School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK
Correspondence
Adrian Whitehouse
a.whitehouse{at}leeds.ac.uk
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ABSTRACT |
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INTRODUCTION |
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Sequencing of the A11-strain L-DNA revealed 75 open reading frames (Albrecht et al., 1992b). A number of these represent homologues of cellular genes. Some (eg ORF 2/vDHFR, ORF 16/vBcl2, ORF 72/vCyclin) are shared by most of the rhadinoviruses, while others (eg ORF 15/vCD59, ORF 13/vIL17) are unique to HVS or carried by only a few viruses. Most of the HVS ORFs are transcribed during the HVS lytic cycle, under the temporal control of ORFs 50 and 57 (Nicholas et al., 1988
, 1991
; Whitehouse et al., 1997
, 1998a
, b
).
In contrast, only three genes (ORFs 73, 72 and 71) are transcribed from a common promoter during an HVS latency model in a non-transforming strain (Hall et al., 2000a). In KSHV, the latent persistence of the herpesvirus episome requires the action of the protein expressed from ORF 73 (LANA) (Ballestas et al., 1999
; Ballestas & Kaye, 2001
), which binds to the TR of the virus to mediate persistence (Ballestas & Kaye, 2001
) and episome replication (Garber et al., 2002
; Grundhoff & Ganem, 2003
; Hu et al., 2002
). ORF 73 of HVS shows considerable sequence homology to LANA in the region responsible for latent replication of the episome (Grundhoff & Ganem, 2003
), and both proteins associate with metaphase chromosomes and exhibit similar nuclear distribution during interphase (Hall et al., 2000b
). The functional relationship of LANA and HVS ORF 73 was confirmed by combining three copies of the HVS terminal repeats with ORF 73 in a plasmid, which is maintained as an episome (Collins et al., 2002
).
One of the major tools for the functional characterization of viral genes and other non-transcribed elements is the creation of mutants. Conventional mutagenesis strategies require the production of viruses that remain replication competent. This therefore precludes the mutation of genes essential for the replication, packaging and infection aspects of the virus life-cycle. Recently, this problem has been overcome by the use of F-factor-based bacterial artificial chromosomes (BACs) for the cloning and mutagenesis of viral genomes (reviewed in Wagner et al., 2002). The initial cloning of murine cytomegalovirus, the prototype betaherpesvirus, into a BAC (Messerle et al., 1997
) has been followed by the generation of BACs carrying the genomes of herpes simplex virus (Horsburgh et al., 1999
; Saeki et al., 1998
; Stavropoulos & Strathdee, 1998
), human CMV (Borst et al., 1999
), and the gammaherpesviruses EBV (Delecluse et al., 1998
), KSHV (Delecluse et al., 2001
; Zhou et al., 2002
) and murine gammaherpesvirus 68 (MHV-68) (Adler et al., 2000
).
BAC elements have recently been cloned into the H-DNA of the HVS C-strain virus C484-77 (Collins et al., 2002). The resultant HVS-C-BACs retained one disrupted copy of the terminal repeat unit, and were able to undergo productive lytic infections, but did not directly persist as latent episomes. Persistent episomes were produced by incorporation of four copies of TR into the BAC.
Here we describe the cloning into a BAC of the herpesvirus saimiri A11-S4 strain (Desrosiers et al., 1984, 1986
), which is a non-transforming mutant of the sequenced strain (Desrosiers et al., 1985
). We elected to target the BAC to ORF 15 (vCD59) of HVS, which lies between conserved gene blocks. This gene is unique among the gammaherpesviruses, and encodes a homologue of mammalian CD59, a complement control protein (Albrecht et al., 1992a
). Human CD59 inhibits complement action by restricting the formation of the membrane attack complex, the last stage of complement-mediated cytolysis. HVS vCD59 also protects against cytolysis, but over a broader host range, protecting cells from lysis by rat serum as well as human and squirrel monkey serum, and is expressed only during the lytic cycle (Rother et al., 1994
). While vCD59 may play a role in vivo in protecting infected cells and/or the virus from immune destruction, it seemed unlikely to be required for in vitro replication and infection. We have investigated the cell culture phenotype of the HVS-BAC and compared it to the parental strain. We have then adapted a RecA-based modification system to allow the introduction of precise modifications into the HVS-BAC, and have used this to both exchange marker genes and to delete the HVS terminal repeats.
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METHODS |
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Virus was titrated as follows. Virus stocks were serially diluted 1 in 10 twice and 1 in 5 twice. Twenty µl of each dilution was added to 1 ml serum-free medium and left on OMK cells in a six-well plate with occasional mixing for 1 h. The medium was replaced with DMEM containing 5 % FCS and left overnight. This medium was replaced with a 1 : 1 mix of DMEM (10 % FCS) and 2 % low melting point agarose in PBS. After the agarose had set, it was overlaid with DMEM (5 % FCS) to prevent drying of the agarose gel. Virus plaques were counted (typically 12 weeks post-infection in the two lowest-concentration wells, which contained 0·2 and 0·04 µl of neat virus) to assess virus titre.
RNA analysis.
Total RNA was isolated using TRIzol (Invitrogen) according to the manufacturer's instructions and reverse transcribed with Superscript II (Invitrogen) for 1 h at 42 °C using an oligo(dT) primer. cDNA was then amplified by PCR using specific primers for ORFs 57 and 73 as appropriate, with primers for GAPDH as a cellular control as described previously (Smith et al., 2001). ORF 15 was amplified with a forward primer, ORF15-Fwd (TGCAAGCCAATACACAGCTTGC), and reverse primer, ORF15-mid-rev (GTCATCAAACTTCCACACCTG).
RecA-mediated recombination.
Recombination plasmids pKOV-KanF and pDF25-Tet are described previously (Lalioti & Heath, 2001). The NotIBbsI fragment containing the chloramphenicol gene was deleted from pKOV-KanF to produce pKOV-Kan-
Cm. The targeting construct pKOV-Kan-GFP was constructed by cloning the CMV-GFP expression cassette (NotIClaI from p5253; Wade-Martins et al., 2000
), via pBSKSII+, into BamHIXhoI of pKOV-Kan-
Cm. pKOV-Kan (and derivatives) and pDF25-Tet (a RecA expression plasmid) were grown at 30 °C in 10 ml LB cultures. DNA was isolated by a standard alkaline lysis miniprep protocol.
The deletion of the terminal repeats was achieved with the construct pKOV-Kan-TR. This contained PCR products from the left end (primers CGCGGTGACCTTACTGCAGCACTATGTGATTC and GCCATCGATGACTACTACATCTTCTGTACCAG) and the right end (primers CGCACGCGTGCAGAGGCTTGTGAGTGCAGAG and CGCGGTCACCGGCTTAATTACTACAGCAGCAG) of the HVS L-DNA. The left end region (positions 186 to 451 and 4271 to 4421 of L-DNA; accession no. X64346) includes the S4 strain deletion site, whereas the right end region (position 109620 to 110029) is in the middle of ORF75. These two regions were cloned together in pBluescript, through the BstEII sites designed into the PCR primers. This targeting region was then subcloned into pKOV-Kan-
Cm with BamHI/SalI to construct pKOV-Kan-
TR.
DH10B cells carrying the BAC pHVS15+.1 were made transformation-competent with calcium chloride. These were co-transformed with 1 µg of targeting construct (pKOV-Kan-GFP) and 5 µg of pDF25-Tet, and plated onto LB agar supplemented with chloramphenicol (cm), tetracycline (tet) and kanamycin (kan) at 30 °C overnight. A streak of colonies (550) was picked into 1 ml LB, and 100 µl plated immediately onto LB agar (cm+kan) and incubated overnight at 43 °C. Larger colonies were picked into 10 ml LB, grown overnight and analysed by restriction enzyme digestion to identify cointegrants.
E. coli carrying the correct cointegrant were made transformation-competent, transformed with 2 µl of pDF25-Tet and grown overnight on LB agar (cm+tet) at 30 °C. Pools of colonies were then picked into 1 ml of LB (cm+tet) supplemented with sucrose to 5 % w/v. This was grown for 8 h at 30 °C and then 110 µl streaked on LB agar supplemented with sucrose (5 %) and cm, and grown at 43 °C overnight. The colonies were replica plated onto cm+kan LB agar plates to screen for colonies surviving due to mutation in the sacB gene. Colonies that failed to grow on kanamycin were picked and BAC DNA analysed by restriction enzyme digestion and PCR to distinguish recombinants from revertants and to check the integrity of the BAC.
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RESULTS |
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Restriction analyses of the BACs produced band sizes that were consistent with those expected from the published sequence (Albrecht et al., 1992b). Bands containing ORF 15 were changed in ways consistent with the incorporation of the BAC elements by homologous recombination at ORF 15. The three BACs differed only in the orientation of the BAC elements (which depended on the targeting construct from which they were derived), and the size of their terminal repeats. pHVS15+1 and +2 had TRs of about 55 kb (pHVS15+2 marginally the larger) whereas that of pHVS15-1 was approximately 25 kb. PCR analysis showed that all three BACs lacked an intact ORF 15, but contained appropriate junctions between ORF 15 and BAC elements. This confirmed the restriction analyses that suggested that the BAC had been incorporated by homologous recombination at both flanking regions.
The S4 variant of the A11 strain of HVS is non-transforming and non-oncogenic as a result of a constructed deletion in the left end of the virus (Desrosiers et al., 1984, 1985
, 1986
). This deletion between SacI sites, where both ORF 1 (STP-A) and ORF 2 (vDHFR) and the region of dyad symmetry that lies in between (Fig. 1B
) have been removed, was confirmed by subcloning and sequencing of the left end of the viral genome.
However, as determined by PCR and restriction analysis, none of the BACs contained the GFP and neomycin cassettes that were in the virus used to create the A549 cell line from which the BACs were cloned. These cassettes were originally recombined into the junction between ORF75 and the terminal repeats. To analyse this region, pHVS+.1 was digested with MfeI and NotI and the right end was subcloned and sequenced. Both restriction analysis and sequencing showed that the ORF75 end of pHVS15+.1 was identical to the published sequence (Albrecht et al., 1992b).
The HVS-BAC produces virus at approximately wild-type levels
The first stage of analysis of the HVS-BAC was to establish whether it was capable of lytic virus replication. In most cell types, HVS infection generates a latent episome, but can be switched to its program of lytic replication and virus production by chemical stimuli such as n-butyrate or TPA. In contrast, OMK cells support the constitutive activity of the HVS lytic cycle. After transfection into OMK cells, all three HVS-BACs efficiently entered a productive lytic cycle, producing characteristic plaques surrounded by cells that expressed dsRed, indicative of a lytic infection by the HVS-BAC-derived virus (Fig. 2A). The supernatant from these cells was harvested and used to infect further OMK cells, in order to scale up the production of virus. Titres of HVS-BAC in the supernatant were approximately 1x106 p.f.u. ml-1, similar to titres attained from wild-type virus. RT-PCR analysis of OMK cells infected with the HVS-BACs was able to detect polyadenylated ORF 15 RNA from parental virus, but could not demonstrate any RNA predicted to produce the vCD59 truncated by insertion of the BAC elements. Other ORFs tested were transcribed normally (Fig. 2B
).
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To determine the impact of the deletion, pHVS15+TR.5 was transfected into OMK cells alongside pHVS15+1. Four days after transfection, pHVS15+.1 had generated viral plaques, whereas pHVS15+
TR.5 formed a few abortive foci of infection (Fig. 6
A), but was unable to generate functional virus or conventional viral plaques. To assess the behaviour of these BACs as latent episomes, pHVS15+
TR.5 and pHVS15+.1 were transfected into SW480 cells, split into 96-well plates after 48 h (250 cells per well) and selected with hygromycin. The majority of wells containing cells transfected with pHVS15+.1 generated colonies, while only a tiny minority of wells containing pHVS15+
TR.5 produced colonies, and did so no more frequently than a BAC vector containing no HVS elements (Fig. 6B
). Furthermore, episome was rescued from pHVS+.1 colonies, but could not be recovered from cells transfected with pHVS15+
TR.5.
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DISCUSSION |
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We chose to introduce the BAC elements into a gene that we hoped would minimally affect the viability of the virus. Since ORF 15 is unique to HVS, and lies within a region that is highly variable between different herpesviruses, it seemed unlikely to be essential for the virus tissue culture phenotype. This proved to be the case, as the HVS-BACs produced virus at levels similar to the parental A11-S4 strain. Like its cellular homologue CD59, ORF 15 is able to down-regulate complement activation in a number of different species (Rother et al., 1994). Undoubtedly this plays a role in vivo, but the nature of the gene suggests that the disruption will have almost no effect on any aspects of its life-cycle except those related to evasion of the complement response. The introduction of the BAC elements into ORF 15 as described here has not impaired the growth of HVS in vitro, and provides a potentially useful model for the future analysis of HVS. The recently reported HVS C488-77 strain BAC has been cloned by insertion of BAC elements in place of the H-DNA (Collins et al., 2002
). This BAC, although capable of creating infectious virions, does not generate latent episomes unless the TR are re-incorporated into the BAC.
The sensitivity of the episome rescue technique permits the analysis of individual latent episomes, and has shown that a number of episomes maintained in A549 cells have undergone rearrangements. Investigations of HVS-transformed cell lines that had lost their ability to produce infectious virus have identified a reduced quantity of unique sequences (L-DNA) when compared to terminal repeats (H-DNA) (Fleckenstein et al., 1977; Kaschka-Dierich et al., 1982
; Werner et al., 1977
). Analysis of these deletions demonstrated that, at their most substantial, they retained only the 15 kb regions at the flanks of the L-DNA (Schirm et al., 1984
). Analysis of three rearranged HVS-BAC episomes suggest that they consistently lose the region of the genome around ORF 50 (data not shown), which is responsible for the switch from the latent to lytic replication cycle, suggesting a growth advantage for cells carrying episomes that are unable to reactivate from latency. Rearranged HVS genomes were also observed in a persisting form of the C484-77 strain BAC (Collins et al., 2002
).
One of the major advantages of BACs is that the DNA insert can be precisely manipulated by recombination (reviewed in Wagner et al., 2002). We have used a RecA-based system previously shown to efficiently mediate homologous recombination with a very low incidence of unwanted rearrangements (Lalioti & Heath, 2001
). Furthermore, this approach, in which a cointegrant intermediate is resolved into a recombinant (see Fig. 4A
) allows any change (substitutions, deletions or insertions) to be achieved. The chief alternative to RecA is the use of ET recombination. This requires only short regions of homology, which may be advantageous for modification strategies but raises problems of illegitimate recombination between repeats within the herpesvirus genome. Use of the recently described GET recombination system (Narayanan et al., 1999
) allowed the targeted deletion of genes from bovine herpesvirus with no such complications apparent, with a requirement for only 50 bp of homology (Mahony et al., 2002
).
We have used the RecA system to modify the HVS-BAC. As with ET recombination, a high degree of precision can be achieved, as shown by the precise replacement of dsRed with GFP. The deletion of the HVS terminal repeats and ORF75 produced a genome that is unable to produce infectious virions or to persist as a latent episome. ORF75 encodes a homologue of FGARAT, an enzyme of purine synthesis. The protein is found in the viral tegument, alongside ORF3, another FGARAT homologue encoded by HVS (Fickenscher & Fleckenstein, 2001). While this enzyme may have a transforming role, the apparent redundancy and enzymatic function of ORF75 makes it seem unlikely that it would be essential to either the lytic or latent cycle of the virus in vitro. Work with KSHV has shown that the terminal repeats, in conjunction with the gene product of ORF73 are sufficient to mediate the persistence of KSHV episomes (Ballestas & Kaye, 2001
; Fejér et al., 2003
; Grundhoff & Ganem, 2003
). The C484-77 HVS-BAC was constructed by cloning the BAC elements, with NotI, into the TR of HVS. This deleted most of the TR, leaving only 80 bp on one side of the BAC elements, and approximately 1350 bp on the other. This BAC is also unable to establish itself as a latent episome, although recombining the TR into the BAC restored latency (Collins et al., 2002
). However, in contrast to pHVS15+
TR.5, three of the five C484-77 HVS-BACs were able to undergo a productive lytic infection. This observation implies that at least one of the junctions between the TR and the remainder of the genome, or a single copy of an element within the C484-BAC's remnant of H-DNA, plays a key role in the packaging of HVS. An interesting comparison can be made with EBV, where a unique 9 bp sequence at the junction between the right end of the terminal repeats and the left end of the unique region of the genome is essential for virus packaging (Zimmermann & Hammerschmidt, 1995
). Deletion of the EBV TR (including this flanking sequence) from an EBV-BAC produced a virus that was unable to package itself into infectious virions, but because latency was unaffected it could be used as a helper virus to package other constructs in trans (Delecluse et al., 1999
). The abrogation of both lytic and latent function of HVS by deletion of TR impedes further characterization of pHVS15+
TR.5, but taken with the data from the C484-77 HVS-BAC provides strong evidence for a role of the TR-flanking regions in generating infectious HVS particles.
In conclusion, we have cloned the strain A11-S4 herpesvirus saimiri genome into a BAC. We have shown that the virus exhibits essentially wild-type function, despite the deletion of ORF 15. We have then demonstrated the use of a RecA-based system for introducing changes into the HVS-BAC. We used recombination to substitute dsRed for GFP, and to delete the viral terminal repeats. This has demonstrated that the TR is essential for both the lytic and latent function of the virus. The use of herpesvirus BACs is set to play a major role in the analysis of viral gene function in the future. The precision with which modifications can be made in E. coli will allow both simple deletions and more complex substitutions and modifications to be made in viral genomes. This promises to accelerate the study of the many herpesvirus genes, with the consequent promise of anti-viral drug design as productive targets are identified. There is further potential in the development of herpesviruses as high capacity gene delivery vectors or as vaccines designed against a wide range of strains.
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ACKNOWLEDGEMENTS |
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Received 2 June 2003;
accepted 15 August 2003.