Homologs In Other Herpesviruses Research Articles

Human Cytomegalovirus Nuclear Egress Complex Subunit, UL53, Associates with Capsids and Myosin Va, but Is Not Important for Capsid Localization towards the Nuclear Periphery.

After herpesviruses encapsidate their genomes in replication compartments (RCs) within the nuclear interior, capsids migrate to the inner nuclear membrane (INM) for nuclear egress. For human cytomegalovirus (HCMV), capsid migration depends at least in part on nuclear myosin Va. It has been reported for certain herpesviruses that the nucleoplasmic subunit of the viral nuclear egress complex (NEC) is important for this migration. To address whether this is true for HCMV, we used mass spectrometry and multiple other methods to investigate associations among the HCMV NEC nucleoplasmic subunit, UL53, myosin Va, major capsid protein, and/or capsids. We also generated complementing cells to derive and test HCMV mutants null for UL53 or the INM NEC subunit, UL50, for their importance for these associations and, using electron microscopy, for intranuclear distribution of capsids. We found modest associations among the proteins tested, which were enhanced in the absence of UL50. However, we found no role for UL53 in the interactions of myosin Va with capsids or the percentage of capsids outside RC-like inclusions in the nucleus. Thus, UL53 associates somewhat with myosin Va and capsids, but, contrary to reports regarding its homologs in other herpesviruses, is not important for migration of capsids towards the INM.

The Kaposi's Sarcoma-Associated Herpesvirus Protein ORF42 Is Required for Efficient Virion Production and Expression of Viral Proteins.

Kaposi’s sarcoma-associated herpesvirus (KSHV), the etiologic agent of Kaposi’s sarcoma and other aggressive AIDS-associated malignancies, encodes over 90 genes, most of which are expressed only during the lytic replication cycle. The role of many of the KSHV lytic proteins in the KSHV replication cycle remains unknown, and many proteins are annotated based on known functions of homologs in other herpesviruses. Here we investigate the role of the previously uncharacterized KSHV lytic protein ORF42, a presumed tegument protein. We find that ORF42 is dispensable for reactivation from latency but is required for efficient production of viral particles. Like its alpha- and beta-herpesviral homologs, ORF42 is a late protein that accumulates in the viral particles. However, unlike its homologs, ORF42 appears to be required for efficient expression of at least some viral proteins and may potentiate post-transcriptional stages of gene expression. These results demonstrate that ORF42 has an important role in KSHV replication and may contribute to shaping viral gene expression.

Domain Interaction Studies of Herpes Simplex Virus 1 Tegument Protein UL16 Reveal Its Interaction with Mitochondria.

The HSV-1-encoded tegument protein UL16 is involved in multiple events of the virus replication cycle, ranging from virus assembly to cell-cell spread of the virus, and hence it can serve as an important drug target. Unfortunately, a lack of both structural and functional information limits our understanding of this protein. The discovery of domain interactions within UL16 and the novel ability of UL16 to interact with mitochondria in HSV-infected cells lays a foundational framework for future investigations aimed at deciphering the structure and function of not just UL16 of HSV-1 but also its homologs in other herpesviruses.

Conserved Tryptophan Motifs in the Large Tegument Protein pUL36 Are Required for Efficient Secondary Envelopment of Herpes Simplex Virus Capsids.

Herpes simplex virus (HSV) replicates in the skin and mucous membranes, and initiates lytic or latent infections in sensory neurons. Assembly of progeny virions depends on the essential large tegument protein pUL36 of 3,164 amino acid residues that links the capsids to the tegument proteins pUL37 and VP16. Of the 32 tryptophans of HSV-1-pUL36, the tryptophan-acidic motifs (1766)WD(1767) and (1862)WE(1863) are conserved in all HSV-1 and HSV-2 isolates. Here, we characterized the role of these motifs in the HSV life cycle since the rare tryptophans often have unique roles in protein function due to their large hydrophobic surface. The infectivity of the mutants HSV-1(17(+))Lox-pUL36-WD/AA-WE/AA and HSV-1(17(+))Lox-CheVP26-pUL36-WD/AA-WE/AA, in which the capsid has been tagged with the fluorescent protein Cherry, was significantly reduced. Quantitative electron microscopy shows that there were a larger number of cytosolic capsids and fewer enveloped virions compared to their respective parental strains, indicating a severe impairment in secondary capsid envelopment. The capsids of the mutant viruses accumulated in the perinuclear region around the microtubule-organizing center and were not dispersed to the cell periphery but still acquired the inner tegument proteins pUL36 and pUL37. Furthermore, cytoplasmic capsids colocalized with tegument protein VP16 and, to some extent, with tegument protein VP22 but not with the envelope glycoprotein gD. These results indicate that the unique conserved tryptophan-acidic motifs in the central region of pUL36 are required for efficient targeting of progeny capsids to the membranes of secondary capsid envelopment and for efficient virion assembly. Herpesvirus infections give rise to severe animal and human diseases, especially in young, immunocompromised, and elderly individuals. The structural hallmark of herpesvirus virions is the tegument, which contains evolutionarily conserved proteins that are essential for several stages of the herpesvirus life cycle. Here we characterized two conserved tryptophan-acidic motifs in the central region of the large tegument protein pUL36 of herpes simplex virus. When we mutated these motifs, secondary envelopment of cytosolic capsids and the production of infectious particles were severely impaired. Our data suggest that pUL36 and its homologs in other herpesviruses, and in particular such tryptophan-acidic motifs, could provide attractive targets for the development of novel drugs to prevent herpesvirus assembly and spread.

Identification and Characterization of the Physiological Gene Targets of the Essential Lytic Replicative Epstein-Barr Virus SM Protein.

Epstein-Barr virus (EBV) SM protein is an essential lytic cycle protein with multiple posttranscriptional mechanisms of action. SM binds RNA and increases accumulation of specific EBV transcripts. Previous studies using microarrays and PCR have shown that SM-null mutants fail to accumulate several lytic cycle mRNAs and proteins at wild-type levels. However, the complete effect of SM on the EBV transcriptome has been incompletely characterized. Here we precisely identify the effects of SM on all EBV transcripts by high-throughput RNA sequencing, quantitative PCR (qPCR), and Northern blotting. The effect of SM on EBV mRNAs was highly skewed and was most evident on 13 late genes, demonstrating why SM is essential for infectious EBV production. EBV DNA replication was also partially impaired in SM mutants, suggesting additional roles for SM in EBV DNA replication. While it has been suggested that SM specificity is based on recognition of either RNA sequence motifs or other sequence properties, no such unifying property of SM-responsive targets was discernible. The binding affinity of mRNAs for SM also did not correlate with SM responsiveness. These data suggest that while target RNA binding by SM may be required for its effect, specific activation by SM is due to differences in inherent properties of individual transcripts. We therefore propose a new model for the mechanism of action and specificity of SM and its homologs in other herpesviruses: that they bind many RNAs but only enhance accumulation of those that are intrinsically unstable and poorly expressed. This study examines the mechanism of action of EBV SM protein, which is essential for EBV replication and infectious virus production. Since SM protein is not similar to any cellular protein and has homologs in all other human herpesviruses, it has potential importance as a therapeutic target. Here we establish which EBV RNAs are most highly upregulated by SM, allowing us to understand why it is essential for EBV replication. By comparing and characterizing these RNA transcripts, we conclude that the mechanism of specific activity is unlikely to be based simply on preferential recognition of a target motif. Rather, SM binding to its target RNA may be necessary but not sufficient for enhancing accumulation of the RNA. Preferential effects of SM on its most responsive RNA targets may depend on other inherent characteristics of these specific mRNAs that require SM for efficient expression, such as RNA stability.

Role of Host Cell p32 in Herpes Simplex Virus 1 De-Envelopment during Viral Nuclear Egress.

To clarify the function(s) of the herpes simplex virus 1 (HSV-1) major virion structural protein UL47 (also designated VP13/14), we screened cells overexpressing UL47 for UL47-binding cellular proteins. Tandem affinity purification of transiently expressed UL47 coupled with mass spectrometry-based proteomics technology and subsequent analyses showed that UL47 interacted with cell protein p32 in HSV-1-infected cells. Unlike in mock-infected cells, p32 accumulated at the nuclear rim in HSV-1-infected cells, and this p32 recruitment to the nuclear rim required UL47. p32 formed a complex(es) with HSV-1 proteins UL31, UL34, Us3, UL47, and/or ICP22 in HSV-1-infected cells. All these HSV-1 proteins were previously reported to be important for HSV-1 nuclear egress, in which nucleocapsids bud through the inner nuclear membrane (primary envelopment) and the enveloped nucleocapsids then fuse with the outer nuclear membrane (de-envelopment). Like viral proteins UL31, UL34, Us3, and UL47, p32 was detected in primary enveloped virions. p32 knockdown reduced viral replication and induced membranous invaginations adjacent to the nuclear rim containing primary enveloped virions and aberrant localization of UL31 and UL34 in punctate structures at the nuclear rim. These effects of p32 knockdown were reduced in the absence of UL47. Therefore, the effects of p32 knockdown in HSV-1 nuclear egress were similar to those of the previously reported mutation(s) in HSV-1 regulatory proteins for HSV-1 de-envelopment during viral nuclear egress. Collectively, these results suggested that p32 regulated HSV-1 de-envelopment and replication in a UL47-dependent manner. IMPORTANCE In this study, we have obtained data suggesting that (i) the HSV-1 major virion structural protein UL47 interacted with host cell protein p32 and mediated the recruitment of p32 to the nuclear rim in HSV-1-infected cells; (ii) p32 was a component of the HSV-1 nuclear egress complex (NEC), whose core components were UL31 and UL34; and (iii) p32 regulated HSV-1 de-envelopment during viral nuclear egress. It has been reported that p32 was a component of human cytomegalovirus NEC and was required for efficient disintegration of nuclear lamina, which has been thought to facilitate HSV-1 primary envelopment during viral nuclear egress. Thus, p32 appeared to be a core component of herpesvirus NECs, like UL31 and UL34 homologs in other herpesviruses, and to play multiple roles in herpesvirus nuclear egress.

KSHV ORF67 encoded lytic protein localizes on the nuclear membrane and alters emerin distribution

p29, a newly identified Kaposi's sarcoma-associated herpesvirus (KSHV) protein, is the product of ORF67, the positional homolog of the conserved herpesvirus protein UL34. Like its homologues in other herpesviruses, p29 is expressed early during viral lytic cycle, and is localized on the nuclear rim. Upon chemical induction of viral replication in primary effusion lymphoma cells, p29 interacts with p33, encoded by ORF69, the positional homolog of the conserved herpesvirus protein UL31, and both proteins colocalize on the nuclear membrane. IFA and biochemical analysis of infected or transfected cells showed that p29 expression resulted in delocalization and hyperphosphorylation of emerin, whereas other nuclear lamin associated proteins, such as LUMA, LB1 and LBR were not affected. Mislocalization of emerin was robustly increased upon combined expression of p29 and p33, suggesting that emerin destabilization might represent the first step in nuclear lamina disassembling, a process necessary for nucleocapsid maturation.

The alcelaphine herpesvirus-1 ORF 57 encodes a nuclear shuttling protein

Alcelaphine herpesvirus-1 (AlHV-1) is a gamma(2) rhadinovirus associated with Malignant Catarrhal Fever (MCF) in cattle. ORF 57 is well conserved among gammaherpesviruses and it has been shown that the ORF 57 gene products of Herpesvirus Saimiri (HVS), Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) play an important role in regulating viral gene expression. The AlHV-1 ORF 57 gene product has not been characterized. In the accompanying paper we have demonstrated that AlHV-1 ORF 57 encodes an immediate early protein that acts as a regulator of gene expression. The ORF 57 gene product has an up-regulatory effect only on another immediate early gene product encoded by ORF 50. Here we show that the ORF 57 gene product is a nuclear protein. When ORF 57 was fused to the gene encoding Enhanced Green Fluorescent Protein (EGFP), the fusion protein exhibited a punctate nuclear distribution that co-localized with the nucleolar phosphoprotein C23. The nuclear localisation signal of ORF 57 gene product was located at the N-terminus. The ORF 57 gene product travels from nucleus to the cytoplasm, where it accumulates during Actinomycin D treatment. The domain involved in nuclear shuttling was also localised at the N-terminal region of the protein. Thus in common with homologues in other herpesviruses the AlHV-1 ORF 57 gene product is a nuclear cytoplasmic shuttling protein which may play a role in export of viral mRNAs from the nucleus of infected cells.

Phylogeny of Duck Enteritis Virus: Evolutionary Relationship in the Family Herpesviridae

Objective: To understand the evolutionary relationship of duck enteritis virus (DEV) in the family Herpesviridae and the genome organization of DEV. Methods: A modified ‘targeted gene walking PCR’ method was used to amplify the unknown gene of DEV Clone-03 strain adjacent to a short stretch of known sequence. The assembled unknown fragment was confirmed to be a homologue of herpesviruses by BLAST analysis and comparison with other herpesviruses homologues. Open reading frames (ORFs) were determined and analyzed by Gene Runner software and an online transcriptional element search engine. Two evolutionary analysis approaches were used to conduct phylogenetic trees of DEV in relation to herpesviruses based on the deduced amino acid sequences. Results: A genomic fragment encompassing 25,625 nt and containing 16 ORFs was amplified, determined, and compared with homologues in other herpesviruses. Phylogenetic trees indicated that DEV and other herpesviruses generated from one ancestor and DEV were grouped into the subfamily Alphaherpesvirinae. In most cases, DEV showed a close relationship with the genus Mardivirus, but formed a single branch. Conclusion: Partial genomic organization and phylogenetic analysis in the present study provides evidence that DEV was a member of the subfamily Alphaherpesvirinae and should be assigned as a single group.

Structural Characterization of the UL25 DNA-Packaging Protein from Herpes Simplex Virus Type 1

Herpesviruses replicate their double stranded DNA genomes as high-molecular-weight concatemers which are subsequently cleaved into unit-length genomes by a complex mechanism that is tightly coupled to DNA insertion into a preformed capsid structure, the procapsid. The herpes simplex virus type 1 UL25 protein is incorporated into the capsid during DNA packaging, and previous studies of a null mutant have demonstrated that its function is essential at the late stages of the head-filling process, either to allow packaging to proceed to completion or for retention of the viral genome within the capsid. We have expressed and purified an N-terminally truncated form of the 580-residue UL25 protein and have determined the crystallographic structure of the region corresponding to amino acids 134 to 580 at 2.1-Angstroms resolution. This structure, the first for any herpesvirus protein involved in processing and packaging of viral DNA, reveals a novel fold, a distinctive electrostatic distribution, and a unique "flexible" architecture in which numerous flexible loops emanate from a stable core. Evolutionary trace analysis of UL25 and its homologues in other herpesviruses was used to locate potentially important amino acids on the surface of the protein, leading to the identification of four putative docking regions for protein partners.

Cleavage of Human Cytomegalovirus Protease pUL80a at Internal and Cryptic Sites Is Not Essential but Enhances Infectivity

The cytomegalovirus (CMV) maturational protease, assemblin, contains an "internal" (I) cleavage site absent from its homologs in other herpesviruses. Blocking this site for cleavage did not prevent replication of the resulting I(-) mutant virus. However, cells infected with the I(-) virus showed increased amounts of a fragment produced by cleavage at the nearby "cryptic" (C) site, suggesting that its replication may bypass the I-site block by using the C site as an alternate cleavage pathway. To test this and further examine the biological importance of these cleavages, we constructed two additional virus mutants-one blocked for C-site cleavage and another blocked for both I- and C-site cleavage. Infectivity comparisons with the parental wild-type virus showed that the I(-) mutant was the least affected for virus production, whereas infectivity of the C(-) mutant was reduced by approximately 40% and when both sites were blocked virus infectivity was reduced by nearly 90%, providing the first evidence that these cleavages have biological significance. We also present and discuss evidence suggesting that I-site cleavage destabilizes assemblin and its fragments, whereas C-site cleavage does not.

Assembly Protein Precursor (pUL80.5 Homolog) of Simian Cytomegalovirus Is Phosphorylated at a Glycogen Synthase Kinase 3 Site and Its Downstream “Priming” Site: Phosphorylation Affects Interactions of Protein with Itself and with Major Capsid Protein

Capsid assembly among the herpes-group viruses is coordinated by two related scaffolding proteins. In cytomegalovirus (CMV), the main scaffolding constituent is called the assembly protein precursor (pAP). Like its homologs in other herpesviruses, pAP is modified by proteolytic cleavage and phosphorylation. Cleavage is essential for capsid maturation and production of infectious virus, but the role of phosphorylation is undetermined. As a first step in evaluating the significance of this modification, we have identified the specific sites of phosphorylation in the simian CMV pAP. Two were established previously to be adjacent serines (Ser156 and Ser157) in a casein kinase II consensus sequence. The remaining two, identified here as Thr231 and Ser235, are within consensus sequences for glycogen synthase kinase 3 (GSK-3) and mitogen-activated protein kinase, respectively. Consistent with Thr231 being a GSK-3 substrate, its phosphorylation required a downstream "priming" phosphate (i.e., Ser235) and was reduced by a GSK-3-specific inhibitor. Phosphorylation of Ser235 converts pAP to an electrophoretically slower-mobility isoform, pAP*; subsequent phosphorylation of pAP* at Thr231 converts pAP* to a still-slower isoform, pAP**. The mobility shift to pAP* was mimicked by substituting an acidic amino acid for either Thr231 or Ser235, but the shift to pAP** required that both positions be phosphorylated. Glu did not substitute for pSer235 in promoting phosphorylation of Thr231. We suggest that phosphorylation of Thr231 and Ser235 causes charge-driven conformational changes in pAP, and we demonstrate that preventing these modifications alters interactions of pAP with itself and with major capsid protein, suggesting a functional significance.

Functional analysis of Epstein-Barr virus SM protein: identification of amino acids essential for structure, transactivation, splicing inhibition, and virion production.

The Epstein-Barr virus (EBV) SM protein is a posttranscriptional regulator of cellular and viral gene expression that binds and stabilizes target mRNAs and shuttles from nucleus to cytoplasm. SM enhances expression of several EBV genes required for lytic replication and is essential for virion production. SM increases accumulation of specific mRNAs but also inhibits expression of several intron-containing transcripts. The mechanism by which SM inhibits gene expression is poorly understood. The experiments described here had several aims: to determine whether specific domains of SM were responsible for activation or inhibition function; whether these functions could be separated; and whether one or more of these functions were essential for virion production. A mutational analysis of SM was performed, focusing on amino acids in SM that are evolutionarily conserved among SM homologs in other herpesviruses. Mutation of the carboxy-terminal region of SM revealed a region that is likely to be structurally important for SM protein conformation. In addition, several amino acids were identified that are critical for activation and inhibition function. A specific mutation of a highly conserved cysteine residue revealed that it was essential for gene inhibition but not for transactivation, indicating that these two functions operate through independent mechanisms. Furthermore, the ability of wild-type SM and the inability of the mutant to inhibit gene expression were shown to correlate with the ability to inhibit splicing of a human target gene and thereby prevent accumulation of its processed mRNA. Surprisingly, some mutations which preserved both activation and inhibition functions in vitro nevertheless abolished virion production, suggesting that other SM functions or protein-protein interactions are also required for lytic replication.

Phosphorylation of simian cytomegalovirus assembly protein precursor (pAPNG.5) and proteinase precursor (pAPNG1): multiple attachment sites identified, including two adjacent serines in a casein kinase II consensus sequence.

The assembly protein precursor (pAP) of cytomegalovirus (CMV), and its homologs in other herpesviruses, functions at several key steps during the process of capsid formation. This protein, and the genetically related maturational proteinase, is distinguished from the other capsid proteins by posttranslational modifications, including phosphorylation. The objective of this study was to identify sites at which pAP is phosphorylated so that the functional significance of this modification and the enzyme(s) responsible for it can be determined. In the work reported here, we used peptide mapping, mass spectrometry, and site-directed mutagenesis to identify two sets of pAP phosphorylation sites. One is a casein kinase II (CKII) consensus sequence that contains two adjacent serines, both of which are phosphorylated. The other site(s) is in a different domain of the protein, is phosphorylated less frequently than the CKII site, does not require preceding CKII-site phosphorylation, and causes an electrophoretic mobility shift when phosphorylated. Transfection/expression assays for proteolytic activity showed no gross effect of CKII-site phosphorylation on the enzymatic activity of the proteinase or on the substrate behavior of pAP. Evidence is presented that both the CKII sites and the secondary sites are phosphorylated in virus-infected cells and plasmid-transfected cells, indicating that these modifications can be made by a cellular enzyme(s). Apparent compartmental differences in phosphorylation of the CKII-site (cytoplasmic) and secondary-site (nuclear) serines suggest the involvement of more that one enzyme in these modifications.

The descent of human herpesvirus 8

Analysis of conserved herpesvirus genes shows that human herpesvirus 8 (HHV8) belongs to a clade of primate herpesviruses within the γ2 sublineage. Overall, this clade has developed by cospeciation with host species. Seventeen genes of HHV8 and its nearest relatives lack simple homologues in other herpesviruses, and eight of these have known mammalian homologues, presumed to be the sources of the HHV8 versions. Comparative analyses yield further insights into the source or time of acquisition for several of these genes. All were acquired by the HHV8 lineage in the very distant past. Two unusual processes are manifest in the contemporary evolution of HHV8. First, the K1 gene is uniquely undergoing a process of extensive and positively selected substitution, of unknown significance. Secondly, the K15 gene has been substituted in some lineages by a distant homologue, presumably by recombination with an unknown herpesvirus. While available data are otherwise compatible with a cospeciational relationship between HHV8 and the human species, they do not exclude relatively recent transfer to humans from another primate host.

Genetic content of a 20.9 kb segment of human herpesvirus 6B strain Z29 spanning the homologs of human herpesvirus 6A genes U40-57 and containing the origin of DNA replication.

A continuous 20.9 kb sequence from human herpesvirus 6 variant B (HHV-6B) strain Z29 (GenBank accession number L16947) is genetically colinear with a discrete segment of the human cytomegalovirus (HCMV) UL region and with HHV-6 variant A (HHV-6A). Short nucleotide sequence determinations at multiple sites within an 8.5 kb region immediately 3' to the 20.9 kb contig revealed additional colinearity between HHV-6B, HCMV and HHV-6A. Homology studies with the predicted peptide sequences from 11 complete and 12 partial HHV-6B open reading frames (ORFs) revealed that most encode proteins conserved to varying degrees in all previously sequenced primate herpesviruses. HHV-6B homologs were identified for the HSV-1 ICP18.5, ICP8, UL52, UL24, UL25 and major capsid protein. Several HHV-6B proteins had limited amino acid similarity to their positional homologs in other herpesviruses. Each gene identified is highly homologous to its HHV-6A counterpart, including two unique HHV-6 genes predicted to encode membrane-associated glycoproteins. However, two regions of substantial divergence were noted, one spanning the origin of replication and the other encoding one of the putative HHV-6-specific glycoprotein genes. Substitutions in the latter region lead to predicted differences in reading frames and protein lengths among HHV-6 isolates.

N-terminal sequence analysis of equine herpesvirus 1 glycoproteins D and B and evidence for internal cleavage of the gene 71 product.

Signal cleavage sites of equine herpesvirus 1 (EHV-1) glycoproteins D and B (gD and gB) and an endoproteolytic cleavage site of EHV-1 gB were determined by N-terminal amino acid sequencing and compared with known cleavage sites of homologues in other herpesvirus. Signal cleavage of EHV-1 gD occurred between Arg35 and Ala36 in a region of basic amino acids resembling the endoproteolytic cleavage sites of viral glycoproteins, nine amino acids downstream of the predicted site, while EHV-1 gB was cleaved as predicted between Ala85 and Val86. Endoproteolytic cleavage of EHV-1 gB occurred between Arg548 and Ala549, 28 amino acids downstream of the cleavage site predicted from conserved sequences of other herpesvirus gB homologous. One interpretation of these data is that EHV-1 gB is cleaved internally at both sites, a possibility which was supported by the apparent molecular masses of the unglycosylated gB subunits produced in the presence of tunicamycin. This double cleavage would release a stretch of amino acids which is not present in sequenced gB molecules of other herpesviruses. Experiments with glycosylation inhibitors indicated that cleavage of EHV-1 gB can occur in the absence of glycosylation. N-terminal sequencing also determined that a 42 kDa EHV-1 glycoprotein was a product of internal cleavage of the protein encoded by gene 71. Staggered endoproteolytic cleavage after adjacent arginine residues 506 and 507 separates the 42 kDa C-terminal subunit containing all the cysteine residues from the serine and threonine rich N-terminal region.

Nucleotide sequence analysis of an infectious laryngotracheitis virus gene corresponding to the US3 of HSV-1 and a unique gene encoding a 67 kDa protein.

The DNA sequence of 4005 nucleotides from the Kpnl O and part of Kpnl K fragments in the short unique region of infectious laryngotracheitis virus (ILTV) was determined. The sequence contained two complete and one partial open reading frames (ORFs). The partial ORF was open at the 5' end of the sequence and represented the NH2-terminal 118 amino acids (aa) of a polypeptide. Its partial predicted protein product exhibited significant homology to the US2 gene product of HSV-1 (herpes simplex virus type 1) and it homologs in other herpesviruses. ORF 2 is 471 aa long and could encode a protein of 53.8 kDa which shared aa homology with the protein kinases encoded by HSV-1 US3 and its gene homologs. Analysis of the ORF 2 aa sequence revealed domains characteristic of protein-serine/threonine (S/T) kinases of cellular and viral origin. The ORF 3 encoded a predicted protein of 601 aa (M(r) 67.5 kDa) which exhibited limited homology (18% overall identity) with the UL47 protein (major tegument protein) of HSV-1. Northern (RNA) blot hybridization and metabolic inhibitors were used to characterize the ILTV protein kinase and the 67K mRNAs. The data revealed that protein kinase is a gamma-1 gene encoding a 1.6 mRNa, while the 67K ORF is a gamma-2 gene encoding a 2 kb mRNA.

DNA Sequence of a Simian Varicella Virus Gene That Encodes a Homologue of Varicella Zoster Virus IE62 and Herpes Simplex Virus ICP4

Clinical and biological studies indicate that simian varicella virus (SVV) infection of primates is the counterpart of human varicella zoster virus (VZV) infection. We have identified an SVV open reading frame (ORF) that is homologous to the VZV protein IE62 and herpes simplex virus (HSV) ICP4. Like the genes encoding the VZV and HSV proteins, the SVV gene is located in the repeat region of the virus genome. Its expression in SVV-infected cells yields a 4.2-kb transcript. DNA sequencing of the SVV gene reveals a coding region 3834 nucleotides in length and a G + C content of 60.3%. Like ICP4 homologues in other herpesviruses, the SVV protein has five regions, two of which (regions 2 and 4) are highly conserved. The SVV protein also contains a run of serine residues in its amino terminus that is characteristic of herpesvirus ICP4 homologues. The SVV protein shows an overall homology of 24% to HSV ICP4 and 53% to VZV IE62.