Sequencing of a 5.5-kb DNA Fragment and Identification of a Gene Coding for a Subunit of the Helicase/Primase Complex of Avian Laryngotracheitis virus (ILTV)

The nucleotide sequence of a 5,520-bp EcoRI restriction fragment of avian infectious laryngotracheitis (ILTV) DNA was reported and submitted to GeneBank with an accession number of AF001078. Computer prediction revealed one large potential open reading frame (ORF) with sequence similar to one subunit of the DNA helicase-primase complex of α-herpesviruses. The DNA helicase/primase complex of HSV-1 consists of three sub-units with molecular weights of 12,000, 97,000 and 70,000, encoded by genes UL52, UL5 and UL8, respectively. This enzyme complex is essential for herpesvirus DNA replication. The UL52 and UL5-equivalent genes of ILTV have been reported previously (Fuchs, W. and Mettenleiter, T.C., J Gen Virol, 1996, 77: 2221–2229; Johnson, M.A. et. al., Arch Virol, 1995, 14: 623–634). Amino acid sequence comparison and homology search revealed that this ORF shares sequence similarity to the UL8-equivalent gene of α-herpesviruses, that is, the ORF 52 of vericura-zoster virus (VZV), the ORF 54 of equine herpesvirus type-1 (EHV-1), as well as the equivalent gene of bovine herpesvirus type 1(BHV-1) and canine herpesvirus (Vlcek, C. et al., Virology, 1995, 210: 100–108; Remond, M. et al., J Gen Virol, 1996, 77: 37–48). This revised version was published online in July 2006 with corrections to the Cover Date.     

Open reading frames encoding a protein kinase, homolog of glycoprotein gX of pseudorabies virus, and a novel glycoprotein map within the unique short segment of equine herpesvirus type 1.

DNA sequence analysis of the unique short (Us) segment of the genome of equine herpesvirus type 1 Kentucky A strain (EHV-1) by our laboratory and strains Kentucky D and AB1 by other workers identifies a total of nine open reading frames (ORF). In this report, we present the DNA sequence of three of these newly identified ORFs, designated EUS 2, EUS 3, and EUS 4. The EUS 2 ORF is 1146 nucleotides (nt) in length and encodes a potential protein of 382 amino acids. Cis-regulatory sequences upstream of the putative ATG start codon include a G/C box 112 nt upstream and two potential TATA-like elements located between 15 and 90 nt before the ATG. The EUS 2 translation product exhibits significant homology to Ser/Thr protein kinases encoded within the Us segments of other herpesviruses, such as herpes simplex virus (26% homology) and pseudorabies virus (PRV), (45% homology), and possesses sequence domains conserved in protein kinases of cellular and viral origin. The EUS 3 ORF begins 127 nt downstream from the EUS 2 stop codon and ends at a stop codon 1119 nt further downstream. A single TATA-like element maps 61 nt upstream of the ORF. This ORF encodes a potential protein of 373 amino acids and is a homolog of glycoprotein gX of PRV, as judged by overall homology of amino acid residues, cysteine displacement, and presence of potential glycosylation sites and signal sequence. Interestingly, the EUS 4 ORF encodes a potential membrane glycoprotein that does not exhibit homology to any reported protein sequence. The EUS 4 ORF encodes a 383 amino acid polypeptide with a sequence indicative of a signal sequence at its amino terminal end, glycosylation sites for N-linked oligosaccharides, and a transmembrane domain near its carboxyl terminus. Several cis-acting regulatory sequences lie upstream of this ORF. These findings support the observation that the short region of alphaherpesviruses show considerable variation in their genetic content and gene organization.     

The complete genome sequence of gill-associated virus of Penaeus monodon prawns indicates a gene organisation unique among nidoviruses

We report here a 5596 nt sequence comprising the 3'-end of the (+) ssRNA genome of gill-associated virus (GAV), an invertebrate nidovirus of Penaeus monodon prawns. The sequence extends from a subgenomic RNA start site 35 nt upstream of the 4923 nt ORF3 gene to a 3'-poly(A) tail of the 26235 nt genome of GAV. The putative 1640 amino acid (aa) ORF3 protein (MW = 182049 Da, pI = 6.62) contains 15 potential N-linked glycosylation sites, 15 potential O-linked glycosylation sites and six highly hydrophobic regions predicted to represent transmembrane (TM) domains. Three of the predicted TM domains occur in the amino-terminal 228 aa, two in the central portion, and one near the carboxy-terminus of ORF3. Only one short (83 aa) open reading frame (ORF4) was identified between ORF3 and the 3'-poly(A) tail. Completion of the genome sequence of GAV has revealed a gene organisation unique among nidoviruses in there is no discrete membrane protein gene and that the putative ORF3 spike glycoprotein gene resides downstream of a gene (ORF2) encoding a structural protein associated with nucleocapsids.     

Nucleotide sequence of the gene encoding infectious laryngotracheitis virus glycoprotein B.

The nucleotide sequence of the infectious laryngotracheitis virus (ILTV) gene encoding the 205K complex glycoprotein (gp205) was determined. The gene is contained within a 3-kb EcoRI restriction fragment mapping at approximately map coordinates 0.23 to 0.25 in the UL region of the ILTV genome and is transcribed from right to left. Nucleotide sequence analysis of the DNA fragment identified a single, long open reading frame capable of encoding 873 amino acids. The predicted precursor polypeptide derived from this open reading frame would have a calculated Mr of 98,895 Da and contains nine potential glycosylation sites. Hydropathic analysis indicates the presence of an amino terminal hydrophobic sequence and hydrophobic carboxyl terminal domain which may function as a signal peptide and a membrane anchor sequence, respectively. Comparison of the predicted ILTV gp205 protein sequence with those of other herpesviruses revealed a significant sequence similarity with gB-like glycoproteins. Extensive homology was observed throughout the molecule except for the amino and carboxyl termini. The high homology in predicted primary and secondary structures is consistent with the essential role of the gB family of proteins for viral infectivity and pathogenesis.     

Structure and evolution of the largest chloroplast gene (ORF2280): internal plasticity and multiple gene loss during angiosperm evolution

We have determined the nucleotide sequence of the Pelargonium x hortorum ORF2280 homolog, the largest gene in the plastid genome of most land plants, and compared it to published homologs from Nicotiana tabacum, Epifagus virginiana, Spinacia oleracea, and Marchantia polymorpha. Multiple alignment of protein sequences requires an extraordinary number of gaps, indicating a very high frequency of insertion/deletion events during the evolution of the protein; however, the overall predicted size of the protein varies relatively little among the five species. At 2 109 codons, the Pelargonium gene is smaller than other land plant ORF2280 homologs and exhibits a rate of nucleotide substitution several times higher relative to Nicotiana, Epifagus, and Spinacia. Southern-blot and restriction-mapping studies were carried out to uncover length variation in ORF2280 homologs from 279 species (representing 111 families) of angiosperms. In many independent angiosperm lineages, this gene has sustained deletions ranging in size from 200 bp to almost 6 kb. Based on the severity of deletions, we postulate that the chloroplast homolog of ORF2280 has become nonfunctional in at least four independent lineages of angiosperms.     

Sequence analysis of aMolluscum contagiosum virus DNA region which includes the gene encoding protein kinase 2 and other genes with unique organization

The nucleotide sequence of a near left-terminal region from the genome ofMolluscum contagiosum virus subtype I (MCVI) was determined. This region was contained within three adjacentBamHI fragments, designated L (2.4 kilobases (kb)), M (1.8 kb), and N (1.6 kb).BamHI cleavage of MCVI DNA produced another 1.6-kb fragment (N), which had been mapped 30–50 kb from the L,M region. The MCVI restriction fragments were cloned and end-sequenced. The N fragment that maps at the L,M region was identified by the polymerase chain reaction, using primers devised from the sequence of each fragment. The results from this analysis led to establish the relative position of these fragments within the MCVI genome. The analysis of 3.6 kb of DNA sequence revealed the presence of ten open reading frames (ORFs). Comparison of the amino acid sequence of these ORFs to the amino acid sequence of vaccinia virus (VAC) proteins revealed that two complete MCVI ORFs, termed N1L and L1L, showed high degree of homology with VAC F9 and F10 genes, respectively. The F10 gene encodes a 52-kDa serine/threonine protein kinase (protein kinase 2), an essential protein involved in virus morphogenesis. The MCVI homologue (L1L) encoded a putative polypeptide of 443 aa, with a calculated molecular mass of 53 kDa, and 60.5/30.2% sequence identity/similarity to VAC F10. The MCV N1L (213 aa, 24 kDa) showed 42.6/40.6% amino acid sequence identity/similarity to VAC F9, a gene of unknown function encoding a 24-kDa protein with a hydrophobic C-terminal domain, which was conserved in MCVI. The genomic arrangement of MCVI N1L and L1L was equivalent to that of the vaccinia and variola virus homologues. However, the ORFs contained within MCVI fragment M (leftward) showed no homology, neither similarity in genetic organization, to the genes encoded by the corresponding regions of vaccinia and variola viruses.The contribution to this paper by Antonia Martin-Gallardo and Marta Moratilla is equal, and the order of authorship is arbitrary.     

Identification, cloning and sequence analysisof the equine adenovirus 1 hexon gene

Summary.  Based on sequence homology with human adenovirus 2 (HAdV2), the hexon gene of equine adenovirus 1 (EAdV1) was identified. HindIII restriction fragments containing the hexon and other viral genes were cloned into the plasmids pUC19 and pBlueScript SK(-) and sequenced. The nucleotide sequence of the hexon gene was completely determined and partial sequence data were obtained for seven other EAdV1 genes. Amino acid (aa) sequence comparison with published adenovirus (AdV) proteins identified the genes for the IIIa, penton, pVII, pVI, 23K proteinase, DNA binding and 100K proteins. The eight EAdV1 genes appeared to be in the same relative order as homologous genes of other AdV. The EAdV1 hexon protein was encoded the hexon-associated pVI upstream and the 23K proteinase gene downstream and comprised 2 742 nucleotides which translated into 913 aa. Similar to other members of the genus Mastadenovirus the EAdV1 hexon yielded two highly conserved genome segments at the N- and C-termini which flanked intermediate variable and hypervariable regions. The majority of the residue differences between EAdV1 and other AdV hexons occurred in two loops that are known for other AdV to protrude from the surface of the nucleocapsid. Amino acid comparisons with other AdV hexons revealed highest homology with HAdV12 hexon with 72% identical and 83% functionally similar residues, followed by bovine AdV3 hexon with 71% identities and 82% functional residue conservation. Phylogenetic analysis suggested that EAdV1 and other AdV do not have an immediate common ancestor. Accepted December 13, 1996 Received October 9, 1996     

Conservation of a gene cluster including glycoprotein B in bovine herpesvirus type 2 (BHV-2) and herpes simplex virus type 1 (HSV-1)

A library of subgenomic fragments of bovine herpesvirus type 2 (BHV-2) DNA was constructed in the expression cloning vector lambda gt11 and screened with monoclonal antibodies to the glycoprotein gb BHV-2, which is homologous to glycoprotein gB (gB-1) of herpes simplex virus type 1 (HSV-1). Lambda gt11 clones containing gB BHV-2-specific sequences were used to identify lambda EMBL3 vectors with DNA inserts which contained the complete gB BHV-2 gene. Nucleotide sequencing revealed that the gB BHV-2 gene is highly conserved compared to gB-1. The amino acid sequences and the predicted secondary structures of both glycoproteins are very similar. Two further open reading frames (ORF) in close vicinity to the gene encoding gB BHV-2 showed considerable homology to HSV-1 genes. They code for the major DNA-binding protein (dbp) of BHV-2 and a putative 72-kDa polypeptide. The gene of the latter protein corresponding to ICP18.5 of HSV-1 is interspersed between the ORFs of gB BHV-2 and the dbp of BHV-2. All three genes map in the unique long region of the genome. Their homology and the colinear arrangement compared to HSV-1 indicate a close relationship between the two viruses.     

Identification of an infectious laryngotracheitis virus equivalent to the herpes simplex virus type 2 major DNA binding protein (ICP8)

A region of homology between the genomic DNA of infectious laryngotracheitis virus (ILTV), an avian herpesvirus, and the region encoding the herpes simplex virus type 2 (HSV-2) major DNA binding protein (ICP8) has been detected by the use of nick translation and Southern blot analysis. Further, a monoclonal antibody directed against the HSV-2 ICP8 protein detected has antigenic cross-reaction with ILTV as demonstrated by indirect immunofluorescence.     

The nucleotide sequence of the gB glycoprotein gene of HSV-2 and comparison with the corresponding gene of HSV-1

The nucleotide sequence of the gB glycoprotein gene of HSV-2 has been determined and compared with the homologous gene of HSV-1. The two genes are specified by the same total number of codons (904); eight additional codons of the HSV-1 gene are found within the signal sequence, and eight additional codons of the HSV-2 gene are found at three different sites in the gene. The signal cleavage, membrane-spanning, and eight potential N-linked oligosaccharide sites, as well as 5'- and 3'-regulatory signals are largely conserved. The overall amino acid homology is 85%; least conserved are the N- and C-terminal regions of the protein. Secondary structure plots were determined for the two proteins, and the structures were compared with each other and with alterations in structure due to several mutations in the HSV-1 gB gene for which sequence analysis is available. The high homology in primary and secondary structure suggests a conserved, essential function for the gene.