In vivo studies of genomic packaging in the dsRNA bacteriophage Φ8

BMC Microbiology BioMed Central Research article Open Access In vivo studies of genomic packaging in the dsRNA bacteriophage Φ8 Jian Qiao, Xueying Qiao and Leonard Mindich* Address: Department of Microbiology, The Public Health Research Institute. Newark, New Jersey 07103, USA Email: Jian Qiao - ; Xueying Qiao - ; Leonard Mindich* - * Corresponding author Published: 11 March 2005 BMC Microbiology 2005, 5:10 doi:10.1186/1471-2180-5-10 Received: 12 January 2005 Accepted: 11 March 2005 This article is available from: http://www.biomedcentral.com/1471-2180/5/10 © 2005 Qiao et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Φ8 is a bacteriophage containing a genome of three segments of double-stranded RNA inside a polyhedral capsid enveloped in a lipid-containing membrane. Plus strand RNA binds and is packaged by empty procapsids. Whereas Φ6, another member of the Cystoviridae, shows high stringency, serial dependence and precision in its genomic packaging in vitro and in vivo, Φ8 packaging is more flexible. Unique sequences (pac) near the 5' ends of plus strands are necessary and sufficient for Φ6 genomic packaging and the RNA binding sites are located on P1, the major structural protein of the procapsid. Results: In this paper the boundaries of the Φ8 pac sequences have been explored by testing the in vivo packaging efficacy of transcripts containing deletions or changes in the RNA sequences. The pac sequences have been localized to the 5' untranslated regions of the viral transcripts. Major changes in the pac sequences are either tolerated or ameliorated by suppressor mutations in the RNA sequence. Changes in the genomic packaging program can be established as a result of mutations in P1, the major structural protein of the procapsid and the determinant of RNA binding specificity. Conclusion: Although Φ8 is distantly related to bacteriophage Φ6, and does not show sequence similarity, it has a similar genomic packaging program. This program, however, is less stringent than that of Φ6. Background The Cystoviridae are a family of bacteriophages having genomes consisting of three segments of double-stranded RNA (dsRNA). The RNA is contained within a polyhedral capsid which is enveloped in a lipid-containing membrane [1]. Φ6 was the first and the most thoroughly studied member of this family. The packaging of the Φ6 genome was found to progress through a program that involves the sequential packaging of the plus strands of segments S, M and L in that order [2]. This program is rather stringent for Φ6 in that segment S can be packaged alone while M requires prior packaging of S and L requires prior packaging of M. Segment L can be packaged to some extent with only prior packaging of S but this is much less efficient than the normal packaging [3,4]. The Cystoviridae are the only family of RNA viruses that are reliably known to package their genomic segments into preformed capsids. Although the Reoviridae have an inner core particle structure strikingly similar to that of the Cystoviridae, their mechanisms of genomic packaging have not yet been determined [5]. Packaging in the Cystoviridae is driven by an NTPase motor comprised of a Page 1 of 10 (page number not for citation purposes) BMC Microbiology 2005, 5:10 http://www.biomedcentral.com/1471-2180/5/10 Table 3: Plasmids used in this study with cDNA copies of Φ8S Plasmid changes oligo plaques 2755 2816 2817 2819 2820 2914 2916 2918 2920 2929 2938 2939 2955 wt ∆137–158 ∆120–158 ∆101–158 ∆75–158 UAG135CUU AG83UU UUCG41∆ UUCG41CCCA UU128CA A166C UAAA163CCCC GG159AU 590 591 592 593 681 683 685 687 693 699 701 708 600 >1000 1 1 2 400 1000 small 200 300 >1000 251 small 207 60 2956 GG159AU A170 ∆ GG159AU A166C GG159AU A166C GG159AU A166C C132U GG159AU ∆137–158 A166C GG159AU ∆137–158 C132U C132U ∆135–143 GAG138CUC GAG138∆ 708 >1000 no +A172C* or U161A no + C167U no + U135C U133A G175A no +A166∆ 708 714 708 714 708 714 722 708 728 714 724 728 722 722 24 no + C132A 6 no + C132U 742 744 3 >1000 C199A 703 >1000 2979 2980 2990 2997 2996 3000 2999 3013 3015 3016 2940 suppressors no no no no >1000 10 small 700 small >1000 0 no or G111A or U114C * suppressor containing plaques were larger than the others hexamer of protein P4 [6-8]. The plus strands of Φ6 have an 18 base consensus sequence at the 5' end and at about 50 bases downstream, a sequence of about 200 nucleotides that is necessary and sufficient for packaging into procapsids. The 200 nucleotide sequence is called pac and it is unique for each segment, with very limited identity in sequence between the three segments. Minus strand synthesis begins after the completion of the plus strand packaging and plus strand synthesis begins after minus strand synthesis is completed. A model has been proposed that involves the programmed changes in the binding sites for plus strands on the surface of the procapsid as a function of the amount of RNA in the particle [2]. The pac sequences of Φ6 were defined by deletion analysis of the plus strand transcripts of plasmids carrying cDNA copies of the genomic segments. In vitro packaging was used to assess the success of packaging of transcripts harboring a series of deletions. It was found that the pac sequences ended about 50 nucleotides before the first open reading frames (orfs) of the segments [9]. The pac sequences showed considerable secondary structure, with a number of stem-loops [10]. Comparison of the pac sequences of close relatives of Φ6 suggested that the stemloop structures were important because base changes preserved the stems in a number of cases [2]. Φ8 is the most distantly related member of the Cystoviridae relative to Φ6. It has no sequence identity or similarity to Φ6 and it has significant differences in genetic and physical struc- Page 2 of 10 (page number not for citation purposes) BMC Microbiology 2005, 5:10 http://www.biomedcentral.com/1471-2180/5/10 NdeI 138 AvrII 306 NsiI 328 SmaI 369 Φ8 segment L 7051 bp gene H gene 1 gene 4 gene 2 gene 7 SfiI 4580 XhoI 191 BamHI 215 SfiI 418 gene14 segment M 4741 bp gene 3b orf F orf G BamHI 2928 SacII 3124 gene 8 orf J gene 12 gene 5 gene 9 orf I SphI 1580 AflII 1290 BamHI 2006 gene 3a BspEI 619 ApaI 869 SacII 963 DraI 160 gene 10 gene 6 segment S 3192 bp Figure cDNA map 1 of the genomic segments of bacteriophage Φ8 cDNA map of the genomic segments of bacteriophage Φ8. An SP6 promoter precedes the cDNA copies in the plasmids. ture [11,12]. Our aim was to determine whether Φ8 genomic packaging uses pac sequences in a manner similar to that found for Φ6. Whereas the pac sequences were defined in Φ6 by in vitro packaging studies, we have (...truncated)