Expression of the herpes simplex virus type-2 major DNA-binding protein (ICP8) gene in COS-1 cells

By the use of an SV40 origin of replication plasmid vector and the COS-1 cell system, expression of the gene encoding the herpes simplex virus type-2 (HSV-2) major DNA binding protein (ICP8) has been achieved. The HSV-2 4.5 kb BgIII 0 DNA fragment containing the ICP8 coding region was inserted into the plasmid vector pSVOd containing the SV40 origin of replication. Transfection of COS-1 cells by the resultant recombinant plasmid (pSVOd20), and subsequent multiplication of this plasmid, led to the production of the HSV-2 major DNA binding protein in sufficient quantities to allow its detection with either monoclonal or polyclonal antibodies as well as sera taken from HSV-2 patients.     

Characterization of the large tegument protein (ICP1/2) of herpes simplex virus type 1.

ICP1/2 (also designated VP1/2) is a 270-kDa structural protein of herpes simplex virus type 1 (HSV-1) which is located in the tegument region of the virion. In this report we describe the production of a polyclonal antiserum specific for ICP1/2 and the use of this antiserum to examine the synthesis, processing, and intracellular localization of the viral polypeptide. Pulse-labeling studies indicated that ICP1/2 is synthesized late during infection, being initially detectable between 8 and 9 hr postinfection with the rate of synthesis continuing to increase until 11 to 12 hr postinfection. Further studies on the expression of ICP1/2 in the presence or absence of viral DNA replication indicated that the synthesis of the polypeptide is absolutely dependent on viral DNA replication. These results suggest that ICP1/2 represents a gamma 2 (true late) gene product. Additionally, we have performed experiments to determine if ICP1/2 is post-translationally modified in HSV-infected cells. These studies indicated that ICP1/2 is phosphorylated on serine residues; however, we found no evidence to suggest that the protein is glycosylated. Using subcellular fractionation and indirect immunofluorescence techniques, we have determined that ICP1/2 is diffusely distributed throughout the nucleus and cytoplasm of HSV-infected cells with no specific compartmentalization of the polypeptide.     

Genomic sequences homologous to the protein kinase region of the bifunctional herpes simplex virus type 2 protein ICP10

The large subunit of herpes simplex virus type 2 (HSV-2) ribonucleotide reductase (ICP10) consists of two functional domains. The amino (N)-terminal domain at residues 1–411 has serine/threonine-specific kinase activity (PK domain) and is encoded by a DNA fragment with transforming potential ( 15,17). The remaining region is required for ribonucleotide reductase activity (RR domain) (14,15). Computer-assisted comparison of the ICP10 sequence to the EMBL database 21 has revealed sequences within the RR domain that are common to all RR1 proteins. Motifs homologous to the catalytic domains of all PKs were identified in the PK region (15). However, based on this database all other sequences were unique. Secondary structure analysis of the PK and RR junction region of ICP10 identified twist angle variations with helical periodicity characteristic of enhancer elements. Sequences homologous to a segment of the PK domain were amplified and cloned from human DNA using the polymerase chain reaction (PCR), suggesting that the PK domain may have originated from a cellular gene.     

The synthesis of the major DNA-binding protein (ICP8) in cells infected with the strain HSZP or KOS of herpes simplex virus type 1.

Synthesis of the major DNA-binding protein (ICP8) was investigated in primary rabbit kidney (RK) and Vero cells infected with the syncytial (syn) strain HSZP or with the non-syn strain KOS of herpes simplex virus type 1 (HSV-1). Results showed the following: 1. In contrast to strain KOS, the rate of viral polypeptide synthesis was accelerated in Vero cells infected with strain HSZP. The ICP8 could be detected in the nuclei of cells by one hour post-infection (hr p. i.) where it became associated with the viral DNA (DNase sensitive form). Later on (7 hr p.i.), the synthesis of viral polypeptides decreased and no further translocation of ICP8 from the cytoplasm into the nucleus was observed. 2. Strain HSZP was approx. three times more resistant to the action of phosphonoacetic acid (PAA) than strain KOS. In order to block the synthesis of HSZP gamma-2 polypeptides, a concentration of 600 micrograms PAA/ml had to be used. Under this condition, the HSZP ICP8 was translocated into the cell nucleus at later interval only (7 hr p.i.), and it was still possible to release this polypeptide from the nucleus by DNase treatment. The failure of the HSZP ICP8 to associate with the nuclear matrix (DNase resistant form) of infected cells in the absence of viral DNA replication may reflect its predominant affinity for the viral DNA which, in turn, may be responsible for the observed accelerated synthesis of the HSZP polypeptides in infected Vero cells. 3. In primary RK cells infected with strain HSZP the ICP8 did not translocate into the cell nucleus. Therefore, no gamma-2 polypeptides were synthesized.     

Isolation of high-molecular-weight infectious DNA from type 1 herpes simplex virus

Isolation of DNA from type 1 herpes simplex virus (strain L2) is described; the DNA possessed the characteristics of an intact molecule: sedimentation rate, physical length, and infectivity. Data on infectivity of preparations of this DNA were obtained in cultures of chick embryonic fibroblasts.D. I. Ivanovskii Institute of Virology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR V. D. Solov'ev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 1, pp. 26–28, January, 1977.     

C-terminal region of herpes simplex virus ICP8 protein needed for intranuclear localization

The herpes simplex virus single-stranded DNA-binding protein, ICP8, localizes initially to structures in the nucleus called prereplicative sites. As replication proceeds, these sites mature into large globular structures called replication compartments. The details of what signals or proteins are involved in the redistribution of viral and cellular proteins within the nucleus between prereplicative sites and replication compartments are poorly understood; however, we showed previously that the dominant-negative d105 ICP8 does not localize to prereplicative sites and prevents the localization of other viral proteins to prereplicative sites (J. Virol. 74 (2000) 10122). Within the residues deleted in d105 (1083 to 1168), we identified a region between amino acid residues 1080 and 1135 that was predicted by computer models to contain two alpha-helices, one with considerable amphipathic nature. We used site-specific and random mutagenesis techniques to identify residues or structures within this region that are required for proper ICP8 localization within the nucleus. Proline substitutions in the predicted helix generated ICP8 molecules that did not localize to prereplicative sites and acted as dominant-negative inhibitors. Other substitutions that altered the charged residues in the predicted alpha-helix to alanine or leucine residues had little or no effect on ICP8 intranuclear localization. The predicted alpha-helix was dispensable for the interaction of ICP8 with the U(L)9 origin-binding protein. We propose that this C-terminal alpha-helix is required for localization of ICP8 to prereplicative sites by binding viral or cellular factors that target or retain ICP8 at specific intranuclear sites.     

HSV-2 ICP34.5 protein modulates herpes simplex virus glycoprotein processing

The ICP34.5 gene from HSV-2 strain 333 was cloned and, when expressed in Vero cells, enhanced the efficiency and extent of glycoprotein processing of glycoprotein C (gC1), a representative viral glycoprotein, during infection with HSV-1 SP7. The ICP34.5 from HSV-1 SP7 limits the extent and efficiency of viral glycoprotein processing. The ability of the HSV-2 ICP34.5 protein to enhance the efficiency and extent of HSV-1 SP7 glycoprotein processing indicates that modulation of viral glycoprotein processing is also a property of the HSV-2 ICP34.5 protein.     

The herpes simplex virus type 2 equivalent of the herpes simplex virus type 1 US7 gene and its flanking sequences

Nucleotide sequencing studies (D. J. McGeoch, A. Dolan, S. Donald, and F. Rixon, 1985, J. Mol. Biol. 181, 1-14) have indicated that herpes simplex virus type 1 (HSV-1) has a coding sequence, referred to as US7, between the genes for the glycoproteins D and E (gD and gE). Northern blot analysis and nucleotide sequencing have been carried out to show that the type 2 virus (HSV-2) has an equivalent to the US7 gene. A comparison with the HSV-1 sequence has revealed some surprising similarities and differences. At the nucleotide level, HSV-2 has inserted a large sequence into the gE promoter, retained a large palindrome present in the coding sequence but not some tandem repeats, and deleted a region beside those repeats. At the amino acid level, the putative transmembrane sequence has been remarkably well conserved, and hydrophobic moment analysis indicates that it could be interacting with polar species within the plane of the membrane. Immediately after the deletion in the HSV-2 sequence, there is an N-glycosylation signal, and HSV-2 has one more such signal than HSV-1. The longest conserved sequence at the nucleotide level codes for a region of polypeptide that is strongly predicted to fold into alpha-helix. Implications of these analyses to the structure and possible function of these molecules are discussed.     

Some characteristics of an early protein (ICP 22) synthesized in cells infected with herpes simplex virus

In Vero cells incubated at 40 degrees C or treated with azetidine at 37 degrees C, synthesis of a polypeptide ('C') of apparent mol. wt. 66000 was stimulated. It was not phosphorylated and was found in the cytoplasmic fraction of cell lysates. In cells infected with herpes simplex virus type 1 (HSV-1) in the presence of azetidine, synthesis of cellular proteins, including polypeptide C, was suppressed and infected cell polypeptides ICP 4, 0, 22 and 27 (apparent mol. wt. 170000, 120000, 75000 and 60000, respectively) were made. All were phosphorylated and accumulated in the nucleus. Messenger RNA for the same four polypeptides was made in cells infected in the presence of cycloheximide. Thus, ICP 22 is distinct from cellular polypeptide C and is probably a virus-specific alpha polypeptide, although it differs from alpha ICP 4, 0 and 27 in that its rate of synthesis does not decline rapidly when later polypeptides are produced. It is modified after synthesis in at least two steps, the second of which may require a later virus-specific polypeptide. In cells infected with HSV-2 the synthesis of a polypeptide analogous to ICP 22 could not be detected.     

Identification and characterization of deoxyribonucleoprotein complexes containing the major DNA-binding protein of herpes simplex virus type 1

The stimulation of host cell DNA synthesis was studied in permissive human embryonic lung (HEL) cells and in nonpermissive rabbit kidney (RK) cells infected with human cytomegalovirus (HCMV). Host cell DNA synthesis was induced by HCMV infection in resting cells of both types. In permissive cultures the stimulation of cellular DNA synthesis was detectable mainly in those cells which had not become productively infected and in which virus antigens were not detectable. In abortively infected RK cells, on the other hand, stimulation of host cell DNA synthesis and the expression of virus antigens were detected in the same cells. Infection of actively growing permissive HEL cells resulted in a shutdown of cellular DNA synthesis beginning approximately 10 hr postinfection. Shutdown of cellular DNA synthesis also occurred when the infected cells were treated with phosphonoacetic acid and was thus classified as an "early" virus function. In actively growing, abortively infected RK cells, on the other hand, host cell DNA synthesis was not affected, indicating that the early virus function(s) responsible for inhibition of cellular DNA synthesis was not expressed in these cells. Virus-encoded DNA polymerase activity, another early virus gene function, was also not detected in these abortively infected cultures. In RK cells the cellular DNA synthesized as a result of infection was capable of undergoing at least one further round of replication, indicating that the HCMV gene expression which occurred in abortively infected RK cells was not lethal for these cells.     

Genetic identification of a portion of the herpes simplex virus ICP8 protein required for DNA-binding

The major DNA-binding protein or infected cell protein 8 (ICP8) encoded by herpes simplex virus exhibits multiple interactions with the cell nucleus in that it interacts with the host cell nuclear matrix and viral DNA molecules as sequential stages in its maturational process (M. P. Quinlan, L. B. Chen, and D. M. Knipe (1984), Cell 36, 857-868). To define the portion(s) of ICP8 required for DNA binding, we have fine-mapped and identified the sequence changes in mutant genes causing changes in the protein that affect DNA binding. These mutations lead to amino acid changes between residues 348 and 450 of ICP8. Construction of a mutant ICP8 gene specifically altered at residues 499 and 502 led to a gene product that was also defective in a nuclear function. Thus, at least part of the region of ICP8 from residues 348 to 450 is required for DNA binding by ICP8. This portion of the protein may be involved in binding to DNA or forming intermolecular contacts needed for cooperative DNA binding. If this region is directly involved in binding of the protein to DNA, the most likely structure predicted for this region involves folding of beta-strands to form a channel for binding to a nucleotide chain.     

Identification of disulfide-linked protein complexes in the nucleocapsids of herpes simplex virus type 2.

High molecular weight disulfide-linked protein complexes in herpes simplex virus type 2 (HSV-2) nucleocapsids were demonstrated by sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS-PAGE) analysis under nonreducing conditions. The appearance of these complexes was accompanied by a reduction in intensity of three major bands (p155, p50, and p40). The major disulfide-linked component (P350) was shown to contain p155 and p50 by diagonal gel electrophoresis. However, we were unable to resolve distinct disulfide-linked species possessing p40 by either SDS-PAGE or gel filtration in the presence of guanidine hydrochoride (GuHCl).     

Monoclonal antibodies to herpes simplex virus type 1 proteins, including the immediate-early protein ICP 4.

Monoclonal antibodies were prepared against herpes simplex virus type 1 (strain 14012) by two immunization procedures. Procedure A utilized infectious virus propagated in mouse cells, and procedure B utilized mouse cells infected with herpes simplex virus in the presence of cycloheximide and harvested 1 h after removal of the inhibitor. A total of 52 monoclonal antibodies were obtained against 10 herpes simplex virus proteins, including four glycosylated proteins (a 110,000-molecular-weight protein, gB, gC, and gD) and six nonglycosylated proteins (a 68,000-molecular-weight protein, ICP 9, ICP 8, ICP 6, ICP 5, and the immediate-early ICP 4). The antibodies were assayed against herpes simplex virus types 1 and 2 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioimmunoprecipitates, immunofluorescence, and neutralization. Using the reagents prepared, we concluded that the 110,000-molecular-weight protein, gD, ICP 9, ICP 9, ICP 6, and the 68,000-molecular-weight protein express both type-specific and cross-reactive antigenic determinants. In contrast, nine antibodies against gB all cross-reacted with herpes simplex virus type 2, whereas eight antibodies to gC all reacted type specifically.     

Sequence analysis of the bovine herpesvirus type 1 genes homologous to the DNA polymerase (UL30), the major DNA-binding protein (UL29) and ICP18.5 assembly protein (UL28) genes of herpes simplex virus

Summary. The nucleotide sequence of a 10.5 kb region (map position 0.332 to 0.410) of bovine herpesvirus type 1 (BHV-1) was determined. This region contained three open reading frames (ORFs) homologous to herpes simplex virus DNA polymerase catalytic subunit (DNApol, UL30), major DNA-binding protein (MDBP, UL29) and ICP18.5 assembly protein (ICP18.5, UL28). The BHV-1 DNApol, MDBP and ICP18.5 ORFs were 1 246, 1 203 and 826 amino acids long with a calculated molecular mass of 134.2 kDa, 124.4 kDa and 86.9 kDa, respectively. They showed a high homology with alphaherpesvirus homologs despite large differences in the G+C content of the UL30-UL28 segment ranging from 44.4% for varicella zoster virus to 71.5% for BHV-1. Particularly well conserved among Alphaherpesvirinae are the putative functional domains of the DNApol and MDBP proteins which are discussed. Phylogenetic analysis revealed that BHV-1 clustered in the Varicellovirus genus with the animal D-type viruses. In this group, the BHV-1 position was shown to vary according to the investigated genes. Indeed, pseudorabies virus clustered with BHV-1 in the DNApol tree but with equine herpesvirus 1 in the ICP18.5 tree. Received May 16, 1996 Accepted August 8, 1996     

Identification of proteins tightly bound to herpes simplex virus DNA

Proteins bound tightly but noncovalently to highly purified herpes simplex virus DNA were radioiodinated in vitro by reaction with Bolton-Hunter reagent. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, four protein bands were seen in autoradiographs of the dried gel. These four proteins had molecular weights of approximately 71K, 50K, 20K, and 16K, respectively.     

单纯疱疹病毒Ⅱ型ICP27对Vero细胞凋亡作用的研究

目的 探讨单纯疱疹病毒Ⅱ型转录前蛋白ICP27对Vero细胞凋亡作用及其参与细胞凋亡的可能途径.方法 用前期构建成功的pcDNA3.0-ICP27质粒瞬时转染Vero细胞,MTT法检测pcDNA3.0-ICP27转染Vero细胞的活性,Giemsa染色及流式细胞术检测转染后ICP27在细胞凋亡中的作用,实时荧光定量PCR检测Bax、Bcl-2 mRNAs的变化和对凋亡执行蛋白Caspase-3的活性的检测以探讨ICP27参与细胞凋亡的途径.结果 pcDNA3.0-ICP27转染Vero细胞后,MTT法检测发现其活性明显低于转染pcDNA3.0及空白转染的Vero细胞活性;Giemsa染色后发现转染pcDNA3.0-ICP27的Vero细胞变圆,细胞核破裂,流式细胞检测发现pcDNA3.0-ICP27转染的Vero细胞发生的凋亡率更高.凋亡执行蛋白Caspase-3的活性明显高于转染pcDNA3.0及空白对照;荧光定量PCR检测Bax、Bcl-2 mRNAs的变化,转染重组质粒的Vero细胞中Bax mRNAs表达量上调,Bcl-2 mRNAs表达降低,Bax/Bcl-2明显高于正常培养的Vero细胞及转染pcDNA3.0质粒的Vero细胞.结论 ICP27对Vero细胞有促进细胞凋亡的作用.而这种作用可能是使Bax及Bcl-2在细胞中的比例发生变化而导致的.     

Myristylation and polylysine-mediated activation of the protein kinase domain of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10)

The amino-terminal domain of the large subunit of herpes simplex virus type 2 (HSV-2) ribonucleotide reductase (ICP10) was previously shown to possess protein kinase (PK) activity that localizes to the cytosolic, cytoskeletal, and plasma membrane fractions. Further studies of the PK domain using computer-assisted sequence analysis have identified a single transmembrane segment and fatty acid incorporation findings indicate that ICP10 is myristylated. Myristylation does not depend on a viral enzyme, since myristic acid is incorporated into ICP10 precipitated from cells transfected with an ICP10 expression vector. It is also incorporated into the 57-kDa protein expressed by the amino-terminal PK expression vector. The myristyl moiety is linked through an amide bond. The basic protein polylysine stimulates the kinase activity and alters its divalent cation requirements resulting in 20- to 40-fold stimulation in the presence of 0.1 mM Mn2+. The PK activity is inhibited by antibody to synthetic peptides corresponding to residues 355-369 and 13-26, respectively, located within, and amino-terminal to, the predicted PK catalytic domain.