In vivo studies of genomic packaging in the dsRNA bacteriophage Φ8
© Qiao et al; licensee BioMed Central Ltd. 2005
Received: 12 January 2005
Accepted: 11 March 2005
Published: 11 March 2005
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.
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.
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.
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 . 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 . 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 . Packaging in the Cystoviridae is driven by an NTPase motor comprised of a 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 .
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 . The pac sequences showed considerable secondary structure, with a number of stem-loops . Comparison of the pac sequences of close relatives of Φ6 suggested that the stem-loop structures were important because base changes preserved the stems in a number of cases . 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 structure [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 found that in vitro packaging for Φ8 is non-specific when evaluated by the uptake of radioactive RNA . We have therefore used in vivo transcript acquisition to define the pac sequences of Φ8 in this report.
The determination of the pac sequences of Φ8
Plasmids used in this study with cDNA copies of Φ8S
no +A172C* or U161A
no + C167U
no + U135C
no + C132A
no + C132U
no or G111A or U114C
Plasmids used in this study with cDNA copies of Φ8L
Plasmids used in this study with cDNA copies of Φ8M
The consequences of replacing the pac sequence of M with that of L
Base changes within the pac regions of the Φ6 genome have resulted in the isolation of suppressor mutations in protein P1, the major structural protein of the procapsid and the determinant of the RNA binding sites. However, in the case of Φ8 we have found less stringency in the pac sequences and we have not found suppressor mutations in P1. In the case of Φ6 it was found that suppressor mutations in P1 could also be isolated when conditions were established that were contrary to the packaging program . Packaging of segments M and L without segment S could be established in carrier states if suppressor mutations were promoted in gene 1 . We had previously found that gene 8 was indispensable in Φ8, thereby making it difficult to establish carrier states in the absence of segment S. We therefore set out to select for a condition where the M segment would have the pac sequence of L and that phage would be selected for with both the M and L segments having the same pac sequences.
Production of phage by electroporation of cDNA plasmids with pac sequence of L in segment M
M has Lpac
1 or 0
Mutant L and M having Lpac
Mutant L and Normal M
Plus strands of the three genomic segments of bacteriophage Φ6 are packaged by core structures composed of proteins P1, P2, P4 and P7 . They bind to protein P1, the major structural protein of the core and are translocated to the interior by hexamers of the NTPase P4. We had worked out the identification of sequences necessary and sufficient for packaging in bacteriophage Φ6 by in vitro packaging experiments. A region near the 5' end designated as pac is necessary and sufficient for packaging. We have established that the pac sequences of Φ6 are located in the 5' untranslated regions of the plus strands. Whereas the in vitro packaging of Φ6 is very stringent and specific, that of Φ8 was found to be non-specific when uptake of radioactive RNA was being assayed. In vivo packaging was more specific . We therefore established a system of in vivo transcript acquisition to test the sequence limits for the packaging of the Φ8 genome.
In this report, we have identified the pac regions for bacteriophage Φ8, a distant relative of Φ6. All three plus strands of Φ8 have a consensus sequence of 9 nucleotides at the 5' end. Again, we have found that the pac regions occupy space in the 5' untranslated regions of the plus strands. Although the pac regions of both Φ6 and Φ8 have many stem-loop structures, there is no sequence or structural similarity between the two sets. There is also no sequence similarity between the pac sequences of the three genomic segments of Φ8. While the Φ6 pac sequences extend approximately to nucleotides 230, 305 and 270 for L, M and S respectively; those of Φ8 extend to near nucleotides 244, 242 and 170. The Φ8 pac sequences end closer to the first orfs than those of Φ6. In the cases of segments S and M the pac sequences might overlap the first orfs. Changes within the pac region of Φ8 segment S are less destructive of packaging than similar changes in pac regions of Φ6. Many changes are tolerated, and in some cases the effects of changes are suppressed by other changes in the pac sequences. In some cases it is possible to rationalize the suppression in terms of the structure shown by mfold ; in other cases it is not clear why the suppression is effective. In Φ6, we found little suppression due to changes in the pac sequences but we found suppressors by mutation in the sequence of P1, the major structural protein of the core. Genomic RNA that is bound to procapsids can be cross-linked to protein P1 . So far, we have not found suppressors of this type in Φ8 for changes in the pac sequences. However, we did obtain a suppressor mutation for another type of packaging program transgression. We found that we could exchange the pac sequence of L with that of M. The L segment could be packaged in this case if an amino acid change was present in P1.
The experiments involving manipulation of the pac sequence of segment S indicate that the Φ8 packaging mechanism has a lower level of stringency than that of Φ6 . Is there a benefit of low stringency in packaging for a viral system? It appears that the answer is yes in many cases. Many bacteriophages with DNA genomes are able to package host or plasmid DNA and consequently carry out transduction of host genes . Low stringency offers the possiblility of the acquisition of host RNA transcripts by RNA viruses. We have seen that Φ6 can acquire plasmid transcripts that do not have pac sequences, albeit at extremely low frequencies . It also allows the acquisition of genomic segments of distantly related phages. Although Φ6 cannot acquire the genomic segments of Φ13 a related cystovirus, Φ13 can acquire the S and M segments of Φ6 . Influenza virus is now believed to have specific packaging of its genomic segments . However the packaging is of low enough stringency that virions can acquire genomic segments from viruses that infect very different host organisms .
The sequences that are necessary for packaging transcripts of the dsRNA genome of bacteriophage Φ8 are located in the 5' untranslated regions of the three genomic segments. Genomic packaging in Φ8 is much less stringent than that found for the distantly related bacteriophage Φ6. Many changes in the pac region of S are tolerated while others are suppressed by compensating changes in the RNA sequence. Changes in the packaging program can be produced by mutations that change the amino acid sequence of P1, the major structural protein of the procapsid.
Bacterial strains and plasmids
Pseudomonas syringae HB10Y derivative LM2489 was the host for Φ8 [1, 19]. Escherichia coli strain JM109 (recA1, endA1, gyrA96, thi, hsdR17, supE44, relA1, λ-, Δ(lac-proAB)), [F', traD36, proAB, lacIq, ΔM15]  was used for the propagation of all plasmids. Epicurian Super Competent cells of E. coli (Stratagene) were used for transformation after directed mutagenesis. Plasmids utilized in this study are listed in tables 1, 2 and 3. All cDNA plasmids used in this study have a SP6 promoter upstream of the cDNA sequences. The SP6 promoter is preceded by a BglII site. The sequences of the Φ8 genomic segments are in GenBank with accession numbers NC_003299, NC_003300 and NC_003299 for L, M and S respectively.
Reverse genetics for the modification of the viral genome
The simplest means of virus construction is to electroporate host cells with SP6 promoter plasmids that contain cDNA copies of the genomic segments along with a plasmid that codes for SP6 RNA polymerase. We generally add about 200 ng of each cDNA plasmid to about 2 × 109 cells. Using colE1 based plasmids such as pT7T319U, that transcribe but do not replicate in pseudomonads we can produce hundreds of plaques. If the polymerase plasmid is resident in the host strain, we can produce tens of thousands of infectious centers with wild type constructions. Plasmid pLM2790 is a derivative of shuttle vector pLM254 with SP6 polymerase gene of PSR3  cloned into the BamH1 site . In cases where we alter the pac sequences, the number of plaques can be reduced drastically. In some cases, the resulting phage contain suppressor mutations or reversions of the directed mutations. Although the 5' sequence of the Φ8 plus strands, GAAA, is more compatible with the specificity of SP6 polymerase, it is possible to use T7 RNA polymerase as well, although with somewhat lower efficacy.
If one of the genomic segments has a gene for kanamycin resistance, kan, inserted into either its non coding region or replacing one or more genes, then electroporation can result in the establishment of a carrier state in which the viral genome is replicated in cells without lysis and with the expression of the drug resistance so as to form stable drug resistant colonies .
Production of deletions in the pac sequences of the Φ8 segments
oligonucleotides used for mutagenesis of pac sequences
The placing of the pac sequence of L on segment M
The BglII/XhoI fragment of plasmid pLM2669 contains the pac sequence of Φ8M as well as the SP6 promoter. This fragment was exchanged with the BglII/SmaI fragment of plasmid pLM2653 which carries the cDNA copy of Φ8L (Fig. 1).
dsRNA was isolated from crude cell lysates by first treating with DNase, then phenol extraction and electroelution of the RNA from agarose gels. The RNA was then denatured and annealed to primer and incubated with AMV reverse transcriptase. The products were then subjected to PCR with Pfu turbo DNA polymerase (Stratagene) . The resulting DNA products were cloned into pT7T319U and sequenced at the Molecular Resources Facility of the University of Medicine and Dentistry of New Jersey.
Mfolds of segment S pac sequences
The secondary structures of the pac sequences were determined on the mfold web server using the mfold 3.1 program of Zuker and Turner . The folds were constrained so that the maximum distance between paired bases was 50. The mfold structural predictions for the Φ6 pac sequences were previously found to be similar to those found by nuclease sensitivity . The structures shown in Figures 2 and 3 are not unique in that there are several structures with similar free energy values. However, stem-loop F in Figure 2 is present in all of them.
This work was supported by grant GM34352 from the National Institutes of Health. Plasmid PSR3 was a gift of Dr. William McAllister.
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