Protein Engineering vol.6 no.8 pp.883-891, 1993 Analysis of RNA phage fr coat protein assembly by insertion, deletion and substitution mutagenesis P.Pushko, T.Kozlovskaya, I.Sominskaya 1 , A.Brede, E.Stankevica, V.Ose 2 , P.Pumpens and E.Grens 3 Institute of Molecular Biology, Latvian Academy of Sciences and University of Latvia, Krustpils street 53, Riga LV 1065, 'Latvian Medical Academy, Dzirciema 16, Riga LV 1007 and 2 Institute of Microbiology, Latvian Academy of Sciences, Kleisti, Riga, Republic of Latvia ^ o wnom correspondence should be addressed A structure-function analysis of the icosahedral RNA bacteriophage/r coat protein (CP) assembly was undertaken using linker-insertion, deletion and substitution mutagenesis. Mutations were specifically introduced into either pre-existing or artificially created restriction enzyme sites within fr CP gene expressed in Escherichia coli from a recombinant plasmid. This directs synthesis of wild type protein that undergoes self-assembly and forms capsid-like particles in- distinguishable morphologically and immunologically from native phage particles. A series of fr CP variants containing sequence alterations in the regions which are (i) exposed on the external surface of capsid or (ii) located on the contac- ting areas between CP subunits were obtained and their assembly properties investigated. The majority of mutants demonstrated reduction of assembly ability and formed either CP dimers (mutations at residues 2, 10, 63 or 129) or both dimer and capsid structures (residue 2 or 69). The exceptions were variants demonstrating normal assembly and containing insertions at residues 2, 50 or 129 of thefr CP. A third type of assembled structure was formed by a variant with a single amino acid substitution I104T. The aA-helix region (residues 97-111) is particularly sensitive to mutation and any alteration in this region decreases accumulation of mutant protein in E.coli. The relative contributions of particular fr CP domains in maintenance of capsid structural integrity as well as the possible capsid assembly mechanism are discussed. Key words: bacteriophage /r/capsid assembly/coat protein/ protein subunit interactions/RNA bacteriophages Introduction The fr phage belongs to serological group I of RNA bacterio- phages (MS2, f2, R17, etc.) infecting male Escherichia coli bacteria [for a review see Fiers (1979) and van Duin (1988)]. The fr phage particle contains the positive-sense, single-stranded RNA molecule, 3575 nucleotides in length (Adhin et al., 1990) encapsulated in the icosahedral shell that protects viral RNA and consists of 180 copies of a single coat protein (CP) subunit of 129 amino acids in length (Zipper et al., 1971). Apart from its structural function, CP acts as a translational repressor of phage replicase (RNA-dependent RNA polymerase) synthesis (Sugiyama and Nakada, 1970). The mature phage particle also contains one molecule of A-protein presumably responsible for attachment to bacterial pili (Weber and Konigsberg, 1975) and —1000 spermidine molecules which neutralize phosphates of the phage RNA (Fukuma and Cohen, 1975). Replicase is not a structural component of the phage particle and appears transiendy during phase replication in bacteria. The structural simplicity of the RNA phages makes them a convenient model system for investigation of different biological events at the molecular level including protein-RNA and protein-protein interactions during the capsid assembly process. It has been shown by in vitro experiments that CP alone without other phage components can form capsid-like particles, similar to native virions (Zinder, 1975), although evidence exists that the presence of RNA, especially of the repressor-binding fragment, stimulates assembly (Beckett et al., 1988). It has been shown that the translational repressor complex I consisting of the CP dimer and the repressor-binding fragment can serve as a nucleation site triggering capsid assembly (Sugiyama et al., 1967; Beckett and Uhlenbeck, 1988). Self-assembly of the CP into capsid-like particles has been observed in vivo after cloning and expression of the^r and MS2 CP genes in E. coli (Kastelein etal., 1983; Kozlovskaya et al., 1986; Peabody, 1990). These experiments indicate that information required for RNA phage capsid assembly is encoded within its CP primary structure. The 3-D structure of RNA phage MS2 (Valegard et al., 1990), the CP of which differs from that offr by 17 amino acid substitutions, shows interactions between CP subunits in the assembled capsid. However, the relative contributions of the different CP domains to the maintenance of phage capsid integrity is not yet completely understood. The assembly mechanism also remains to be investigated since die folding pattern of the MS2 CP differs significandy from that of capsid proteins of the other RNA viruses for which assembly mechanisms have been proposed (Rossmann et al., 1983; Savithri and Erickson, 1983; Sorger et al., 1986). In order to define the specific role of different fr CP domains involved in subunit interactions, as well as possible subassembly structures of the fr capsids, we have conducted insertion, deletion and substitution mutagenesis of the cloned fr CP gene to probe for coding sequences that change the primary structure and assembly properties of the CP monomer. Correlations between alterations within different fr CP primary structure regions and self-assembly properties of fr CP monomers were found. The locations of these mutations define the relative contribution of different CP domains to the maintenance of capsid integrity and the possible mechanism of capsid assembly. Materials and methods Restriction enzymes were purchased from 'Fermentas' (Vilnius, Lithuania) and Bal-31 exonuclease, Klenow fragment of E. coli DNA polymerase, T4 DNA ligase and polynucleotide kinase were purchased from Boehringer-Mannheim (Mannheim, Germany). DNA preparation, restriction enzyme cleavage, cloning of the oligonucleotides, bacterial transformation and growth were performed essentially as described in Maniatis et al. (1982) and Sambrook et al. (1988). Rabbit antisera against phage fr and against /r-denatured CP were a gift from Dr Baumanis. Plasmid pFAN15 was kindly provided to us by Dr Berzin. © Oxford University Press 883 at Ryerson University on February 8, 2013 http://peds.oxfordjournals.org/ Downloaded from