Virology 276, 376–387 (2000) doi:10.1006/viro.2000.0565, available online at http://www.idealibrary.com on View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector The Structural Protein E of the Archaeal Virus ␾Ch1: Evidence for Processing in Natrialba magadii during Virus Maturation Reinhard Klein,1 Barbara Greineder, Ulrike Baranyi,2 and Angela Witte3 Institute of Microbiology and Genetics, University of Vienna, Dr. Bohr-Gasse 9, A-1030 Vienna, Austria Received March 1, 2000; accepted July 31, 2000 ␾Ch1 is a lysogenic virus for the haloalkalophilic archaeon Natrialba magadii. The virus morphology resembles other members of Myoviridae infecting Halobacterium species. The gene of the major capsid protein E of virus ␾Ch1 was cloned and the DNA sequence was determined. Gene E was mapped to a 3.2-kbp ClaI fragment, localized to the 5Ј-end of the ␾Ch1 genome. The complete nucleotide sequence of this region was determined and the identity of gene E was confirmed by comparing the experimentally determined N-terminal amino acid sequence of the purified protein to the translated DNA sequence of its open reading frame. We present evidence that the gene E product is proteolytically cleaved between Lys16 and Asn17 to yield the 305 residue polypeptides found in the mature viral capsid. Processing of the protein itself during virus development was determined by 2D gel electrophoresis using protein E-specific antibodies. Sequence similarity studies revealed an 80% identity to capsid protein Hp32 of ␾H, infecting Halobacterium salinarum. RT-PCR analysis as well as Western blot studies revealed gene E as a late gene. Transcripts and proteins could be detected shortly before onset of lysis of the lysogenic strain N. magadii L11. © 2000 Academic Press Key Words: virus; protein processing; Natrialba; capsid; Archaea; halophilic. INTRODUCTION of virus-encoded insertion sequences (Schnabel et al., 1982; Schnabel, 1984), mapping of early and late tran- Besides Bacteria and Eukarya, Archaea have been scripts (Gropp et al., 1989, 1992; Stolt and Zillig, 1992), suggested to form the third domain of life (Woese and analysis of lytic control by the action of a transcriptional Fox, 1977). Viruses which infect members of each of the repressor as well as antisense RNA control (Ken and three major phenotypic groups of Archaea, the extreme Hackett, 1991; Stolt and Zillig, 1993a,b,c, 1994), and the thermophiles, the methanogens, and the halophiles, identification of genes for structural proteins and putative have been isolated (for reviews see Zillig et al., 1986, methyltransferases (Stolt et al., 1994). 1988; Reiter et al., 1988). Virus ␾Ch1 was the first one isolated from a member Most archaeal viruses known so far have been iso- of the haloalkalophilic branch of Archaea (Witte et al., lated during the last years from the group of extreme 1997). Haloalkalophilic Archaea require not only high salt halophilic Archaea. These viruses include Hs1 (Torsvik concentrations but also high pH for growth and therefore ␾ and Dundas, 1974, 1980), Ja1 (Wais et al., 1975), H differ from the other genera of the Halobacteriales, living (Schnabel et al., 1982), Hh-1 and Hh-3 (Pauling, 1982; at a neutral pH. ␾Ch1 is a head–tail virus belonging to ␾ Rohrmann et al., 1983), and N (Vogelsang-Wenke and the family of Myoviridae and infecting Archaea of the Oesterhelt, 1988), all of them infecting the genus species Natrialba magadii, thereby resulting in lysis of Halobacterium. HF1 and HF2 (Nuttall and Dyall-Smith, the cells. Two strains of N. magadii are available: the 1993) were found to infect Halorubrum and Haloferax. lysogenic strain L11 and strain L13 which has been His1, the first lemon-shaped virus within this group of cured of the virus (Witte et al., 1997). The latter one was halophilic Archaea, was isolated from Haloarcula his- used for infection with ␾Ch1. Nucleic acid preparations panica (Bath and Dyall-Smith, 1998). of purified ␾Ch1 particles have been found to contain Halovirus ␾H is the most intensively studied virus DNA as well as RNA, which is a highly unusual feature of within this group. Studies included the characterization viruses (Witte et al., 1997). The only comparable situation has been found for Bacillus subtilis bacteriophage ␾29 (Guo et al., 1987a,b), as well as for other B. subtilis 1 Present address: Department of Biology, University of Pennsylvania, phages (Wichitwechkarn et al., 1989), in which a certain 415 South University Avenue, Philadelphia, PA 19103. RNA is associated with phage proheads. For phage ␾29 2 Present address: Department of Vascular Biology and Thrombosis Research, VIRCC, University of Vienna, A-1235 Vienna, Austria. it was shown that this RNA is necessary for the DNA 3 ␾ To whom correspondence should be addressed. E-mail: emily@ packaging process (Guo et al., 1987a,b). 29 RNA is gem.univie.ac.at. thought to form a circular, hexameric structure which is 0042-6822/00 $35.00 Copyright © 2000 by Academic Press 376 All rights of reproduction in any form reserved. CAPSID PROTEIN OF ␾Ch1 377 associated with the phage proheads and is not found in mature phage particles contrary to the ␾Ch1 RNA. Fur- thermore, the RNA fraction isolated from ␾Ch1 virus particle preparations is a mixture of primarily host-en- coded but also virus-encoded RNAs varying in length from 100 to 800 nucleotides. Since ␾Ch1 was the first virus detected in haloalkalo- philic Archaea and due to the fact that DNA as well as RNA was found in purified virus particles, ␾Ch1 was FIG. 1. Schematically presentation of the localization of gene E. Part analyzed in more detail. Here we describe studies of the of the restriction map of ␾Ch1 DNA is given with different restriction major capsid protein E of virus ␾Ch1. We have mapped fragments named by their size. Gene E is indicated on the top by a box. and sequenced gene E and expression has been moni- tored on the RNA as well as on the protein level. Two- dimensional protein gel electrophoresis was used to ferred onto PVDF membranes by semidry electroblotting show that protein E is processed in vivo during virus and stained with Coomassie Brilliant Blue. The band maturation. We present evidence that protein E might be containing protein E was excised and subjected to N- associated with the cell envelope of N. magadii during terminal amino acid sequencing using standard Edman virus development. degradation techniques. The N-terminal amino acid se- quence determined for protein E was NALTVDDL, lack- RESULTS AND DISCUSSION ing at least the first methionine. To localize the corre- sponding gene on the ␾Ch1 genome, a degenerate oli- Identification of the gene encoding the major capsid gonucleotide deduced from this sequence was designed protein E of virus ␾Ch1 (oligonucleotide E-deg, Table 1). ␾Ch1 DNA was cut with Purified virus particles were precipitated with 10% TCA restriction enzymes ClaI, PstI, and EcoRV, separated on and separated by 12% SDS-PAGE. Proteins were trans- 0.8% agarose gels, blotted onto nylon membranes, and hybridized with the 32P-labeled oligonucleotide. Hybrid- ization with oligonucleotide E-deg revealed a signal cor- TABLE 1 responding to a 3.2-kbp ClaI fragment (fragment D), a 13-kbp PstI fragment (fragment B), and a 2-kbp EcoRV Strains, Plasmids, and Primers fragment (fragment G). The localization of the gene de- scribed within this study is shown in a partial restriction Strain Relevant marker Source/reference map of the virus genome (Fig. 1). N. magadii L11 wt, ␾Ch1 prophage Witte et al., 1997 Sequence analysis L13 cured for ␾Ch1 Witte et al., 1997 E. coli The ClaI fragment D was isolated and cloned into Ϫ ϩ ϩ XL1-Blue endA1, gyrA96, hsdR17 (r k m K ), Stratagene pBlueskript II KS , resulting in clone pC-E. The fragment lac, recA1, relA1, supE44, was subjected to DNA sequencing using the dideoxy thi, (FЈ, lacIq, lacZ⌬M15, ϩ method of Sanger et al. (1977). An open reading frame of proAB , tet) 966 nt coding for a 321 aa protein, which included the KT950 placUV5-T7 gene 1-kan::l`acZ Tedin et al., 1995 (FЈ, lacIq, lacZ⌬M15, proABϩ, sequence NALTVDDL as determined by Edman degra- tet) dation, could be identified on ClaI fragment D. This open reading frame was therefore termed gene E (Fig. 2). As Plasmids Relevant marker Source/reference shown in Fig. 2, the N-terminal aa sequence NALTVDDL is in a distance of 16 codons from the first methionine ϩ pKS-II mcs, bla, ColE1 Stratagene start codon of the open reading frame. This indicates pC-E ClaI-E-fragment cloned into pKS-IIϩ This study pET-KH His-tag, mcs, bla, ColE1 Novagen (modified) posttranslational processing of protein E. The size of the pET-E E cloned into pET-KH This study mature protein was calculated to be 34.0 kDa, which is lower than inferred from SDS-PAGE (Table 2). Protein E is Primers Sequence an acidic protein as reflected by a calculated isoelectric point of 3.74. A surplus of acidic over basic residues has E-deg 5Ј-AAYGCXYTXACXGTXGAYGAYYTX-3Ј E-HIS-5 5Ј-GAGATTTCAATGAGATCTCGAACCATCAAC-3Ј also been observed for several H. salinarum proteins E-HIS-3 5Ј-GATGGTGCGGCTAGCCATCGTTCACCG-3Ј (Zaccai et al., 1989; Eisenberg and Wachtel, 1987), as CD-2 5Ј-TCGACTGACAACCAACACACCC-3Ј well as for capsid proteins of virus ␾H infecting H. CD-4 5Ј-GCGTTCAGCCATCGTTCACC-3Ј salinarum (Stolt et al., 1994), a characteristic feature of Ј Ј Nb16F 5 -GGAGACCATTCCGG-3 halophilic proteins in general (Lanyi, 1974; Eisenberg et Nb16R 5Ј-GGATCCGTCTTCCAG-3Ј al., 1992; Frolow et al., 1996). In contrast to the low 378 KLEIN ET AL. FIG. 2. Sequence of gene E encoding the major capsid protein of ␾Ch1.