This report examines the role of African swine fever virus (ASFV) structural protein pE120R in virus replication. Immunoelectron microscopy revealed that protein pE120R localizes at the surface of the intracellular virions. Consistent with this, coimmunoprecipitation assays showed that protein pE120R binds to the major capsid protein p72. Moreover, it was found that, in cells infected with an ASFV recombinant that inducibly expresses protein p72, the incorporation of pE120R into the virus particle is dependent on p72 expression. Protein pE120R was also studied using an ASFV recombinant in which E120R gene expression is regulated by the Escherichia coli lac repressor-operator system. In the absence of inducer, pE120R expression was reduced about 100-fold compared to that obtained with the parental virus or the recombinant virus grown under permissive conditions. One-step virus growth curves showed that, under conditions that repress pE120R expression, the titer of intracellular progeny was similar to the total virus yield obtained under permissive conditions, whereas the extracellular virus yield was about 100-fold lower than in control infections. Immunofluorescence and electron microscopy demonstrated that, under restrictive conditions, intracellular mature virions are properly assembled but remain confined to the replication areas. Altogether, these results indicate that pE120R is necessary for virus dissemination but not for virus infectivity. The data also suggest that protein pE120R might be involved in the microtubule-mediated transport of ASFV particles from the viral factories to the plasma membrane.     

Molecular cloning and expression of key gene encoding hypothetical DNA polymerase from B. mori parvo-like virus

BmPLV-Z is the abbreviation for Bombyx mori parvo-like virus (China isolate). This is a novel virus with two single-stranded linear DNA molecules, viz., VD1 (6543 bp) and VD2 (6022 bp), which are encapsidated respectively into separate virions. Analysis of the deduced amino acid sequence of VD1-ORF4 indicated the existence of a putative DNA-polymerase with exonuclease activity, possibly involved in the replication of BmPLV-Z. In the present study, a recombinant baculovirus was constructed to express the full length of the protein encoded by the VD1-ORF4 gene (3318 bp). In addition, a 2163-bp fragment amplified from the very same gene was cloned into prokaryotic expression vector pET-30a and expressed in E.coli Rosetta 2 (DE3) pLysS. The expressed fusion protein was employed to immunize New Zealand white rabbits for the production of an antiserum, afterwards used for examining the expression of the protein encoded by VD1-ORF4 gene in Sf-9 cells infected with recombinant baculovirus. Western blot analysis of extracts from thus cells infected revealed a specific band of about 120 kDa, thereby indicating that the full length protein encoded by the VD1-ORF4 gene had been successfully and stably expressed in Sf-9 cells.     

口蹄疫病毒P1基因在大肠杆菌中的高效表达及其生物活性的初步分析

将口蹄疫病毒 (FMDV)结构蛋白基因P1的完整cDNA序列插入原核表达性载体pGEX KG中 ,使P1基因与GST融合 ,获得融合表达质粒pKG P1,转化E .coliBL₂₁ (DE3) ,经IPTG诱导 ,SDS PADE结果表明GST P1融合蛋白获得高效表达 ,Western blot检测证实表达的融合蛋白具有免疫学活性 ,表达产物主要存在于细菌裂解液上清中。进一步采用GST纯化试剂盒纯化P1蛋白并作为诊断抗原 ,建立了P1 ELISA诊断方法 ,与FMD间接血凝 (IHA)检测方法平行检测 86 4份血清样品 ,总的符合率达87%。     

人免疫缺陷病毒早期蛋白Nef的表达、纯化及免疫原性研究

目的：通过原核细胞表达人免疫缺陷病毒（HIV）Nef抗原,制备特异抗血清,为Nef抗原检测提供技术方法。方法：以HIVBotswana毒株基因组为模板,用PCR法获得Nef蛋白编码基因,将其克隆到pET30a载体中,在大肠杆菌中表达Nef融合蛋白;用纯化的融合蛋白免疫BALB／c小鼠获得抗血清,用真核表达的Nef抗原对其特异性进行分析。结果：构建的Nef融合基因在大肠杆菌中获得表达,相对分子质量约为36x103,免疫BALB／c小鼠获得针对融合蛋白的高效价抗血清,ELISA抗体滴度为1：6400;免疫荧光和Westemblot检测表明,该抗血清能特异地与重组痘苗病毒表达的Nef抗原反应。结论：在大肠杆菌中表达了HIVNef融合蛋白,制备了Nef融合蛋白的高效价小鼠免疫血清,该血清能特异性识别HIVNef抗原,为HlVNef抗原检测提供了技术方法。     

Vaccinia virus E2L null mutants exhibit a major reduction in extracellular virion formation and virus spread

The vaccinia virus E2L (VACWR058) gene is conserved in all sequenced chordopoxviruses and is predicted to encode an 86-kDa protein with no recognizable functional motifs or nonpoxvirus homologs. Although the region immediately upstream of the open reading frame lacked optimal consensus promoter motifs, expression of the E2 protein occurred after viral DNA replication. Transfection studies, however, indicated that the promoter was weak compared to well-characterized intermediate and late promoters. The E2 protein was present in mature virions purified from infected cells but was more abundant in extracellular enveloped forms. Despite the conservation of the E2L gene in chordopoxviruses, deletion mutants could be isolated from both the WR and IHD-J strains of vaccinia virus. These null mutants produced very small plaques in all cell lines tested, reduced amounts of mature infectious virions, and very low numbers of extracellular virions. Nevertheless, viral protein synthesis appeared qualitatively and quantitatively normal. The defect in extracellular virus formation was corroborated by electron microscopy, which also showed some aberration in the wrapping of virions by cisternal membranes. Extracellular virions that did form, however, were able to induce actin tail formation.