Molecular Characterization of the Duck Enteritis Virus UL4 Gene

Duck enteritis virus (DEV) is a herpesvirus that causes an acute, contagious and fatal disease. In the present article, the DEV UL4 gene was cloned and sequenced from a vaccine virus. A degenerate oligonucleotide primer for the consensus site of herpesvirus UL3 gene and a specific primer located in UL5 were used in the polymerase chain reaction (PCR) to amplify a DNA product 2 086 bp in size. DNA sequence analysis revealed that a 714 bp open reading frame (ORF) of DEV encoding a 237 amino acid polypeptide is homologous to the family of herpesvirus UL4 proteins and therefore has been characterized as a DEV UL4 gene. Alignment of the DEV UL4 protein sequence with those of other alphaherpesviruses showed that 10 amino acid residues are completely conserved. Phylogenetic tree analysis showed that the seventeen alphaherpesviruses viruses analyzed were classified into four large groups, and the duck enteritis virus branched separately, closely related to the Mardiviruses group comprising Gallid herpesvirus 2 (GaHV-2), Gallid herpesvirus 3 (GaHV-3) and Meleagrid herpesvirus 1 (MeHV-1). The present study showed that the evolutionary relationship of the UL4 protein could be used for classification of alphaherpesviruses.     

Molecular characterization of the duck enteritis virus <Emphasis Type="Italic">UL4</Emphasis> gene

Duck enteritis virus (DEV) is a herpesvirus that causes an acute, contagious and fatal disease. In the present article, the DEV UL4 gene was cloned and sequenced from a vaccine virus. A degenerate oligonucleotide primer for the consensus site of herpesvirus UL3 gene and a specific primer located in UL5 were used in the polymerase chain reaction (PCR) to amplify a DNA product 2 086 bp in size. DNA sequence analysis revealed that a 714 bp open reading frame (ORF) of DEV encoding a 237 amino acid polypeptide is homologous to the family of herpesvirus UL4 proteins and therefore has been characterized as a DEV UL4 gene. Alignment of the DEV UL4 protein sequence with those of other alphaherpesviruses showed that 10 amino acid residues are completely conserved. Phylogenetic tree analysis showed that the seventeen alphaherpesviruses viruses analyzed were classified into four large groups, and the duck enteritis virus branched separately, closely related to the Mardiviruses group comprising Gallid herpesvirus 2 (GaHV-2), Gallid herpesvirus 3 (GaHV-3) and Meleagrid herpesvirus 1 (MeHV-1). The present study showed that the evolutionary relationship of the UL4 protein could be used for classification of alphaherpesviruses.      

鸭肠炎病毒UL6基因的分子特征

为了研究鸭肠炎病毒(Duck enteritis virus,DEV)UL6的特征,以DEV Clone-03株基因组DNA为模板,用靶基因步移PCR方法扩增得到UL6基因区域2534 bp的DNA片段。结果发现,DEV Clone-03 UL6基因由2373个核苷酸组成,编码一条790个氨基酸残基组成的多肽。与14株已报道的具有全UL6同源基因序列的疱疹病毒参考株进行比较,DEV Clone-03株与α疱疹病毒的核苷酸和氨基酸同源性较之与β疱疹病毒和γ疱疹病毒要高。氨基酸序列比较显示,DEV Clone-03 UL6基因具有α疱疹病毒UL6同源基因5个保守区域。UL6基因推导的氨基酸系统发育进化树分析发现,DEV Clone-03与α疱疹病毒属一个群,在α疱疹病毒中与马立克氏病病毒(Marek′sdisease herpesvirus,MDV)更接近。同时,序列分析发现,在201~222和425~441等氨基酸位,α疱疹病毒参考毒株均有不同程度的缺失,而DEV Clone-03表现出独有的完整性。     

Common and specific properties of herpesvirus UL34/UL31 protein family members revealed by protein complementation assay

The proteins encoded by the UL34 and UL31 genes of herpes simplex virus are conserved among herpesviruses. They form a complex that is essential for the egress of the herpesvirus nucleocapsids from the nucleus. In previous work on the homologous protein complex in murine cytomegalovirus (MCMV), we defined their mutual binding domains. Here, we started to map binding domains within the UL34/UL31 proteins of alpha-, beta-, and gammaherpesviruses and to locate other functional properties. A protein complementation assay (PCA) using the TEM-1 beta-lactamase fragments fused to UL31 and UL34 protein homologues was used to study protein-protein interactions in cells. Wild-type MCMV M50 and M53 provided a strong reaction in the PCA, whereas mutants unable to form a complex did not. The homologous pairs of herpes simplex virus type 1, pseudorabies virus, human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and murine herpes virus 68 proteins also reacted, with the exception of the EBV proteins. Cross-complementation was found to be positive only within the same herpesvirus subfamily. Moreover, the HCMV homologues rescued replication-defective MCMV genomes lacking one or the other gene. We identified the binding site of M53 for M50 in the first conserved region (CR1) (M. Loetzerich, Z. Ruzsics, and U. H. Koszinowski, J. Virol. 80:73-84). Here we show that the CR1 of all tested UL31 proteins contains the UL34 binding site, and chimeric proteins carrying the subfamily-specific CR1 rescued the ability to cross-complement in the PCA.     

Identification and characterization of the pseudorabies virus UL3.5 protein, which is involved in virus egress.

Alphaherpesvirus genomes exhibit a generally collinear gene arrangement, and most of their genes are conserved among the different members of the subfamily. Among the exceptions is the UL3.5 gene of pseudorabies virus (PrV) for which positional homologs have been detected in the genomes of varicella-zoster virus, equine herpesvirus 1, and bovine herpesvirus 1 but not in the genomes of herpes simplex virus types 1 and 2. To identify and characterize the predicted 224 amino acid UL3.5 protein of PrV, a rabbit antiserum was prepared against a UL3.5 fusion protein expressed in Escherichia coli. In Western blot (immunoblot) analyses the antiserum detected a 30-kDa protein in the cytoplasm of PrV infected cells which was absent from purified virions. For functional analysis, UL3.5-expressing cell lines were established and virus mutants were isolated after the rescue of defective, glycoprotein B-negative PrV by insertion of the complementing glycoprotein B-encoding gene of bovine herpesvirus 1 at two sites within the UL3.5 locus. A PrV mutant carrying the insertion at codon 159 and expressing a truncated UL3.5 protein was still capable of efficient productive replication in noncomplementing cells. In contrast, a PrV mutant carrying the insertion at codon 10 of the UL3.5 gene did not express detectable UL3.5 protein and exhibited a dramatic growth deficiency on non-complementing cells with regard to plaque formation and one-step replication. Electron microscopical studies showed an accumulation of unenveloped capsids in the vicinity of the Golgi apparatus. This defect could be compensated by propagation on complementing UL3.5-expressing cell lines. Our results thus demonstrate that the PrV UL3.5 gene encodes a nonstructural protein which plays an important role in virus replication, presumably during virus egress. The functionally relevant domains appear to be located within the N-terminal part of the UL3.5 protein which also comprises the region exhibiting the highest level of homology between the predicted UL3.5 homologous proteins of other alphaherpesviruses.     

Linker insertion mutations in the herpes simplex virus type 1 UL28 gene: effects on UL28 interaction with UL15 and UL33 and identification of a second-site mutation in the UL15 gene that suppresses a lethal UL28 mutation

The UL28 protein of herpes simplex virus type 1 (HSV-1) is one of seven viral proteins required for the cleavage and packaging of viral DNA. Previous results indicated that UL28 interacts with UL15 and UL33 to form a protein complex (terminase) that is presumed to cleave concatemeric DNA into genome lengths. In order to define the functional domains of UL28 that are important for DNA cleavage/packaging, we constructed a series of HSV-1 mutants with linker insertion and nonsense mutations in UL28. Insertions that blocked DNA cleavage and packaging were found to be located in two regions of UL28: the first between amino acids 200 to 400 and the second between amino acids 600 to 740. Insertions located in the N terminus or in a region located between amino acids 400 and 600 did not affect virus replication. Insertions in the carboxyl terminus of the UL28 protein were found to interfere with the interaction of UL28 with UL33. In contrast, all of the UL28 insertion mutants were found to interact with UL15 but the interaction was reduced with mutants that failed to react with UL33. Together, these observations were consistent with previous conclusions that UL15 and UL33 interact directly with UL28 but interact only indirectly with each other. Revertant viruses that formed plaques on Vero cells were detected for one of the lethal UL28 insertion mutants. DNA sequence analysis, in combination with genetic complementation assays, demonstrated that a second-site mutation in the UL15 gene restored the ability of the revertant to cleave and package viral DNA. The isolation of an intergenic suppressor mutant provides direct genetic evidence of an association between the UL28 and UL15 proteins and demonstrates that this association is essential for DNA cleavage and packaging.     

Molecular cloning and characterization of the UL31 gene from Duck enteritis virus

Using a combination of bioinformation analysis and Dot blot technique, a gene, designated hereafter as the duck enteritis virus (DEV) UL31 gene (GenBank accession number EF643559), was identified from the DEV CHv genomic library. Then, the UL31 gene was cloned and sequenced, which was composed of 933 nucleotides encoding 310 amino acids. Multiple sequence alignment suggested that the UL31 gene was highly conserved in Alphaherpesvirinae and similar to the other herpesviral UL31. Phylogenetic analysis showed that the gene had a close evolutionary relationship with the avian herperviruses, and DEV should be placed into a single cluster within the subfamily Alphaherpesvirinae. Antigen prediction indicated that several potential B-cell epitopes sites located in the UL31 protein. To further study, the UL31 gene was cloned into a pET prokaryotic expression vector and transformed into Escherichia coli BL21 (DE3). A 55 kDa fusion protein was induced by the further culture at 37°C after addition of 0.8 mM IPTG. Polyclonal antibody raised against the recombinant UL31 from rabbit was prepared. A protein about 55 kDa that reacted with the antibody was detected in immunoblots of bacterial proteins, suggesting that the 55 kDa protein was the product of the UL31 gene. Immunofluorescence analysis revealed that the protein was localized in very fine punctate forms in the nuclei of infected cells. Our results may provide some insight for further research about the gene and also enrich the database of herpesvirus.     

Assembly of the herpes simplex virus capsid: requirement for the carboxyl-terminal twenty-five amino acids of the proteins encoded by the UL26 and UL26.5 genes.

Herpes simplex virus type 1 (HSV-1) intermediate capsids are composed of seven proteins, VP5, VP19C, VP21, VP22a, VP23, VP24, and VP26, and the genes that encode these proteins, UL19, UL38, UL26, UL26.5, UL18, UL26, and UL35, respectively. The UL26 gene encodes a protease that cleaves itself and the product of the UL26.5 gene at a site (M site) 25 amino acids from the C terminus of these two proteins. In addition, the protease cleaves itself at a second site (R site) between amino acids 247 and 248. Cleavage of the UL26 protein gives rise to the capsid proteins VP21 and VP24, and cleavage of the UL26.5 protein gives rise to the capsid protein VP22a. Previously we described the production of HSV-1 capsids in insect cells by infecting the cells with recombinant baculoviruses expressing the six capsid genes (D. R. Thomsen, L. L. Roof, and F. L. Homa, J. Virol. 68:2442-2457, 1994). Using this system, we demonstrated that the products of the UL26 and/or UL26.5 genes are required as scaffolds for assembly of HSV-1 capsids. To better understand the functions of the UL26 and UL26.5 proteins in capsid assembly, we constructed baculoviruses that expressed altered UL26 and UL26.5 proteins. The ability of the altered UL26 and UL26.5 proteins to support HSV-1 capsid assembly was then tested in insect cells. Among the specific mutations tested were (i) deletion of the C-terminal 25 amino acids from the proteins coded for by the UL26 and UL26.5 genes; (ii) mutation of His-61 of the UL26 protein, an amino acid required for protease activity; and (iii) mutation of the R cleavage site of the UL26 protein. Analysis of the capsids formed with wild-type and mutant proteins supports the following conclusions: (i) the C-terminal 25 amino acids of the UL26 and UL26.5 proteins are required for capsid assembly; (ii) the protease activity associated with the UL26 protein is not required for assembly of morphologically normal capsids; and (iii) the uncleaved forms of the UL26 and UL26.5 proteins are employed in assembly of 125-nm-diameter capsids; cleavage of these proteins occurs during or subsequent to capsid assembly. Finally, we carried out in vitro experiments in which the major capsid protein VP5 was mixed with wild-type or truncated UL26.5 protein and then precipitated with a VP5-specific monoclonal antibody.(ABSTRACT TRUNCATED AT 400 WORDS)     

Herpes simplex virus type 1 portal protein UL6 interacts with the putative terminase subunits UL15 and UL28

The herpes simplex virus type 1 (HSV-1) UL6, UL15, and UL28 proteins are essential for cleavage of replicated concatemeric viral DNA into unit length genomes and their packaging into a preformed icosahedral capsid known as the procapsid. The capsid-associated UL6 DNA-packaging protein is located at a single vertex and is thought to form the portal through which the genome enters the procapsid. The UL15 protein interacts with the UL28 protein, and both are strong candidates for subunits of the viral terminase, a key component of the molecular motor that drives the DNA into the capsid. To investigate the association of the UL6 protein with the UL15 and UL28 proteins, the three proteins were produced in large amounts in insect cells with the baculovirus expression system. Interactions between UL6 and UL28 and between UL6 and UL15 were identified by an immunoprecipitation assay. These results were confirmed by transiently expressing wild-type and mutant proteins in mammalian cells and monitoring their distribution by immunofluorescence. In cells expressing the single proteins, UL6 and UL15 were concentrated in the nuclei whereas UL28 was found in the cytoplasm. When the UL6 and UL28 proteins were coexpressed, UL28 was redistributed to the nuclei, where it colocalized with UL6. In cells producing either of two cytoplasmic UL6 mutant proteins and a functional epitope-tagged form of UL15, the UL15 protein was concentrated with the mutant UL6 protein in the cytoplasm. These observed interactions of UL6 with UL15 and UL28 are likely to be of major importance in establishing a functional DNA-packaging complex at the portal vertex of the HSV-1 capsid.     

The open reading frames UL3, UL4, UL10, and UL16 are dispensable for the replication of herpes simplex virus 1 in cell culture.

By means of insertion and deletion mutagenesis, we have constructed four herpes simplex virus 1 recombinants, each lacking most sequences encoding a different open reading frame. The deleted genes are located in the unique sequences of the long component and include those designated UL3, UL4, UL10, and UL16. The recombinant virus R7211 lacks 579 of the 696 bp of UL3. The recombinant virus R7217 lacks 307 of the 597 bp of the UL4 open reading frame. R7216 contains a 972-bp deletion within the 1,419-bp open reading frame of UL10, whereas R7210 lacks 988 bp of the 1,119-bp UL16 open reading frame. Growth curves indicated that the yields of these viruses in Vero and BHK cell cultures were only slightly reduced from or in some instances equivalent to that of the parent virus. The function of the gene products is not known. It is of interest to note that (i) the UL16 open reading frame maps entirely within the single intron of UL15 and (ii) on the basis of the extent and size of hydrophobic domains, the UL3 and UL10 gene products were predicted to be membrane proteins.     

Novel class of thiourea compounds that inhibit herpes simplex virus type 1 DNA cleavage and encapsidation: resistance maps to the UL6 gene

In our search for novel inhibitors of herpes simplex virus type 1 (HSV-1), a new class of thiourea inhibitors was discovered. N-(4-[3-(5-Chloro-2,4-dimethoxyphenyl)-thioureido]-phenyl)-acetamide and its 2-fluoro-benzamide derivative inhibited HSV-1 replication. HSV-2, human cytomegalovirus, and varicella-zoster virus were inhibited to a lesser extent. The compounds acted late in the replication cycle by impairing both the cleavage of concatameric viral DNA into progeny genome length and the packaging of the DNA into capsids, indicative of a defect in the encapsidation process. To uncover the molecular target of the inhibition, resistant HSV-1 isolates were generated, and the mutation responsible for the resistance was mapped using marker transfer techniques. Each of three independent isolates had point mutations in the UL6 gene which resulted in independent single-amino-acid changes. One mutation was located in the N terminus of the protein (E121D), while two were located close together in the C terminus (A618V and Q621R). Each of these point mutations was sufficient to confer drug resistance when introduced into wild-type virus. The UL6 gene is one of the seven HSV-1 genes known to play a role in DNA packaging. This novel class of inhibitors has provided a new tool for dissection of HSV-1 encapsidation mechanisms and has uncovered a new viable target for the treatment of herpesviral diseases.     

A mutation in the human herpes simplex virus type 1 UL52 zinc finger motif results in defective primase activity but can recruit viral polymerase and support viral replication efficiently

Herpes simplex virus type 1 (HSV-1) encodes a heterotrimeric helicase/primase complex consisting of UL5, UL8, and UL52. UL5 contains conserved helicase motifs, while UL52 contains conserved primase motifs, including a zinc finger motif. Although HSV-1 and HSV-2 UL52s contain a leucine residue at position 986, most other herpesvirus primase homologues contain a phenylalanine at this position. We constructed an HSV-1 UL52 L986F mutation and found that it can complement a UL52 null virus more efficiently than the wild type (WT). We thus predicted that the UL5/8/52 complex containing the L986F mutation might possess increased primase activity; however, it exhibited only 25% of the WT level of primase activity. Interestingly, the mutant complex displayed elevated levels of DNA binding and single-stranded DNA-dependent ATPase and helicase activities. This result confirms a complex interdependence between the helicase and primase subunits. We previously showed that primase-defective mutants failed to recruit the polymerase catalytic subunit UL30 to prereplicative sites, suggesting that an active primase, or primer synthesis, is required for polymerase recruitment. Although L986F exhibits decreased primase activity, it can support efficient replication and recruit UL30 efficiently to replication compartments, indicating that a partially active primase is capable of recruiting polymerase. Extraction with detergents prior to fixation can extract nucleosolic proteins but not proteins bound to chromatin or the nuclear matrix. We showed that UL30 was extracted from replication compartments while UL42 remained bound, suggesting that UL30 may be tethered to the replication fork by protein-protein interactions.     

Identification and characterization of the pseudorabies virus tegument proteins UL46 and UL47: role for UL47 in virion morphogenesis in the cytoplasm

Proteins encoded by the UL46 and UL47 genes of herpes simplex virus type 1 (HSV-1) constitute major components of the viral tegument. However, their functions have so far not been elucidated in detail. By use of monospecific antisera directed against bacterially expressed glutathione-S-transferase fusion proteins, the homologous UL46 and UL47 proteins of the alphaherpesvirus pseudorabies virus (PrV) were identified in virus-infected cells and in virions. The PrV UL46 gene product of 693 amino acids (aa) exhibits an apparent molecular mass of 95 kDa, whereas the UL47 product of 750 aa was identified as a 97-kDa protein. Both are present in purified virions, correlating with their role as tegument proteins. Immunofluorescence analysis by confocal laser scan microscopy showed that late in infection the UL46 product is detectable in the cytoplasm, whereas the UL47 product was observed to be diffuse in the cytoplasm and speckled in the nucleus. Virus mutants lacking either the UL46 or the UL47 gene or both were isolated on noncomplementing cells, demonstrating that these genes either singly or in combination are not required for productive viral replication. However, plaque sizes were decreased. Interestingly, in one-step growth analysis, UL47 deletion mutants exhibited an approximately 10-fold decrease in final titers, whereas the UL46 deletion mutant was not affected. This finding correlated with ultrastructural observations which showed unimpaired virion morphogenesis in the absence of the UL46 protein, whereas in the absence of the UL47 protein intracytoplasmic aggregates of partially tegumented capsids were observed. In summary, we identified the PrV UL46 and UL47 proteins and show that the UL47 protein plays an important role in virion assembly in the cytoplasm.     

The bovine herpesvirus 1 maturational proteinase and scaffold proteins can substitute for the homologous herpes simplex virus type 1 proteins in the formation of hybrid type B capsids.

We determined the nucleotide sequence of a 3.5-kb region of the bovine herpesvirus 1 (BHV-1) genome which contained the complete BHV-1 homologs of the herpes simplex virus type 1 (HSV-1) UL26 and UL26.5 genes. In HSV-1, the UL26 and UL26.5 open reading frames encode scaffold proteins upon which viral capsids are assembled. The UL26-encoded protein is also a proteinase and specifically cleaves both itself and the UL26.5-encoded protein. The overall BHV-1-encoded amino acid sequence showed only 41% identity to the HSV-1 sequences and was most divergent in the regions defined to be involved in the scaffolding function. We substituted the proteins encoded by the BHV-1 homologs of the UL26 and UL26.5 open reading frames, expressed in baculovirus, for the corresponding HSV-1 proteins in an in vitro HSV-1 capsid assembly system. The proteins expressed from the BHV-1 UL26 and UL26.5 homologs facilitated the formation of hybrid type B capsids indistinguishable from those formed entirely with HSV-1-encoded proteins.     

Herpes simplex virus type 1 gene UL14: phenotype of a null mutant and identification of the encoded protein

Herpes simplex virus type 1 (HSV-1) gene UL14 is located between divergently transcribed genes UL13 and UL15 and overlaps the promoters for both of these genes. UL14 also exhibits a substantial overlap of its coding region with that of UL13. It is one of the few HSV-1 genes for which a phenotype and protein product have not been described. Using mass spectrometric and immunological approaches, we demonstrated that the UL14 protein is a minor component of the virion tegument of 32 kDa which is expressed late in infection. In infected cells, the UL14 protein was detected in the nucleus at discrete sites within electron-dense nuclear bodies and in the cytoplasm initially in a diffuse distribution and then at discrete sites. Some of the UL14 protein was phosphorylated. A mutant with a 4-bp deletion in the central region of UL14 failed to produce the UL14 protein and generated small plaques. The mutant exhibited an extended growth cycle at low multiplicity of infection and appeared to be compromised in efficient transit of virus particles from the infected cell. In mice injected intracranially, the 50% lethal dose of the mutant was reduced more than 30,000-fold. Recovery of the mutant from the latently infected sacral ganglia of mice injected peripherally was significantly less than that of wild-type virus, suggesting a marked defect in the establishment of, or reactivation from, latent infection.     

Role of the UL25 gene product in packaging DNA into the herpes simplex virus capsid: location of UL25 product in the capsid and demonstration that it binds DNA

Recent studies have suggested that the herpes simplex type 1 (HSV-1) UL25 gene product, a minor capsid protein, is required for encapsidation but not cleavage of replicated viral DNA. This study set out to investigate the potential interactions of UL25 protein with other virus proteins and determine what properties it has for playing a role in DNA encapsidation. The UL25 protein is found in 42 +/- 17 copies per B capsid and is present in both pentons and hexons. We introduced green fluorescent protein (GFP) as a fluorescent tag into the N terminus of UL25 protein to identify its location in HSV-1-infected cells and demonstrated the relocation of UL25 protein from the cytoplasm into the nucleus at the late stage of HSV-1 infection. To clarify the cause of this relocation, we analyzed the interactions of UL25 protein with other virus proteins. The UL25 protein associates with VP5 and VP19C of virus capsids, especially of the penton structures, and the association with VP19C causes its relocation into the nucleus. Gel mobility shift analysis shows that UL25 protein has the potential to bind DNA. Moreover, the amino-terminal one-third of the UL25 protein is particularly important in DNA binding and forms a homo-oligomer. In conclusion, the UL25 gene product forms a tight connection with the capsid being linked with VP5 and VP19C, and it may play a role in anchoring the genomic DNA.