Nucleotide sequence of the herpes simplex virus type 2 thymidine kinase gene.

We have determined the complete nucleotide sequence of the thymidine kinase gene of herpes simplex virus (HSV) type 2 strain 333. The sequence of the thymidine kinase gene exhibits an open translational reading frame of 1,128 nucleotides encoding a protein of 376 amino acids. The DNA sequence was compared with that of the HSV type 1 thymidine kinase gene from strain MP (S. L. McKnight, Nucleic Acids Res. 8:5949-5964, 1980) and from strain CL 101 (M. J. Wagner, J. A. Sharp, and W. C. Summers, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445, 1981) to assess the extent of intra- and intertypic variation for one viral gene. The nucleotides encoding the structural gene varied 1.7% between the two HSV type 1 strains and 19% between HSV type 1 and HSV type 2, which translated to differences in the amino acid sequence of the two proteins of 1.9 and 27%, respectively. The DNA encoding the 5' regulatory sequences appeared to be more conserved than the DNA coding for the structural gene, and the DNA at the 3' end of the gene was the least homologous.     

Genetic analysis of temperature-sensitive mutants which define the gene for the major herpes simplex virus type 1 DNA-binding protein.

We have assigned eight temperature-sensitive mutants of herpes simplex virus type 1 to complementation group 1-1. Members of this group fail to complement mutants in herpes simplex virus type 2 complementation group 2-2. The mutation of one member of group 1-1, tsHA1 of strain mP, has been shown to map in or near the sequence which encodes the major herpes simplex virus type 1 DNA-binding protein (Conley et al., J. Virol. 37:191-206, 1981). The mutations of five other members of group 1-1 map in or near the sequence in which the tsHA1 mutation maps, a sequence which lies near the center of UL between the genes for the viral DNA polymerase and viral glycoprotein gAgB. These mutants can be divided into two groups; the mutations of one group map between coordinates 0.385 and 0.398, and the mutations of the other group map between coordinates 0.398 and 0.413. At the nonpermissive temperature mutants in group 1-1 are viral DNA negative, and mutant-infected cells fail to react with monoclonal antibody to the 130,000-dalton DNA-binding protein. Taken together, these data indicate that mutants in complementation groups 1-1 and 2-2 define the gene for the major herpes simplex virus DNA-binding protein, an early gene product required for viral DNA synthesis.     

Identification of proteins encoded by a fragment of herpes simplex virus type 2 DNA that has transforming activity.

Cloned BglII fragment N (map units 0.58 to 0.625) of herpes simplex virus type 2 DNA has been shown to transform rodent cells to an oncogenic phenotype (Galloway and McDougall, J. Virol. 38: 749-760, 1981). RNA homologous to this fragment directs the synthesis of five polypeptides in a cell-free translation system. The approximate molecular weights of these proteins are 140,000, 61,000, 56,000, 35,000, and 23,500. The 35,000-dalton protein is the major species late in infection and is the only species detected before the onset of viral DNA replication. The arrangement of the sequences encoding these proteins along the herpes simplex virus type 2 genome was determined by hybridization of the RNA to cloned PstI fragment of BglII-N and to single-stranded DNA segments cloned into M13mp7. Both the hybridization experiments and immunoprecipitation with monoclonal antibodies suggested that the 140,000- and 35,000-dalton proteins are at least partially colinear and share antigenic determinants.     

Transcription initiation sites and nucleotide sequence of a herpes simplex virus 1 gene conserved in the Epstein-Barr virus genome and reported to affect the transport of viral glycoproteins

Earlier reports have localized mutations which affect the processing and transport of herpes simplex virus 1 glycoproteins to a region located between the genes specifying glycoprotein B and the major viral DNA-binding protein (beta 8). The nucleotide sequence of this region contains a single long open reading frame encoding a 780-amino-acid protein with a predicted molecular weight of 83,845. To confirm the existence of this protein, rabbit polyclonal antibody was made against a synthetic peptide made according to the predicted sequence of a hydrophilic domain near the carboxy terminal of the protein. This antibody reacted with an infected cell protein of an apparent molecular weight of 95,500. We designated this protein infected cell protein 18.5 (ICP18.5). S1 nuclease analysis suggested that the 5.6-kilobase mRNA encoding ICP18.5 is initiated predominantly from one site, but three weaker initiation sites also seemed to occur within a 74-base-pair stretch of DNA. This gene appears to be conserved in the Epstein-Barr virus (EBV) genome, inasmuch as 174 of the 780 amino acids of ICP18.5 align with corresponding amino acids predicted by the EBV open reading frame BALF3. The EBV gene is located adjacent to the gene specifying a homolog of the herpes simplex virus 1 glycoprotein B.     

中国西藏小反刍兽疫病毒野生株China/Tib/Gej/07-30基质蛋白基因和融合蛋白基因的分子特征分析

对我国西藏小反刍兽疫病毒野生株China/Tib/Gej/07-30进行基质蛋白(M)和融合蛋白(F)基因序列测定,并进行分子生物学特征分析。首先应用逆转录聚合酶链式反应扩增出M和F基因片段,对聚合酶链式反应产物进行直接测序,然后对测定的核苷酸和推测的氨基酸序列进行比较分析。China/Tib/Gej/07-30的M基因由1483个核苷酸组成,编码335个氨基酸,与其他分离株核苷酸和氨基酸序列同源性分别为92.4%～97.7%和97.0%～98.2%。F基因由2411个核苷酸组成,编码546个氨基酸,与其他分离株核苷酸和氨基酸序列同源性分别为85.5%～96.1%和94.3%～98.2%。China/Tib/Gej/07-30的F蛋白含有信号肽序列和跨膜结构域,序列高度变异。F蛋白第104～108位和第109～133位氨基酸位点分别是高度保守的裂解位点和融合肽结构域。F蛋白还含有序列高度保守的三个七肽重复区。China/Tib/Gej/07-30的M基因3′端的非编码区(UTR)长度为443个核苷酸,GC含量高达68.4%,与其他PPRV毒株的同源性为82.4%～93.5%。China/Tib/Gej/07-30的F基因5′UTR区长度为634个核苷酸,GC含量高达70.0%,与其他PPRV毒株序列相似性为76.2%～91.7%。     

The herpes simplex virus 1 gene for ICP34.5, which maps in inverted repeats, is conserved in several limited-passage isolates but not in strain 17syn+.

In a previous study, it was reported that herpes simplex virus 1 (HSV-1) strain F contains a transcribed open reading frame situated in the inverted repeats of the L component between the terminal a sequence and the open reading frame that encodes the alpha 0 gene (J. Chou and B. Roizman, J. Virol. 57: 629-637, 1986). By means of an antibody to repeats of the trimer Ala-Thr-Pro predicted to be specified by the open reading frame, it was shown that the open reading frame specifies a protein (M. Ackermann, J. Chou, M. Sarmiento, R. A. Lerner, and B. Roizman, J. Virol. 58: 843-850, 1986). This open reading frame is absent from the reported sequence of HSV-1(17)syn+ (D. J. McGeoch, M. A. Dalrymple, A. J. Davison, A. Dolan, M. C. Frame, D. McNab, L. J. Perry, J. E. Scott, and P. Taylor, J. Gen. Virol. 69: 1531-1574, 1988; L. J. Perry and D. J. McGeoch, J. Gen. Virol. 69: 2831-2846, 1988). To define the extent of variability in this open reading frame, we compared the sequences of the ICP34.5-encoding open reading frames of the genomes of three strains characterized by limited passage in cell culture with that of the HSV-1(17)syn+ strain. Furthermore, to establish unambiguously that the antibody to the Ala-Thr-Pro repeats reacts with the product of this open reading frame, we inserted a short sequence that encodes a known epitope in frame at the 5' terminus of the coding domain. Our results indicate that with minor variations, the open reading frame is conserved in the three HSV-1 genomes analyzed but not in HSV-1(17)syn+. Thus, two strains contain an inserted amino acid and one strain, isolated from a case of human encephalitis, lacks a seven-amino-acid sequence. The recombinant virus carrying the foreign epitope expressed a slightly slower-migrating protein which reacted with both the rabbit polyclonal antibody to the Ala-Thr-Pro trimer repeats and the monoclonal antibody to the inserted epitope. The implications of the results are discussed.     

Extensive homology between the herpes simplex virus type 2 glycoprotein F gene and the herpes simplex virus type 1 glycoprotein C gene.

The region of the herpes simplex virus type 2 (HSV-2) genome which maps colinearly with the HSV-1 glycoprotein C (gC) gene has been cloned, and the DNA sequence of a 2.29-kilobase region has been determined. Contained within this sequence is a major open reading frame of 479 amino acids. The carboxyterminal three-fourths of the derived HSV-2 protein sequence showed a high degree of sequence homology to the HSV-1 gC amino acid sequence reported by Frink et al. (J. Virol. 45:634-647, 1983). The amino-terminal region of the HSV-2 sequence, however, showed very little sequence homology to HSV-1 gC. In addition, the HSV-1 gC sequence contained 27 amino acids in the amino-terminal region which were missing from the HSV-2 protein. Computer-assisted analysis of the hydrophilic and hydrophobic properties of the derived HSV-2 sequence demonstrated that the protein contained structures characteristic of membrane-bound glycoproteins, including an amino-terminal signal sequence and carboxy-terminal hydrophobic transmembrane domain and charged cytoplasmic anchor. The HSV-2 protein sequence also contained seven putative N-linked glycosylation sites. These data, in conjunction with mapping studies of Para et al. (J. Virol. 45:1223-1227, 1983) and Zezulak and Spear (J. Virol. 49:741-747, 1984), suggest that the protein sequence derived from the HSV-2 genome corresponds to gF, the HSV-2 homolog of HSV-1 gC.     

Identification and characterization of the herpes simplex virus type 2 gene encoding the essential capsid protein ICP32/VP19c.

We describe the characterization of the herpes simplex virus type 2 (HSV-2) gene encoding infected cell protein 32 (ICP32) and virion protein 19c (VP19c). We also demonstrate that the HSV-1 UL38/ORF.553 open reading frame (ORF), which has been shown to specify a viral protein essential for capsid formation (B. Pertuiset, M. Boccara, J. Cebrian, N. Berthelot, S. Chousterman, F. Puvian-Dutilleul, J. Sisman, and P. Sheldrick, J. Virol. 63: 2169-2179, 1989), must encode the cognate HSV type 1 (HSV-1) ICP32/VP19c protein. The region of the HSV-2 genome deduced to contain the gene specifying ICP32/VP19c was isolated and subcloned, and the nucleotide sequence of 2,158 base pairs of HSV-2 DNA mapping immediately upstream of the gene encoding the large subunit of the viral ribonucleotide reductase was determined. This region of the HSV-2 genome contains a large ORF capable of encoding two related 50,538- and 49,472-molecular-weight polypeptides. Direct evidence that this ORF encodes HSV-2 ICP32/VP19c was provided by immunoblotting experiments that utilized antisera directed against synthetic oligopeptides corresponding to internal portions of the predicted polypeptides encoded by the HSV-2 ORF or antisera directed against a TrpE/HSV-2 ORF fusion protein. The type-common immunoreactivity of the two antisera and comparison of the primary amino acid sequences of the predicted products of the HSV-2 ORF and the equivalent genomic region of HSV-1 provided evidence that the HSV-1 UL38 ORF encodes the HSV-1 ICP32/VP19c. Analysis of the expression of the HSV-1 and HSV-2 ICP32/VP19c cognate proteins indicated that there may be differences in their modes of synthesis. Comparison of the predicted structure of the HSV-2 ICP32/VP19c protein with the structures of related proteins encoded by other herpes viruses suggested that the internal capsid architecture of the herpes family of viruses varies substantially.     

A novel herpes simplex virus 1 gene, UL43.5, maps antisense to the UL43 gene and encodes a protein which colocalizes in nuclear structures with capsid proteins.

An open reading frame mapping antisense to the UL43 gene of herpes simplex virus 1 encodes a protein with an apparent Mr of 38,000. The protein was detected in wild-type-infected cells with rabbit monospecific polyclonal antibody directed against a fusion protein containing all of the sequences encoded by the open reading frame. The antibody did not react with mutants from which the open reading frame was deleted. Expression of this gene, designated UL43.5, was grossly decreased or abolished in infected cells incubated in medium containing inhibitory concentrations of phosphonoacetic acid, suggesting that it is regulated as a gamma gene. UL43.5 is dispensable in cell culture. UL43.5 protein colocalized with the major capsid protein (infected cell protein 5) and the capsid scaffolding proteins (infected cell protein 35) in nuclear structures situated at the periphery of the nucleus. The predicted amino acid sequence indicates that the UL43.5 protein is a highly hydrophilic protein. The colocalization of UL43.5 protein with capsid proteins in discrete nuclear structures suggests that the former may be involved in assembly of viral particles in an accessory role in cells in culture.     

UL27.5 Is a Novel γ2 Gene Antisense to the Herpes Simplex Virus 1 Gene Encoding Glycoprotein B

An antibody made against the herpes simplex virus 1 U_S5 gene predicted to encode glycoprotein J was found to react strongly with two proteins, one with an apparent M_r of 23,000 and mapping in the S component and one with a herpes simplex virus protein with an apparent M_r of 43,000. The antibody also reacted with herpes simplex virus type 2 proteins forming several bands with apparent M_rs ranging from 43,000 to 50,000. Mapping studies based on intertypic recombinants, analyses of deletion mutants, and ultimately, reaction of the antibody with a chimeric protein expressed by in-frame fusion of the glutathione S-transferase gene to an open reading frame antisense to the gene encoding glycoprotein B led to the definitive identification of the new open reading frame, designated U_L27.5. Sequence analyses indicate the conservation of a short amino acid sequence common to U_S5 and U_L27.5. The coding sequence of the herpes simplex virus U_L27.5 open reading frame is strongly homologous to the sequence encoding the carboxyl terminus of the herpes simplex virus 2 U_L27.5 sequence. However, both open reading frames could encode proteins predicted to be significantly larger than the mature U_L27.5 proteins accumulating in the infected cells, indicating that these are either processed posttranslationally or synthesized from alternate, nonmethionine-initiating codons. The U_L27.5 gene expression is blocked by phosphonoacetate, indicating that it is a γ₂ gene. The product accumulated predominantly in the cytoplasm. U_L27.5 is the third open reading frame found to map totally antisense to another gene and suggests that additional genes mapping antisense to known genes may exist.     

Herpes simplex virus type 1 Angelotti and a defective viral genotype: analysis of genome structures and genetic relatedness by DNA-DNA reassociation kinetics.

DNA-DNA reassociation kinetics of herpes simplex virus type 1 Angelotti DNA and a class of defective viral DNA revealed that the viral standard genome has a total sequence complexity of about 93 X 10(6) daltons and that a portion of 11 X 10(6) daltons occurs twice on the viral genome. These results agree with structural features of herpes simplex virus type 1 DNA derived from electron microscopic studies and restriction enzyme analyses by several investigators. The defective viral DNA (molecular weight, about 97 X 10(6)) displays a sequence complexity of about 11 X 10(6) daltons, suggesting that the molecule is built up by repetitions of standard DNA sequences comprising about 15,000 base pairs. A 2 X 10(6)-dalton portion of these sequences maps in the redundant region and a 9 X 10(6)-dalton portion maps in the unique part of the standard herpes simplex virus type 1 Angelotti DNA, as could be shown by reassociation of viral standard DNA in the presence of defective DNA and vice versa. No cellular DNA sequences could be detected in defective DNA. A 12% molar fraction of the defective DNA consists of highly repetitive sequences of about 350 to 500 base pairs in length.