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Transcriptional and structural analyses of Amsacta moorei entomopoxvirus protein kinase gene (AMV197, pk)

Transcriptional and structural analyses of Amsacta moorei entomopoxvirus protein kinase gene... Ann Microbiol (2010) 60:523–530 DOI 10.1007/s13213-010-0082-8 ORIGINAL ARTICLE Transcriptional and structural analyses of Amsacta moorei entomopoxvirus protein kinase gene (AMV197, pk) Hacer Muratoğlu & Remziye Nalçacıoğlu & Zihni Demirbağ Received: 13 April 2010 /Accepted: 21 May 2010 /Published online: 16 June 2010 Springer-Verlag and the University of Milan 2010 Abstract The Amsacta moorei entomopoxvirus (AMEV) Keywords Amsacta moorei entomopoxvirus (AMEV) genome has 279 open reading frames (ORFs) among which Protein kinase Transcriptional analysis is the AMV197, composed of 900 nt and potentially encoding a protein of 299 amino acids. Sequence-derived amino acid analysis suggested it to be a serine/threonine Introduction protein kinase (PK) having conserved PK and serine/ threonine PK domains. For transcriptional analysis of the Poxviruses are a family of large DNA genome viruses AMV197 pk gene, Ld652 cells were infected with AMEV pathogenic for many species of mammals, birds and insects. and mRNA was isolated at different times thereafter. RT– Their genomes are dsDNA molecules with hairpin termini PCR analysis indicated that the transcription of the AMV197 (Moss 2001). The International Committee on the Taxon- pk gene started at 4 h post infection (h p.i.) and continued to omy of Viruses has divided Poxviridae family into two be expressed through 24 h p.i. Infection of Ld cells in the subfamilies: the Chordopoxvirinae (poxviruses of verte- presence of Ara-C (inhibits DNA replication), followed by brates) and the Entomopoxvirinae (insect poxviruses). The RT–PCR showed that AMV197 pk is transcribed as an early subfamily Entomopoxvirinae is a related but distinct gene. Transcription was initiated at 54 nt upstream of the member of the Poxviridae family. These viruses share translation start site. The vaccinia virus early promoter many biological features of the poxviruses of chordates, but element G was also found at the correct position (−21) in instead infect the larvae of a number of insect families. the AMV197 pk gene. Rapid amplification of the 3′ ends of Entomopoxviruses (EPVs) have been isolated from several the AMV197 pk transcript showed that there are two insect orders including Coleoptera, Lepidoptera, Orthop- polyadenylation start points. They are located at 22 and 32 tera, and Diptera (Arif 1995). EPVs have structural nucleotides downstream of translation stop site. Also, the similarities to orthopoxviruses and a number of vertebrate translational stop site and poly (A) signal of AMV197 pk are poxvirus gene homologues have been found in entomo- overlapped. The termination signal TTTTTGT sequence of poxviruses (Bawden et al. 2000). Because of this homol- vaccinia virus early genes was found just upstream of the 3′ ogy, it is reasonable to expect similarities in gene regulation end of AMV197 pk gene. Conserved amino acid subdomains at both groups. of the AMV197 PK were found by sequence comparisons Amsacta moorei entomopoxvirus (AMEV), genus with PK’s from other organisms. Analysis of the protein betaentomopoxvirus, has been reported to infect agricul- sequence of AMV197 pk gene reveals close identity with PK turally important pests, such as Estigmena acrea and genes of other organisms. Lymantria dispar included lepidopteran insects. A char- acteristic pathology is associated with each of the four orders of insects infected. Thecourseofinfectionin : : H. Muratoğlu R. Nalçacıoğlu Z. Demirbağ (*) lepidopteran larvae is relatively rapid, ranging from 1 to Faculty of Arts and Sciences, Department of Biology, 3 weeks. Symptoms of the disease vary among hosts. For Karadeniz Technical University, example, Estigmena acrea larvae infected with AMEV 61080 Trabzon, Turkey show little signs until late in the infection, when motility e-mail: zihni@ktu.edu.tr 524 Ann Microbiol (2010) 60:523–530 and coordination are adversely affected. EPV-infected Our results show that the AMV197 pk gene is an early Elasmopalpus ligosellus larvae change color, from gene whose transcripts are detected at 4–24 h post infection brown-striped to red, with hemolymph becoming (h p.i.). It has 54 nt region as 5′ UTR and 32/22 nt regions whitish-blue, possibly because of the accumulation of as 3′ UTRs. We also investigated the positions of the spheroid (a single major structural polypeptide) occlusion protein kinase subdomains in the PK sequence. bodies (Roberts and Granados 1968). The 232-kb AMEV genome has been completely se- quenced and contains 292 open reading frames (ORFs). Materials and methods Among these ORFs, AMV153 and AMV197 suggested it to be a serine/threonine protein kinase (PK) having conserved Cell culture and virus Lymantria dispar (Ld652) cell line PK and serine/threonine PK domains (Bawden et al. 2000). used in this study was obtained from Basil Arif and Serine/threonine protein kinases are phospho-transferases maintained in Excell-420 (SAFC Biosciences) and Grace’s that transfer the phosphate groups onto serine and/or threonine Insect serum-free medium (Gibco) supplemented with 10% residues of protein substrates. Improper functioning of these heat-treated fetal bovine serum (Gibco). Penicillin (50 IU) enzymes is often manifested in several malignancies since and streptomycin (50 µg/ml) were added to cell line growth modification of proteins by phosphorylation at a limited media to prevent microbial contamination. AMEV was number of amino acid residues is identified as a general kindly supplied by Basil Arif. Replication of AMEV in the method of controlling activities of protein synthesis, cell cell line has been described previously (Goodwin et al. division and modulation of metabolic enzymes (Hunter 1987) 1990). For virus production, T75 flasks containing Ld652 in eukaryotes and prokaryotes. Phosphorylation of numerous cells at a density of 9 × 10 cells/ml were infected with wild cellular and viral proteins is also observed in virally infected type AMEV at a low m.o.i (0.5 p.f.u./cell) The virus was cells, which suggests that protein kinases may have a role in harvested when the cytopathic effect was complete at 4-day regulating a wide variety of viral infections (Leader and post infection (h p.i.). Subsequently, the supernatant was Katan 1988;Kannet al. 1999). centrifuged at 1,000 × g for 5 min to remove intact cells The PK of some vertebrate viruses is known to be and cellular debris. The resulting supernatant contained the packaged within the virion particle and shown to phosphor- extra-cellular virus was stored at 4°C. Virus titer was ylate a variety of proteins of viral and non-viral origin (Lin et determined by end point dilution assay (EPDA) (Darling et al. 1992; Rempel and Traktman 1992; Lin and Broyles al. 1998). 1994). Viruses often utilize many of the regulatory mecha- nisms as cells, and this is particularly evident for the Messenger RNA (mRNA) isolation Ld652 cells were seeded poxviruses, such as vaccinia virus. DNA sequence analysis at a density of 1×10 /T75 flask and infected at an m.o.i of 2 suggests that vaccinia virus may encode a protein kinase. with AMEV. At 2 h p.i, the medium was removed and The 34-kDa protein predicted from the sequence of the viral replaced with fresh medium. Cells were harvested at gene BlR bears a striking similarity to the catalytic domains various times after infection (0, 1, 2, 4, 7, 12, and 24 h) of known protein kinases (Howard and Smith 1989; Trakt- and pelleted at 300 × g for 5 min. The pellet was washed man et al. 1989; Goebeletal. 1990). Most of the amino acids with phosphate-buffered saline (1×PBS). Messenger RNA known to be conserved in the protein kinase family are (mRNA) was isolated with the PolyATtract System 1000 kit located in the appropriate sites of the sequence predicted for (Promega) following the manufacturer’s instructions and the B1R protein. This protein kinase is expressed early in quantified at 260 nm. An aliquot of 10 µg mRNA was infections, is found in the virosomes, and is also packaged treated with 200 U of RNAse free DNase I at 37°C for into virions (Banham and Smith 1992). It appears to be an 30 min to remove any residual DNA and then extracted essential viral protein, and temperature-sensitive mutations with phenol-chloroform and quantified after precipitation. that map to the B1R gene produce virus that cannot replicate its DNA at the restrictive temperature (Rempel et al. 1990; Time course analysis of AMV197 pk specific transcripts D- Rempel and Traktman 1992). Nase I treated mRNA preparations were screened for the Amino acid alignment of AMV197 encoding a protein of presence of AMV197 pk gene-specific transcripts by RT– 299 amino acids showed that this gene is an ortholog of PCR. Briefly, 2 µg of mRNA and 75 µM reverse oligo (dT) B1R. However, there are no data on PKs of entomopoxvi- anchor primer (Roche) were denatured at 70°C for 5 min ruses in literature. As part of our continuing work on and then placed on ice. First-strand cDNA was synthesized functional analysis of AMEV genome, we report here on by the addition of 2.5 µl M-MuLV 10X buffer, 2 µl of structural and transcriptional analyses of AMV197 pk gene 10 mM dNTPs, 100 U M-MuLV reverse transcriptase as part of basic studies to understand its function in the (BioLabs). DEPC-treated water was added to make a final context of the infection process. volume of 25 µl. The reaction proceeded at 37°C for 1 h, Ann Microbiol (2010) 60:523–530 525 followed by heating at 70°C for 15 min. PCR was dT anchor primer (Roche). PKSP3R primer was used for performed in a 50 ml volume containing 75 mM Tris-HCl the second PCR in combination with a PCR anchor primer (pH 8.8 at 25°C), 20 mM (NH ) SO , 0.1% Tween 20, (Roche). The amplified fragments were cloned into 4 2 4 1.5 mM MgCl , 0.2 mM each deoxynucleoside triphos- pGEMT-Easy (Promega) and 11 clones were analyzed by phate, 0.2 mM primers PKSP4 and PKR (Table 1), 2.5 U of automated sequencing. Taq DNA polymerase, and 1 µl of completed RT-PCR reaction mixture. Amplification consisted of 1 cycle of Determination of the 3′ UTR of AMV197 pk transcript To denaturation at 95°C for 3 min, 35 cycles of denaturation at determine the 3′ terminus of the AMV197 pk transcript, 3′ 95°C for 1 min, annealing at 55°C for 30 s, and extension at RACE was performed. The first strand cDNA was 72°C for 1 min, and a final cycle of elongation at 72°C for synthesized from mRNA using oligo (dT) anchor primer 7 min. PCR amplification was also done on mRNA isolated and M-MuLV reverse transcriptase. Then, the first strand from non-infected cells to confirm that there was no cellular cDNA was amplified by PCR using a gene specific primer contamination. PKSP4 and a PCR anchor primer corresponding to the oligo (dT) anchor primer (Table 1). The first amplification The temporal class analysis of the AMV197 pk tran- product was then used as a template for the second PCR script Ld652 cells were infected with AMEV at a multi- amplification with the second gene specific primer PKSP5R plicity of infection of 2. In order to inhibit DNA synthesis, and PCR anchor primer. The PCR product was isolated cells were continuously exposed to cytosine arabinoside from gel, cloned in pGEM-T (Promega) and 13 clones were (Ara-C) (100 µg/ml; Sigma) starting at 1 h prior to virus sequenced. infection. The inhibitor was maintained at this level throughout the infection. At 0 and 16 h p.i., cells were Amino acid sequence comparison The predicted amino harvested, and mRNAs were isolated from virus-infected acid sequence of AMEV was examined by using Expasy- cells in the presence and absence of Ara-C by using Prosite program and compared to those of other PK family PolyATtract System 1000 kit (Promega) according to the members (2 viral PKs, 2 PKs from vertebrates and a PK manufacturer’s protocol. The isolated mRNAs were DNase from S. cerevisiae) with the aid of Clustal W program. treated, quantified at 260 nm, subjected to RT-PCR and subsequently to PCR as above. RNA samples from mock infected cells and 0 h p.i. were processed as control. Results Determination of the 5′ UTR of AMV197 pk transcript The Time course transcriptional analysis of AMV197 pk gene To determine whether the putative AMV197 gene 5′ untranslated region of the AMV197 pk mRNA was obtained by rapid amplification of the cDNA ends (Froh- was expressed during viral infection, mRNA was extracted man et al. 1988) using a 5′ RACE kit (Roche) following the from AMEV infected Ld652 cells at 0, 1, 2, 4, 7, 12, and procedure supplied by the manufacturer with a set of three 24 h p.i. and from mock infected cells. The samples were specific primers. First strand cDNA was synthesized from analyzed by RT-PCR. A single band with an expected size 2 µg mRNA isolated at 16 h p.i. using gene-specific primer of 521 bp as pk transcript was first detected at 4 h p.i., (PKSP1R; see Table 1). The first-strand cDNA was then isolated and tailed with dA. This was followed by two consecutive nested PCRs with specific primer (see Table 1). PKSP2R primer was used for the first PCR with an oligo Table 1 Primers used for 5′ RACE (PKSP1R, PKSP2R, and PKSP3R), 3′ RACE (PKSP4, PKSP5) and RT–PCR (PKSP4, PKR) experiments Primer name Sequences PKSP1 5′-CAT AAT AAT CTA TTA CTA TAT ATC-3′ Fig. 1 Transciptional pattern of AMV197 pk gene detected byRT– PKSP2 5′-CCA TTA TGA TAT AGT AAT GGT ATT CC-3′ PCR analysis. M represents a 100-bp ladder DNA marker (Promega). PKSP3 5′-GCA TTT CTA CAA ACA ACG G-3′ Ld indicates DNA amplified from the RNA from mock infected PKSP4 5′-CCG TTG TTT GTA GAA ATG C-3′ Ld652 cells. Numbers show time points hours (h) post-infection for PKSP5 5′-CAA CAC TTG TTT AAC TAA TTC ACC-3′ each RNA samples extracted from infected cells. C shows negative control for the PCR. The amplicons were electrophoresed in a 1.2% PKR 5′-GGC AAA CTT CCA CTA TAC C-3′ agarose gel, and their sizes (in base pairs) are indicated at the right 526 Ann Microbiol (2010) 60:523–530 infected with AMEV in the presence or absence of Ara-C, an inhibitor of DNA replication. mRNA was extracted from cells at 16 h p.i. and analyzed for the presence of AMV197 transcript by RT–PCR (Fig. 2). AMV197 transcript was observed at this time point (Fig. 2, lane 3) and, apparently, it was not affected by the presence of inhibitor of DNA (Fig. 2, lane 5). Because viral DNA replication is not required for the expression of the AMV197 gene, it belongs by definition to an early gene class. This result is in agreement with other viral pk gene’s transcription classes (Liu et al. 2001; Gershburg et al. 2007). Transcription initiation site of AMV197 pk gene The transcription start site for AMV197 gene was identified by 5′ RACE analysis using mRNA extracted from infected cells at 16 h p.i. 5′ RACE was performed using three Fig. 2 Temporal class of AMV197 pk gene determined by RT PCR specific primers (PKSP1R, PKSP2R, and PKSP3R) analysis in the presence of DNA synthesis inhibitor. M represents a (Table 1). Amplified 5′ ends of cDNA fragments were 100-bp ladder DNA marker (Promega; lane 1). mRNA’s extracted cloned in pGEMT-Easy vector and 11 clones were from Ld652 cells in the absence (lane 2) or presence (lane 4) of Ara-C and from AMEV-infected Ld652 cells in the absence (lane 3)or sequenced. The 9 clones obtained for AMV197 showed presence (lane 5) of Ara-C were subjected to RT–PCR analysis using that the transcription initiation site (+1) is at the adenine primers specific for the AMV197 pk gene. The amplicons were (A) located 54 nt upstream of the translational start site electrophoresed in a 1.2 % agarose gel, and their sizes (in base pairs) (Fig. 3). are indicated at the right Transcription termination site of AMV197 pk gene To determine the 3′ terminus site of the AMV197 gene, a 3′ reached to maximum level at 7 h p.i. and started to decrease after this point (Fig. 1). RACE was performed on mRNA. The first strand cDNA was synthesized using the oligo(dT)-anchor primer and M- The transcriptional class analysis of the AMV197 pk MuLV reverse transcriptase. The amplification of the 3′ gene In order to determine the expression class, we RACE PCR products were cloned and 13 clones were employed an RT–PCR approach to detect the transcription- sequenced. The polyadenylation starts at 22 nt downstream al products of AMV197. To that aim, Ld652 cells were of the translational stop point in 8 clones and 32 nt Fig. 3 The locations of transcription initiation and termination sites of at the promoter region of vaccinia virus early genes. The poly (A) AMV197 pk gene. The translation start and stop codons are shaded addition sites that are obtained by sequencing of 3′RACE clones are gray. The dashed arrow indicates the 5′ termini (transcriptional start indicated by the straight arrows. The box indicates poly (A) signal points) of AMV197 pk gene revealed by sequencing of 5′ RACE which is overlapped with the translation stop codon and the dashed clones. The bold G at −21 shows the universal G residue that is found box indicates transcription termination signal Ann Microbiol (2010) 60:523–530 527 Fig. 4 Comparison of the amino acid sequence of AMV197 PK with poxvirus protein kinase (Accession no: AF063866); PIM-1, Mus other PK’s of other organisms. The sites of the conserved catalytic musculus protein kinase (Accession no: AAA39930); MOS, serine/ subdomains are numbered with Roman numbers and shaded. AMEV- threonine-protein kinase-transforming protein (mos) of Mouse (Ac- PK, Amsacta moorei entomopoxvirus protein kinase (Accession no: cession no: P00538); CDC28, cell division control protein 28 of S. NC002520); VACV-B1-RPK, Vaccinia virus protein kinase (Acces- cerevisiae (Accession no: P00546) sion no: YP233065); MSEV-PK, Melanoplus sanguinipes entomo- downstream of the translational stop point in 5 clones of B1R). Major subdomains conserved in various PKs were sequenced 13 clones as shown in Fig. 3. also readilyidentifiedonthe AMV197,but afew subdomains were difficult to be ascertained (Fig. 4). Amino acid sequence alignment of AMV197 pk gene When the deduced amino acid sequence of AMV197 pk gene was At the N-terminal extremity of the catalytic domain, there is a analyzed in Expasy-Prosite, it was observed that the region (subdomain I) containing a glycine (G)-rich stretch of location of protein kinase catalytic domain is between residues (21GFGTVY26) and nearby there is the lysine residues of 12 and 299. residue (42 K) of subdomain II (39–42). The region from The amino acid sequence of AMV197 PK was also subdomain I to II has been shown to be involved in ATP compared with other PK family members (with especially binding (Hanks and Hunter 1995). In the PROSITE database, 528 Ann Microbiol (2010) 60:523–530 this ATP binding region is one of only two regions that have methyltransferase (Martin et al. 1975; Barbosa and Moss been selected to build a protein kinase signature. The 1978) into virions, retention of a protein kinases had not sequence (aa 21–42) in this region matches (although not previously been shown. The studies on poxvirus protein exactly) the protein kinase signature consensus pattern I: kinases were initially conducted on vaccinia virus B1R and [LIV]-G-{P}-G-{P}-[FYWMGSTNH]-[SGA]-{PW}-[LIV- F10L protein kinases. They were followed by investigations CAT]-{PD}-x-[GSTACLIVMFY]-x-(5,18)-[LIVMFYWC- on other poxvirus PKs; Fowlpox virus (FWPV-PK, Poxvir- STAR]-[AIVP]-[LIVM FAGCKR]-K, where K is the residue idae; Afonso et al. 2000) Myxoma virus (MYXV-PK, that binds ATP (square brackets are used to explain the Poxviridae; Cameron et al. 1999), Molluscum contagiosum acceptable alternative amino acids for a given position and virus subtype 1 (MOCV1-PK, Poxviridae; Senkevich et al. the curly brackets explain the amino acids that are not 1996), and Variola major virus (VARV-PK, Poxviridae; acceptable at a given position). The sequences of 54E and Massung et al. 1993). However, to date, data on the 98YIV100 show homology to subdomains III and IV, Amsacta moorei Entomopoxvirus (AMEV) protein kinase respectively. Subdomain V is not clearly discernable. (pk) gene have not been reported. Subdomain VI is highly conserved among protein In order to study the mechanisms controlling AMEV kinases and is used to build the protein kinase signature gene regulation, we have, as a first step, initiated consensus pattern II. The sequences 142YTHNDIKKN- transcriptional and structural analyses of the pk gene. NIMF154 matches well with the protein kinase signature Based on the temporal expression, the AMV197 pk gene consensus pattern II, [LIVMFYC]-x-[HY]-x-D-[LIVMFY]- was classified as an early gene, just as B1R belongs to vaccinia K-x-(2)-N-[LIVMFYCT] (3), where the aspartic acid virus. The transcripts were detected early after infection (by 4 h residue (146D) is probably the residue important for the p.i. and maximized at 7 h p.i.) a time relegated primarily to the catalytic activity of the enzyme (Knighton et al. 1991; Liu synthesis of non-structural proteins, including enzymes in- et al. 2001). The residue at position 148 is K, indicating that volved in replicating the genome and modifying DNA, RNA, it is a serine/threonine specific protein kinase. Subdomain and proteins (http://www.virustaxonomyonline.com/virtax/ VII is found in a highly conserved region, 163LIDYG167. lpext.dll?f=templates&fn=main-h.htm). Subdomain VIII has a highly conserved triplet, A-P-E, in The transcriptional start and stop sites were determined most protein kinases. This region is thought to lie close to the by 5′ and 3′ RACE analysis. 5′ RACE showed that catalytic site and to flank a common autophosphorylation AMV197 pk gene has a short 5′ UTR of 54 nt. Also, the receptor site (Selten et al. 1986; Hunter 1987). No identical promoter region has a universal G residue at −21 flanked by sequence is found within the AMV197 PK sequence. Another variable and A-T-rich sequence (Fig. 4). This sequence sequence, T-L-E, in the appropriate region has been found and matches well with the vaccinia virus early promoter thought to be subdomain VIII in accordance with the vaccinia element identified by Davison and Moss (1989). 34-kDa protein kinase subdomain VIII (Traktman et al. 1989). The 3′ UTR results revealed the transcripts with In this study, the possible change of the APE motif to TLE is heterogeneous 3′ ends were derived from cleavage sites clearly explained. It is found that this motif within published starting 22 nt and 32 nt downstream of the translation kinase sequences reveals some heterogeneity and demon- stop site, which overlaps the polyadenylation signal strates that the final glutamic acid is the only invariant residue AAUAAA. RNA polyadenylation plays an important (Selten et al. 1986). Replacement of alanine with other neutral role in the control of viral and eukaryotic gene expression residues (glycine, leucine and proline) has been observed. and involves cleavage of the nascent transcript and Alanine (neutral), leucine, and isoleucine (nonpolar) have addition of a poly (A) tail with 150–200 adenylate been found as substitutes for the central proline. Subdomain residues (Gilmartin 2005). One cleavage site for the IX is thought to be placed at the sequences 206DIESLM211, AMV197 transcripts was mapped to nt 170167. The other but may also include the 212YNIIEWYSG220 sequence. cleavage site was mapped downstream of the first one, Subdomains X and XI are not so clearly defined in AMV197 with only ten nucleotides separating them at nt 170177 PK but amino acid residues 221 to 225 may potentially (Fig. 4). The heterogeneity of the cleavage site usage in consititute subdomain X. AMV197 transcripts may simply reflect selection flexi- bility, which is common in mammals (Pauws et al. 2001). We examined the AMV197 gene sequence for the Discussion presence of termination signal (TTTTTGT) normally found just upstream of the 3′ ends of early poxvirus mRNAs Although poxviruses are known to incorporate enzymes (Yuen and Moss 1987). However, no termination signal was such as DNA-dependent RNA polymerase (Baroudy and found at that region. Further analysis of the AMV197 pk Moss 1980; Shchelkunov et al. 1993), poly (A) polymerase gene sequence showed that the termination consensus (Moss et al. 1975), mRNA guanyltransferase and mRNA sequence is located in the coding sequence of AMV197 Ann Microbiol (2010) 60:523–530 529 Arif MB (1995) Recent advances in the molecular biology of (Fig. 4) as in the case of vaccinia virus early gene and entomopoxviruses. J Gen Virol 76:1–13 flanked by the polyadenylation signal. Banham AH, Smith GL (1992) Vaccinia virus gene B1R encodes a Protein kinase catalytic domains range from 250 to 300 34-kDa serine/threonine protein kinase that localizes in amino acid residues, corresponding to 30 kDa. Fairly precise cytoplasmic factories and is packaged into virions. Virology 191:803–812 boundaries for the catalytic domains have been defined Barbosa E, Moss B (1978) mRNA (nucleoside-29-)-methyltransferase through an analysis of conserved sequences as well as by from vaccinia virus. Characteristics and substrate specificity. J assays of truncated enzymes (Hanks et al. 1988). The location Biol Chem 253:7698–7702 of the catalytic domain within the protein is not fixed but, in Baroudy BM, Moss B (1980) Purification and characterization of DNA-dependent RNA polymerase from vaccinia virions. J Biol most single subunit enzymes, it lies near the carboxyl Chem 225:4372–4380 terminus, the amino terminus being devoted to a regulatory Bawden AL, Glassberg KJ, Diggans J, Shaw R, Farmerie W, Moyer role. In protein kinases having a multiple subunit structure, RW (2000) Complete genomic sequence of the Amsacta moorei subunit polypeptides consisting almost entirely of a catalytic entomopoxvirus: analysis and comparison with other poxviruses. Virology 274:120–139 domain are common. The AMEV catalytic domain also Cameron C, Hota-Mitchell S, Chen L, Barrett J, Cao JX, Macaulay C, covers almost the entire sequence of the PK gene, starting Willer D, Evans D, McFadden G (1999) The complete DNA from the 12th amino acid. Also, the amino terminus of sequence of myxoma virus. Virology 264:298–318 AMV197 PK catalytic domain lies as close as ten residues Darling AJ, Boose JA, Spaltro J (1998) Virus assay methods: accuracy and validation. Biologicals 26:105–110 from the first conserved glycine, as indicated for some Davison AJ, Moss B (1989) Structure of vaccinia virus early catalytic domain polypeptides by Hanks et al. (1988). promoters. J Mol Biol 210:749–769 A large number of pk-related genes have been sequenced Frohman MA, Dush MK, Martin GR (1988) Rapid production of full- and analyzed for their amino acid sequence patterns. Hanks length cDNAs from rare transcripts: Amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci USA et al. (1988) have reported the identification of 11 conserved 85:8998–9002 regions in the catalytic domains of PKs on the basis of an Gershburg E, Salvatore R, Torrisi MR, Pagano JS (2007) Epstein-Barr alignment of 65 different members of the PK family. In virus-encoded protein kinase (BGLF4) is involved in production AMV197 PK, we clearly identified the subdomains I, II, VI, of infectious virus. J Gen Virol 81:5407–5412 Gilmartin GM (2005) Eukaryotic mRNA 3′ processing: a common VII, VIII and predicted subdomains III, IV, IX and X. means to different ends. Genes Dev 19:2517–2521 However, the subdomains V and XI were difficult to locate. Goebel SJ, Johnson GP, Perkus ME, Davis SW, Winslow JP, Paoletti In this paper, we showed that AMV197 gene belongs to E (1990) The complete DNA sequence of vaccinia virus. early viral gene class by RT–PCR analysis. The vaccinia early Virology 179:247–266 Goodwin RH, Adams JR, Shapiro M (1990) Replication of the promoter region and the transcription termination signal entomopoxvirus from Amsacta moorei in serum-free cultures of a confirm this result. Thus, this gene is transcribed by enzymes gypsy-moth cell line. J Invertebr Pathol 56:190–205 derived and packaged within the virion core (Banham and Hanks SK, Hunter T (1995) Protein kinases 6. The eukaryotic protein Smith 1992). Early gene expression is independent of viral kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J 9:576–596 replication and depends only on previous assembly of a Hanks SK, Quinn AM, Hunter T (1988) The protein kinase family: competent virus particle and a subsequent partial uncoating conserved features and deduced phylogeny of the catalytic of the infecting virus. When the predicted amino acid domains. Science 241:42–52 sequence was compared with those of other PKs, major Howard ST, Smith GL (1989) Two early vaccinia virus genes encode polypeptides related to protein kinases. J Gen Virol 70:3187– subdomains which are conserved in various PKs could be readily identified, but a few subdomains were difficult Hunter T (1987) A thousand and one protein kinases. Cell 50:823– exactly assign. The identified subdomains were homologous to counterparts of PKs of other organisms. However, these Kann M, Sodeik B, Vlachou A, Gerlich WH, Helenius A (1999) Phosphorylation-dependent binding of hepatitis B virus core subdomain sequences presented here are only preliminary particles to the nuclear pore complex. J Cell Biol 145:45–55 data. In order to confirm the validity of these results, genetic Knighton DR, Zheng JH, Ten Eyck LF, Xuong NH, Taylor SS, studies will have to follow. Sowadski JM (1991) Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science 253:414–420 Acknowledgment This work was supported by the Scientific and Leader DP, Katan M (1988) Viral aspects of protein phosphorylation. J Technological Research Council of Turkey, TÜBİTAK with Project Gen Virol 69:1441–1464 No: 106T265. Lin S, Broyles SS (1994) Vaccinia protein kinase 2: a second essential serine/threonine protein kinase encoded by vaccinia virus. Proc Natl Acad Sci USA 91:7653–7657 Lin SQ, Wen C, Broyles SS (1992) The vaccinia virus B1R gene References product is a serine/threonine protein kinase. J Virol 66:2717– Liu WJ, Yu HT, Peng SE, Chang YS, Pien HW, Lin CJ, Huang CJ, Afonso CL, Tulman ER, Lu Z, Zsak L, Kutish GF, Rock DL (2000) Tsai MF, Huang CJ et al (2001) Cloning, characterization, and The genome of fowlpox virus. J Virol 74:3815–3831 530 Ann Microbiol (2010) 60:523–530 phylogenetic analysis of a shrimp white spot syndrome virus Rempel RE, Anderson MK, Evans E, Traktman P (1990) gene that encodes a protein kinase. Virology 289:362–377 Temperature-sensitive vaccinia virus mutants identify a gene Martin SA, Paoletti E, Moss B (1975) Purification of mRNA with an essential role in viral replication. J Virol 64:574–583 guanylytransferase and mRNA (guanine-7-)-methyltransferase Roberts DW, Granados RR (1968) A poxlike virus from Amcasta from vaccinia virions. J Biol Chem 250:9322–9329 moorei (Lepidaptera: Arctiidae). J Invertebr Pathol 12:141 Massung RF, Esposito JJ, Liu LI, Qi J, Utterback TR, Knight JC, Selten G, Cuypers HT, Boelens W, Robanus-Maandag E, Verbeek J, Aubin L, Yuran TE, Parsons JM et al (1993) Potential virulence Domen J, van Beveren C, Berns A (1986) The primary structure determinants in terminal regions of Variola smallpox virus of the putative oncogene pim-1 shows extensive homology with genome. Nature 366:748–751 protein kinases. Cell 32:603–611 Moss B (2001) Poxviridae: the viruses and their replication. In: Knipe Senkevich TG, Bugert JJ, Sisler JR, Koonin EV, Darai G, Moss B (1996) DM, Howley PM (eds) Fields virology. Lippincott Williams & Genome sequence of a human tumorigenic poxvirus: prediction of Wilkins, Philadelphia, pp 2849–2883 specific host response-evasion genes. Science 273:813–816 Moss B, Rosenblum EW, Gershowitz A (1975) Characterization of a Shchelkunov SN, Marennikova SS, Blinov VM, Resenchuk SM, polyadenylate polymerase from vaccinia virions. J Biol Chem Totmenin AV, Chizhikov VE, Guturov VV, Safronov PF, 250:4722–4729 Kurmanov RK, Sandaknchiev LS (1993) Entire coding sequence Pauws E, van Kampen AH, van de Graaf SA, de Vijlder JJ, Ris- of the variola virus. Dokl Akad Nauk 328:629–632 Stalpers C (2001) Heterogeneity in polyadenylation cleavage Traktman P, Anderson MK, Rempel RE (1989) Vaccinia virus encodes sites in mammalian mRNA sequences: implications for SAGE an essential gene with strong homology to protein kinases. J Biol analysis. Nucleic Acids Res 29:1690–1694 Chem 264:21458–21461 Rempel RE, Traktman P (1992) Vaccinia virus B1 kinase:henotypic Yuen L, Moss B (1987) Oligonucleotide sequence signaling transcrip- analysis of temerature-sensitive mutants and enzymatic charac- tional termination of vaccinia virus early genes. Biochem terization of recombinant proteins. J Virol 66:4413–4426 84:6417–6421 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Transcriptional and structural analyses of Amsacta moorei entomopoxvirus protein kinase gene (AMV197, pk)

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Springer Journals
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Copyright © Springer-Verlag and the University of Milan 2010
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
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1590-4261
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10.1007/s13213-010-0082-8
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Abstract

Ann Microbiol (2010) 60:523–530 DOI 10.1007/s13213-010-0082-8 ORIGINAL ARTICLE Transcriptional and structural analyses of Amsacta moorei entomopoxvirus protein kinase gene (AMV197, pk) Hacer Muratoğlu & Remziye Nalçacıoğlu & Zihni Demirbağ Received: 13 April 2010 /Accepted: 21 May 2010 /Published online: 16 June 2010 Springer-Verlag and the University of Milan 2010 Abstract The Amsacta moorei entomopoxvirus (AMEV) Keywords Amsacta moorei entomopoxvirus (AMEV) genome has 279 open reading frames (ORFs) among which Protein kinase Transcriptional analysis is the AMV197, composed of 900 nt and potentially encoding a protein of 299 amino acids. Sequence-derived amino acid analysis suggested it to be a serine/threonine Introduction protein kinase (PK) having conserved PK and serine/ threonine PK domains. For transcriptional analysis of the Poxviruses are a family of large DNA genome viruses AMV197 pk gene, Ld652 cells were infected with AMEV pathogenic for many species of mammals, birds and insects. and mRNA was isolated at different times thereafter. RT– Their genomes are dsDNA molecules with hairpin termini PCR analysis indicated that the transcription of the AMV197 (Moss 2001). The International Committee on the Taxon- pk gene started at 4 h post infection (h p.i.) and continued to omy of Viruses has divided Poxviridae family into two be expressed through 24 h p.i. Infection of Ld cells in the subfamilies: the Chordopoxvirinae (poxviruses of verte- presence of Ara-C (inhibits DNA replication), followed by brates) and the Entomopoxvirinae (insect poxviruses). The RT–PCR showed that AMV197 pk is transcribed as an early subfamily Entomopoxvirinae is a related but distinct gene. Transcription was initiated at 54 nt upstream of the member of the Poxviridae family. These viruses share translation start site. The vaccinia virus early promoter many biological features of the poxviruses of chordates, but element G was also found at the correct position (−21) in instead infect the larvae of a number of insect families. the AMV197 pk gene. Rapid amplification of the 3′ ends of Entomopoxviruses (EPVs) have been isolated from several the AMV197 pk transcript showed that there are two insect orders including Coleoptera, Lepidoptera, Orthop- polyadenylation start points. They are located at 22 and 32 tera, and Diptera (Arif 1995). EPVs have structural nucleotides downstream of translation stop site. Also, the similarities to orthopoxviruses and a number of vertebrate translational stop site and poly (A) signal of AMV197 pk are poxvirus gene homologues have been found in entomo- overlapped. The termination signal TTTTTGT sequence of poxviruses (Bawden et al. 2000). Because of this homol- vaccinia virus early genes was found just upstream of the 3′ ogy, it is reasonable to expect similarities in gene regulation end of AMV197 pk gene. Conserved amino acid subdomains at both groups. of the AMV197 PK were found by sequence comparisons Amsacta moorei entomopoxvirus (AMEV), genus with PK’s from other organisms. Analysis of the protein betaentomopoxvirus, has been reported to infect agricul- sequence of AMV197 pk gene reveals close identity with PK turally important pests, such as Estigmena acrea and genes of other organisms. Lymantria dispar included lepidopteran insects. A char- acteristic pathology is associated with each of the four orders of insects infected. Thecourseofinfectionin : : H. Muratoğlu R. Nalçacıoğlu Z. Demirbağ (*) lepidopteran larvae is relatively rapid, ranging from 1 to Faculty of Arts and Sciences, Department of Biology, 3 weeks. Symptoms of the disease vary among hosts. For Karadeniz Technical University, example, Estigmena acrea larvae infected with AMEV 61080 Trabzon, Turkey show little signs until late in the infection, when motility e-mail: zihni@ktu.edu.tr 524 Ann Microbiol (2010) 60:523–530 and coordination are adversely affected. EPV-infected Our results show that the AMV197 pk gene is an early Elasmopalpus ligosellus larvae change color, from gene whose transcripts are detected at 4–24 h post infection brown-striped to red, with hemolymph becoming (h p.i.). It has 54 nt region as 5′ UTR and 32/22 nt regions whitish-blue, possibly because of the accumulation of as 3′ UTRs. We also investigated the positions of the spheroid (a single major structural polypeptide) occlusion protein kinase subdomains in the PK sequence. bodies (Roberts and Granados 1968). The 232-kb AMEV genome has been completely se- quenced and contains 292 open reading frames (ORFs). Materials and methods Among these ORFs, AMV153 and AMV197 suggested it to be a serine/threonine protein kinase (PK) having conserved Cell culture and virus Lymantria dispar (Ld652) cell line PK and serine/threonine PK domains (Bawden et al. 2000). used in this study was obtained from Basil Arif and Serine/threonine protein kinases are phospho-transferases maintained in Excell-420 (SAFC Biosciences) and Grace’s that transfer the phosphate groups onto serine and/or threonine Insect serum-free medium (Gibco) supplemented with 10% residues of protein substrates. Improper functioning of these heat-treated fetal bovine serum (Gibco). Penicillin (50 IU) enzymes is often manifested in several malignancies since and streptomycin (50 µg/ml) were added to cell line growth modification of proteins by phosphorylation at a limited media to prevent microbial contamination. AMEV was number of amino acid residues is identified as a general kindly supplied by Basil Arif. Replication of AMEV in the method of controlling activities of protein synthesis, cell cell line has been described previously (Goodwin et al. division and modulation of metabolic enzymes (Hunter 1987) 1990). For virus production, T75 flasks containing Ld652 in eukaryotes and prokaryotes. Phosphorylation of numerous cells at a density of 9 × 10 cells/ml were infected with wild cellular and viral proteins is also observed in virally infected type AMEV at a low m.o.i (0.5 p.f.u./cell) The virus was cells, which suggests that protein kinases may have a role in harvested when the cytopathic effect was complete at 4-day regulating a wide variety of viral infections (Leader and post infection (h p.i.). Subsequently, the supernatant was Katan 1988;Kannet al. 1999). centrifuged at 1,000 × g for 5 min to remove intact cells The PK of some vertebrate viruses is known to be and cellular debris. The resulting supernatant contained the packaged within the virion particle and shown to phosphor- extra-cellular virus was stored at 4°C. Virus titer was ylate a variety of proteins of viral and non-viral origin (Lin et determined by end point dilution assay (EPDA) (Darling et al. 1992; Rempel and Traktman 1992; Lin and Broyles al. 1998). 1994). Viruses often utilize many of the regulatory mecha- nisms as cells, and this is particularly evident for the Messenger RNA (mRNA) isolation Ld652 cells were seeded poxviruses, such as vaccinia virus. DNA sequence analysis at a density of 1×10 /T75 flask and infected at an m.o.i of 2 suggests that vaccinia virus may encode a protein kinase. with AMEV. At 2 h p.i, the medium was removed and The 34-kDa protein predicted from the sequence of the viral replaced with fresh medium. Cells were harvested at gene BlR bears a striking similarity to the catalytic domains various times after infection (0, 1, 2, 4, 7, 12, and 24 h) of known protein kinases (Howard and Smith 1989; Trakt- and pelleted at 300 × g for 5 min. The pellet was washed man et al. 1989; Goebeletal. 1990). Most of the amino acids with phosphate-buffered saline (1×PBS). Messenger RNA known to be conserved in the protein kinase family are (mRNA) was isolated with the PolyATtract System 1000 kit located in the appropriate sites of the sequence predicted for (Promega) following the manufacturer’s instructions and the B1R protein. This protein kinase is expressed early in quantified at 260 nm. An aliquot of 10 µg mRNA was infections, is found in the virosomes, and is also packaged treated with 200 U of RNAse free DNase I at 37°C for into virions (Banham and Smith 1992). It appears to be an 30 min to remove any residual DNA and then extracted essential viral protein, and temperature-sensitive mutations with phenol-chloroform and quantified after precipitation. that map to the B1R gene produce virus that cannot replicate its DNA at the restrictive temperature (Rempel et al. 1990; Time course analysis of AMV197 pk specific transcripts D- Rempel and Traktman 1992). Nase I treated mRNA preparations were screened for the Amino acid alignment of AMV197 encoding a protein of presence of AMV197 pk gene-specific transcripts by RT– 299 amino acids showed that this gene is an ortholog of PCR. Briefly, 2 µg of mRNA and 75 µM reverse oligo (dT) B1R. However, there are no data on PKs of entomopoxvi- anchor primer (Roche) were denatured at 70°C for 5 min ruses in literature. As part of our continuing work on and then placed on ice. First-strand cDNA was synthesized functional analysis of AMEV genome, we report here on by the addition of 2.5 µl M-MuLV 10X buffer, 2 µl of structural and transcriptional analyses of AMV197 pk gene 10 mM dNTPs, 100 U M-MuLV reverse transcriptase as part of basic studies to understand its function in the (BioLabs). DEPC-treated water was added to make a final context of the infection process. volume of 25 µl. The reaction proceeded at 37°C for 1 h, Ann Microbiol (2010) 60:523–530 525 followed by heating at 70°C for 15 min. PCR was dT anchor primer (Roche). PKSP3R primer was used for performed in a 50 ml volume containing 75 mM Tris-HCl the second PCR in combination with a PCR anchor primer (pH 8.8 at 25°C), 20 mM (NH ) SO , 0.1% Tween 20, (Roche). The amplified fragments were cloned into 4 2 4 1.5 mM MgCl , 0.2 mM each deoxynucleoside triphos- pGEMT-Easy (Promega) and 11 clones were analyzed by phate, 0.2 mM primers PKSP4 and PKR (Table 1), 2.5 U of automated sequencing. Taq DNA polymerase, and 1 µl of completed RT-PCR reaction mixture. Amplification consisted of 1 cycle of Determination of the 3′ UTR of AMV197 pk transcript To denaturation at 95°C for 3 min, 35 cycles of denaturation at determine the 3′ terminus of the AMV197 pk transcript, 3′ 95°C for 1 min, annealing at 55°C for 30 s, and extension at RACE was performed. The first strand cDNA was 72°C for 1 min, and a final cycle of elongation at 72°C for synthesized from mRNA using oligo (dT) anchor primer 7 min. PCR amplification was also done on mRNA isolated and M-MuLV reverse transcriptase. Then, the first strand from non-infected cells to confirm that there was no cellular cDNA was amplified by PCR using a gene specific primer contamination. PKSP4 and a PCR anchor primer corresponding to the oligo (dT) anchor primer (Table 1). The first amplification The temporal class analysis of the AMV197 pk tran- product was then used as a template for the second PCR script Ld652 cells were infected with AMEV at a multi- amplification with the second gene specific primer PKSP5R plicity of infection of 2. In order to inhibit DNA synthesis, and PCR anchor primer. The PCR product was isolated cells were continuously exposed to cytosine arabinoside from gel, cloned in pGEM-T (Promega) and 13 clones were (Ara-C) (100 µg/ml; Sigma) starting at 1 h prior to virus sequenced. infection. The inhibitor was maintained at this level throughout the infection. At 0 and 16 h p.i., cells were Amino acid sequence comparison The predicted amino harvested, and mRNAs were isolated from virus-infected acid sequence of AMEV was examined by using Expasy- cells in the presence and absence of Ara-C by using Prosite program and compared to those of other PK family PolyATtract System 1000 kit (Promega) according to the members (2 viral PKs, 2 PKs from vertebrates and a PK manufacturer’s protocol. The isolated mRNAs were DNase from S. cerevisiae) with the aid of Clustal W program. treated, quantified at 260 nm, subjected to RT-PCR and subsequently to PCR as above. RNA samples from mock infected cells and 0 h p.i. were processed as control. Results Determination of the 5′ UTR of AMV197 pk transcript The Time course transcriptional analysis of AMV197 pk gene To determine whether the putative AMV197 gene 5′ untranslated region of the AMV197 pk mRNA was obtained by rapid amplification of the cDNA ends (Froh- was expressed during viral infection, mRNA was extracted man et al. 1988) using a 5′ RACE kit (Roche) following the from AMEV infected Ld652 cells at 0, 1, 2, 4, 7, 12, and procedure supplied by the manufacturer with a set of three 24 h p.i. and from mock infected cells. The samples were specific primers. First strand cDNA was synthesized from analyzed by RT-PCR. A single band with an expected size 2 µg mRNA isolated at 16 h p.i. using gene-specific primer of 521 bp as pk transcript was first detected at 4 h p.i., (PKSP1R; see Table 1). The first-strand cDNA was then isolated and tailed with dA. This was followed by two consecutive nested PCRs with specific primer (see Table 1). PKSP2R primer was used for the first PCR with an oligo Table 1 Primers used for 5′ RACE (PKSP1R, PKSP2R, and PKSP3R), 3′ RACE (PKSP4, PKSP5) and RT–PCR (PKSP4, PKR) experiments Primer name Sequences PKSP1 5′-CAT AAT AAT CTA TTA CTA TAT ATC-3′ Fig. 1 Transciptional pattern of AMV197 pk gene detected byRT– PKSP2 5′-CCA TTA TGA TAT AGT AAT GGT ATT CC-3′ PCR analysis. M represents a 100-bp ladder DNA marker (Promega). PKSP3 5′-GCA TTT CTA CAA ACA ACG G-3′ Ld indicates DNA amplified from the RNA from mock infected PKSP4 5′-CCG TTG TTT GTA GAA ATG C-3′ Ld652 cells. Numbers show time points hours (h) post-infection for PKSP5 5′-CAA CAC TTG TTT AAC TAA TTC ACC-3′ each RNA samples extracted from infected cells. C shows negative control for the PCR. The amplicons were electrophoresed in a 1.2% PKR 5′-GGC AAA CTT CCA CTA TAC C-3′ agarose gel, and their sizes (in base pairs) are indicated at the right 526 Ann Microbiol (2010) 60:523–530 infected with AMEV in the presence or absence of Ara-C, an inhibitor of DNA replication. mRNA was extracted from cells at 16 h p.i. and analyzed for the presence of AMV197 transcript by RT–PCR (Fig. 2). AMV197 transcript was observed at this time point (Fig. 2, lane 3) and, apparently, it was not affected by the presence of inhibitor of DNA (Fig. 2, lane 5). Because viral DNA replication is not required for the expression of the AMV197 gene, it belongs by definition to an early gene class. This result is in agreement with other viral pk gene’s transcription classes (Liu et al. 2001; Gershburg et al. 2007). Transcription initiation site of AMV197 pk gene The transcription start site for AMV197 gene was identified by 5′ RACE analysis using mRNA extracted from infected cells at 16 h p.i. 5′ RACE was performed using three Fig. 2 Temporal class of AMV197 pk gene determined by RT PCR specific primers (PKSP1R, PKSP2R, and PKSP3R) analysis in the presence of DNA synthesis inhibitor. M represents a (Table 1). Amplified 5′ ends of cDNA fragments were 100-bp ladder DNA marker (Promega; lane 1). mRNA’s extracted cloned in pGEMT-Easy vector and 11 clones were from Ld652 cells in the absence (lane 2) or presence (lane 4) of Ara-C and from AMEV-infected Ld652 cells in the absence (lane 3)or sequenced. The 9 clones obtained for AMV197 showed presence (lane 5) of Ara-C were subjected to RT–PCR analysis using that the transcription initiation site (+1) is at the adenine primers specific for the AMV197 pk gene. The amplicons were (A) located 54 nt upstream of the translational start site electrophoresed in a 1.2 % agarose gel, and their sizes (in base pairs) (Fig. 3). are indicated at the right Transcription termination site of AMV197 pk gene To determine the 3′ terminus site of the AMV197 gene, a 3′ reached to maximum level at 7 h p.i. and started to decrease after this point (Fig. 1). RACE was performed on mRNA. The first strand cDNA was synthesized using the oligo(dT)-anchor primer and M- The transcriptional class analysis of the AMV197 pk MuLV reverse transcriptase. The amplification of the 3′ gene In order to determine the expression class, we RACE PCR products were cloned and 13 clones were employed an RT–PCR approach to detect the transcription- sequenced. The polyadenylation starts at 22 nt downstream al products of AMV197. To that aim, Ld652 cells were of the translational stop point in 8 clones and 32 nt Fig. 3 The locations of transcription initiation and termination sites of at the promoter region of vaccinia virus early genes. The poly (A) AMV197 pk gene. The translation start and stop codons are shaded addition sites that are obtained by sequencing of 3′RACE clones are gray. The dashed arrow indicates the 5′ termini (transcriptional start indicated by the straight arrows. The box indicates poly (A) signal points) of AMV197 pk gene revealed by sequencing of 5′ RACE which is overlapped with the translation stop codon and the dashed clones. The bold G at −21 shows the universal G residue that is found box indicates transcription termination signal Ann Microbiol (2010) 60:523–530 527 Fig. 4 Comparison of the amino acid sequence of AMV197 PK with poxvirus protein kinase (Accession no: AF063866); PIM-1, Mus other PK’s of other organisms. The sites of the conserved catalytic musculus protein kinase (Accession no: AAA39930); MOS, serine/ subdomains are numbered with Roman numbers and shaded. AMEV- threonine-protein kinase-transforming protein (mos) of Mouse (Ac- PK, Amsacta moorei entomopoxvirus protein kinase (Accession no: cession no: P00538); CDC28, cell division control protein 28 of S. NC002520); VACV-B1-RPK, Vaccinia virus protein kinase (Acces- cerevisiae (Accession no: P00546) sion no: YP233065); MSEV-PK, Melanoplus sanguinipes entomo- downstream of the translational stop point in 5 clones of B1R). Major subdomains conserved in various PKs were sequenced 13 clones as shown in Fig. 3. also readilyidentifiedonthe AMV197,but afew subdomains were difficult to be ascertained (Fig. 4). Amino acid sequence alignment of AMV197 pk gene When the deduced amino acid sequence of AMV197 pk gene was At the N-terminal extremity of the catalytic domain, there is a analyzed in Expasy-Prosite, it was observed that the region (subdomain I) containing a glycine (G)-rich stretch of location of protein kinase catalytic domain is between residues (21GFGTVY26) and nearby there is the lysine residues of 12 and 299. residue (42 K) of subdomain II (39–42). The region from The amino acid sequence of AMV197 PK was also subdomain I to II has been shown to be involved in ATP compared with other PK family members (with especially binding (Hanks and Hunter 1995). In the PROSITE database, 528 Ann Microbiol (2010) 60:523–530 this ATP binding region is one of only two regions that have methyltransferase (Martin et al. 1975; Barbosa and Moss been selected to build a protein kinase signature. The 1978) into virions, retention of a protein kinases had not sequence (aa 21–42) in this region matches (although not previously been shown. The studies on poxvirus protein exactly) the protein kinase signature consensus pattern I: kinases were initially conducted on vaccinia virus B1R and [LIV]-G-{P}-G-{P}-[FYWMGSTNH]-[SGA]-{PW}-[LIV- F10L protein kinases. They were followed by investigations CAT]-{PD}-x-[GSTACLIVMFY]-x-(5,18)-[LIVMFYWC- on other poxvirus PKs; Fowlpox virus (FWPV-PK, Poxvir- STAR]-[AIVP]-[LIVM FAGCKR]-K, where K is the residue idae; Afonso et al. 2000) Myxoma virus (MYXV-PK, that binds ATP (square brackets are used to explain the Poxviridae; Cameron et al. 1999), Molluscum contagiosum acceptable alternative amino acids for a given position and virus subtype 1 (MOCV1-PK, Poxviridae; Senkevich et al. the curly brackets explain the amino acids that are not 1996), and Variola major virus (VARV-PK, Poxviridae; acceptable at a given position). The sequences of 54E and Massung et al. 1993). However, to date, data on the 98YIV100 show homology to subdomains III and IV, Amsacta moorei Entomopoxvirus (AMEV) protein kinase respectively. Subdomain V is not clearly discernable. (pk) gene have not been reported. Subdomain VI is highly conserved among protein In order to study the mechanisms controlling AMEV kinases and is used to build the protein kinase signature gene regulation, we have, as a first step, initiated consensus pattern II. The sequences 142YTHNDIKKN- transcriptional and structural analyses of the pk gene. NIMF154 matches well with the protein kinase signature Based on the temporal expression, the AMV197 pk gene consensus pattern II, [LIVMFYC]-x-[HY]-x-D-[LIVMFY]- was classified as an early gene, just as B1R belongs to vaccinia K-x-(2)-N-[LIVMFYCT] (3), where the aspartic acid virus. The transcripts were detected early after infection (by 4 h residue (146D) is probably the residue important for the p.i. and maximized at 7 h p.i.) a time relegated primarily to the catalytic activity of the enzyme (Knighton et al. 1991; Liu synthesis of non-structural proteins, including enzymes in- et al. 2001). The residue at position 148 is K, indicating that volved in replicating the genome and modifying DNA, RNA, it is a serine/threonine specific protein kinase. Subdomain and proteins (http://www.virustaxonomyonline.com/virtax/ VII is found in a highly conserved region, 163LIDYG167. lpext.dll?f=templates&fn=main-h.htm). Subdomain VIII has a highly conserved triplet, A-P-E, in The transcriptional start and stop sites were determined most protein kinases. This region is thought to lie close to the by 5′ and 3′ RACE analysis. 5′ RACE showed that catalytic site and to flank a common autophosphorylation AMV197 pk gene has a short 5′ UTR of 54 nt. Also, the receptor site (Selten et al. 1986; Hunter 1987). No identical promoter region has a universal G residue at −21 flanked by sequence is found within the AMV197 PK sequence. Another variable and A-T-rich sequence (Fig. 4). This sequence sequence, T-L-E, in the appropriate region has been found and matches well with the vaccinia virus early promoter thought to be subdomain VIII in accordance with the vaccinia element identified by Davison and Moss (1989). 34-kDa protein kinase subdomain VIII (Traktman et al. 1989). The 3′ UTR results revealed the transcripts with In this study, the possible change of the APE motif to TLE is heterogeneous 3′ ends were derived from cleavage sites clearly explained. It is found that this motif within published starting 22 nt and 32 nt downstream of the translation kinase sequences reveals some heterogeneity and demon- stop site, which overlaps the polyadenylation signal strates that the final glutamic acid is the only invariant residue AAUAAA. RNA polyadenylation plays an important (Selten et al. 1986). Replacement of alanine with other neutral role in the control of viral and eukaryotic gene expression residues (glycine, leucine and proline) has been observed. and involves cleavage of the nascent transcript and Alanine (neutral), leucine, and isoleucine (nonpolar) have addition of a poly (A) tail with 150–200 adenylate been found as substitutes for the central proline. Subdomain residues (Gilmartin 2005). One cleavage site for the IX is thought to be placed at the sequences 206DIESLM211, AMV197 transcripts was mapped to nt 170167. The other but may also include the 212YNIIEWYSG220 sequence. cleavage site was mapped downstream of the first one, Subdomains X and XI are not so clearly defined in AMV197 with only ten nucleotides separating them at nt 170177 PK but amino acid residues 221 to 225 may potentially (Fig. 4). The heterogeneity of the cleavage site usage in consititute subdomain X. AMV197 transcripts may simply reflect selection flexi- bility, which is common in mammals (Pauws et al. 2001). We examined the AMV197 gene sequence for the Discussion presence of termination signal (TTTTTGT) normally found just upstream of the 3′ ends of early poxvirus mRNAs Although poxviruses are known to incorporate enzymes (Yuen and Moss 1987). However, no termination signal was such as DNA-dependent RNA polymerase (Baroudy and found at that region. Further analysis of the AMV197 pk Moss 1980; Shchelkunov et al. 1993), poly (A) polymerase gene sequence showed that the termination consensus (Moss et al. 1975), mRNA guanyltransferase and mRNA sequence is located in the coding sequence of AMV197 Ann Microbiol (2010) 60:523–530 529 Arif MB (1995) Recent advances in the molecular biology of (Fig. 4) as in the case of vaccinia virus early gene and entomopoxviruses. J Gen Virol 76:1–13 flanked by the polyadenylation signal. Banham AH, Smith GL (1992) Vaccinia virus gene B1R encodes a Protein kinase catalytic domains range from 250 to 300 34-kDa serine/threonine protein kinase that localizes in amino acid residues, corresponding to 30 kDa. Fairly precise cytoplasmic factories and is packaged into virions. Virology 191:803–812 boundaries for the catalytic domains have been defined Barbosa E, Moss B (1978) mRNA (nucleoside-29-)-methyltransferase through an analysis of conserved sequences as well as by from vaccinia virus. Characteristics and substrate specificity. J assays of truncated enzymes (Hanks et al. 1988). The location Biol Chem 253:7698–7702 of the catalytic domain within the protein is not fixed but, in Baroudy BM, Moss B (1980) Purification and characterization of DNA-dependent RNA polymerase from vaccinia virions. J Biol most single subunit enzymes, it lies near the carboxyl Chem 225:4372–4380 terminus, the amino terminus being devoted to a regulatory Bawden AL, Glassberg KJ, Diggans J, Shaw R, Farmerie W, Moyer role. In protein kinases having a multiple subunit structure, RW (2000) Complete genomic sequence of the Amsacta moorei subunit polypeptides consisting almost entirely of a catalytic entomopoxvirus: analysis and comparison with other poxviruses. Virology 274:120–139 domain are common. The AMEV catalytic domain also Cameron C, Hota-Mitchell S, Chen L, Barrett J, Cao JX, Macaulay C, covers almost the entire sequence of the PK gene, starting Willer D, Evans D, McFadden G (1999) The complete DNA from the 12th amino acid. Also, the amino terminus of sequence of myxoma virus. Virology 264:298–318 AMV197 PK catalytic domain lies as close as ten residues Darling AJ, Boose JA, Spaltro J (1998) Virus assay methods: accuracy and validation. Biologicals 26:105–110 from the first conserved glycine, as indicated for some Davison AJ, Moss B (1989) Structure of vaccinia virus early catalytic domain polypeptides by Hanks et al. (1988). promoters. J Mol Biol 210:749–769 A large number of pk-related genes have been sequenced Frohman MA, Dush MK, Martin GR (1988) Rapid production of full- and analyzed for their amino acid sequence patterns. Hanks length cDNAs from rare transcripts: Amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci USA et al. (1988) have reported the identification of 11 conserved 85:8998–9002 regions in the catalytic domains of PKs on the basis of an Gershburg E, Salvatore R, Torrisi MR, Pagano JS (2007) Epstein-Barr alignment of 65 different members of the PK family. In virus-encoded protein kinase (BGLF4) is involved in production AMV197 PK, we clearly identified the subdomains I, II, VI, of infectious virus. J Gen Virol 81:5407–5412 Gilmartin GM (2005) Eukaryotic mRNA 3′ processing: a common VII, VIII and predicted subdomains III, IV, IX and X. means to different ends. Genes Dev 19:2517–2521 However, the subdomains V and XI were difficult to locate. Goebel SJ, Johnson GP, Perkus ME, Davis SW, Winslow JP, Paoletti In this paper, we showed that AMV197 gene belongs to E (1990) The complete DNA sequence of vaccinia virus. early viral gene class by RT–PCR analysis. The vaccinia early Virology 179:247–266 Goodwin RH, Adams JR, Shapiro M (1990) Replication of the promoter region and the transcription termination signal entomopoxvirus from Amsacta moorei in serum-free cultures of a confirm this result. Thus, this gene is transcribed by enzymes gypsy-moth cell line. J Invertebr Pathol 56:190–205 derived and packaged within the virion core (Banham and Hanks SK, Hunter T (1995) Protein kinases 6. The eukaryotic protein Smith 1992). Early gene expression is independent of viral kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J 9:576–596 replication and depends only on previous assembly of a Hanks SK, Quinn AM, Hunter T (1988) The protein kinase family: competent virus particle and a subsequent partial uncoating conserved features and deduced phylogeny of the catalytic of the infecting virus. When the predicted amino acid domains. Science 241:42–52 sequence was compared with those of other PKs, major Howard ST, Smith GL (1989) Two early vaccinia virus genes encode polypeptides related to protein kinases. J Gen Virol 70:3187– subdomains which are conserved in various PKs could be readily identified, but a few subdomains were difficult Hunter T (1987) A thousand and one protein kinases. Cell 50:823– exactly assign. The identified subdomains were homologous to counterparts of PKs of other organisms. However, these Kann M, Sodeik B, Vlachou A, Gerlich WH, Helenius A (1999) Phosphorylation-dependent binding of hepatitis B virus core subdomain sequences presented here are only preliminary particles to the nuclear pore complex. J Cell Biol 145:45–55 data. In order to confirm the validity of these results, genetic Knighton DR, Zheng JH, Ten Eyck LF, Xuong NH, Taylor SS, studies will have to follow. Sowadski JM (1991) Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science 253:414–420 Acknowledgment This work was supported by the Scientific and Leader DP, Katan M (1988) Viral aspects of protein phosphorylation. J Technological Research Council of Turkey, TÜBİTAK with Project Gen Virol 69:1441–1464 No: 106T265. Lin S, Broyles SS (1994) Vaccinia protein kinase 2: a second essential serine/threonine protein kinase encoded by vaccinia virus. Proc Natl Acad Sci USA 91:7653–7657 Lin SQ, Wen C, Broyles SS (1992) The vaccinia virus B1R gene References product is a serine/threonine protein kinase. J Virol 66:2717– Liu WJ, Yu HT, Peng SE, Chang YS, Pien HW, Lin CJ, Huang CJ, Afonso CL, Tulman ER, Lu Z, Zsak L, Kutish GF, Rock DL (2000) Tsai MF, Huang CJ et al (2001) Cloning, characterization, and The genome of fowlpox virus. J Virol 74:3815–3831 530 Ann Microbiol (2010) 60:523–530 phylogenetic analysis of a shrimp white spot syndrome virus Rempel RE, Anderson MK, Evans E, Traktman P (1990) gene that encodes a protein kinase. Virology 289:362–377 Temperature-sensitive vaccinia virus mutants identify a gene Martin SA, Paoletti E, Moss B (1975) Purification of mRNA with an essential role in viral replication. J Virol 64:574–583 guanylytransferase and mRNA (guanine-7-)-methyltransferase Roberts DW, Granados RR (1968) A poxlike virus from Amcasta from vaccinia virions. J Biol Chem 250:9322–9329 moorei (Lepidaptera: Arctiidae). J Invertebr Pathol 12:141 Massung RF, Esposito JJ, Liu LI, Qi J, Utterback TR, Knight JC, Selten G, Cuypers HT, Boelens W, Robanus-Maandag E, Verbeek J, Aubin L, Yuran TE, Parsons JM et al (1993) Potential virulence Domen J, van Beveren C, Berns A (1986) The primary structure determinants in terminal regions of Variola smallpox virus of the putative oncogene pim-1 shows extensive homology with genome. Nature 366:748–751 protein kinases. Cell 32:603–611 Moss B (2001) Poxviridae: the viruses and their replication. In: Knipe Senkevich TG, Bugert JJ, Sisler JR, Koonin EV, Darai G, Moss B (1996) DM, Howley PM (eds) Fields virology. Lippincott Williams & Genome sequence of a human tumorigenic poxvirus: prediction of Wilkins, Philadelphia, pp 2849–2883 specific host response-evasion genes. Science 273:813–816 Moss B, Rosenblum EW, Gershowitz A (1975) Characterization of a Shchelkunov SN, Marennikova SS, Blinov VM, Resenchuk SM, polyadenylate polymerase from vaccinia virions. J Biol Chem Totmenin AV, Chizhikov VE, Guturov VV, Safronov PF, 250:4722–4729 Kurmanov RK, Sandaknchiev LS (1993) Entire coding sequence Pauws E, van Kampen AH, van de Graaf SA, de Vijlder JJ, Ris- of the variola virus. Dokl Akad Nauk 328:629–632 Stalpers C (2001) Heterogeneity in polyadenylation cleavage Traktman P, Anderson MK, Rempel RE (1989) Vaccinia virus encodes sites in mammalian mRNA sequences: implications for SAGE an essential gene with strong homology to protein kinases. J Biol analysis. Nucleic Acids Res 29:1690–1694 Chem 264:21458–21461 Rempel RE, Traktman P (1992) Vaccinia virus B1 kinase:henotypic Yuen L, Moss B (1987) Oligonucleotide sequence signaling transcrip- analysis of temerature-sensitive mutants and enzymatic charac- tional termination of vaccinia virus early genes. Biochem terization of recombinant proteins. J Virol 66:4413–4426 84:6417–6421

Journal

Annals of MicrobiologySpringer Journals

Published: Sep 1, 2010

Keywords: Amsacta moorei entomopoxvirus (AMEV); Protein kinase; Transcriptional analysis

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