MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK
Correspondence
Nigel D. Stow
n.stow{at}vir.gla.ac.uk
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ABSTRACT |
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Present address: Division of Gene Expression and Regulation, MSI/WTB, University of Dundee, Dundee DD1 5EH, UK.
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INTRODUCTION |
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In addition to the proteins described above, HSV-1 also encodes an alkaline nuclease, the product of the UL12 gene, which acts upon the viral DNA during the replication and packaging processes (Martinez et al., 1996a). Although the alkaline nuclease is not absolutely essential for virus replication in tissue culture, HSV-1 null mutants produce 100- to 1000-fold less infectious virus than their wild-type (wt) parents, and require complementing cell lines for efficient propagation (Gao et al., 1998
; Martinez et al., 1996b
; Patel et al., 1996
; Weller et al., 1990
). The nuclease activity per se of the UL12 protein appears to perform an important replicative function (Goldstein & Weller; 1998
; Henderson et al., 1998
), but the reasons for the reduced growth of mutants in non-complementing cells remain incompletely understood. Weller and colleagues reported only a small reduction (less than 2-fold) in viral DNA synthesis, and cleavage of DNA concatemers occurred with close to wt efficiency. However, an apparent instability of DNA-containing capsids resulted in only approximately half of the cleaved genomes being recovered in the DNase-resistant (i.e. packaged) fraction. In addition, the mutants exhibited an increased accumulation of A capsids and concomitant decrease in C capsids in the nucleus of infected cells, and very few C capsids were detectable in the cytoplasm. These results led to the proposals that, in the absence of the alkaline nuclease, aberrant genomes were generated and packaged, abortive packaging events occurred frequently and that DNA-containing capsids were defective in their ability to mature into the cytoplasm (Martinez et al., 1996b
; Shao et al., 1993
; Weller et al., 1990
).
The use of gel electrophoretic techniques subsequently demonstrated that, in cells infected with wt HSV-1, the replicative concatemers comprise branched networks containing many X- and Y-type junctions, which are present as often as once per genome (Severini et al., 1994, 1996
; Zhang et al., 1994
). These structures almost certainly reflect the high frequency of recombination known to occur in HSV-1-infected cells (Brown et al., 1992
), and indicate that some mechanism must exist for their resolution prior to DNA packaging. Analysis by pulsed-field gel electrophoresis (PFGE) of DNA from cells infected with a UL12 null mutant demonstrated that the replicative intermediates produced by this virus were more complex than those of wt HSV-1, and apparently contained a higher level of branched structures (Martinez et al., 1996a
), suggesting a key role for the UL12 protein in their processing.
The viral alkaline nuclease has also been implicated in a second recombination-related activity. Database searches revealed that the UL12 protein is conserved throughout the herpesvirus family and homologues are widely distributed in other organisms, including certain double-stranded DNA bacteriophage and baculoviruses (Aravind et al., 2000; Bujnicki & Rychlewski, 2001
; Li & Rohrmann, 2000
; Mikhailov et al., 2003
; Vellani & Myers, 2003
). Several of the related proteins, exemplified by bacteriophage lambda exonuclease, are 5'3' exonucleases which, in association with a single-stranded DNA-binding protein, function as recombinases (Poteete, 2001
; Vellani & Myers; 2003
). The UL12 protein is known to interact with the HSV-1 single-stranded DNA-binding protein UL29 (Thomas et al., 1992
), and recent biochemical studies demonstrating that, together, the two proteins can mediate DNA strand exchange suggest that the UL12 protein may have a role in initiating viral recombination events (Reuven et al., 2003
). The alkaline nuclease is not, however, essential for viral DNA recombination, probably due to the activity of host enzymes (Martinez et al., 1996a
; Weber et al., 1988
).
In order to examine further how failure to express the viral alkaline nuclease impacts on the replicative cycle, we have performed a detailed characterization of the UL12 null mutant ambUL12 (Patel et al., 1996). The impairments in its ability to replicate and package the viral genome were quantified, but these only accounted for a small proportion of the reduction in the yield of infectious progeny virus. Since egress of DNA from the nucleus did not seem to be greatly reduced, we extended our investigation to the virus particles released from cells infected with ambUL12, and now demonstrate that these are greatly reduced in their ability to initiate new rounds of infection.
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METHODS |
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Analysis of DNA replication and packaging.
DNA replication and packaging assays were performed as described previously (Stow, 2001). Cells were infected with 3 p.f.u. virus per cell, incubated at 37 °C and harvested 16 h p.i. (or as stated). Total and DNase-resistant (encapsidated) DNAs were prepared from duplicate samples of whole cells, and nuclear and cytoplasmic DNase-resistant DNAs were isolated from separated fractions. The DNAs were analysed by restriction enzyme digestion and Southern blot hybridization. Phosphorimages were acquired by using a Personal Molecular Imager and analysed with QUANTITY ONE software (Bio-Rad).
PFGE.
Total infected cell DNA was analysed by embedding cells in 1 % agarose blocks (CleanCut; Bio-Rad) prior to in situ lysis and proteinase K digestion as recommended by the manufacturer. DNase digestion of whole cells or nuclear and cytoplasmic fractions was performed as described previously (Stow, 2001). Reactions were terminated by addition of EDTA to a final concentration of 40 mM prior to embedding, lysis and proteinase K digestion. The blocks were washed and pieces corresponding to one-quarter of the cells from a 35 mm Petri dish were inserted into the wells of a precast 1 % agarose gel. Electrophoresis and blotting were performed as described previously (Stow, 2001
).
Preparation of extracellular virus and viral DNA.
Cell monolayers in 175 cm2 tissue culture flasks were infected with 3 p.f.u. virus per cell. The medium was collected at 16 h p.i., and centrifuged at 800 g for 5 min to remove cellular debris, and then the virus particles were pelleted at 100 000 g for 30 min. Virus stocks were prepared by resuspending the pellets in 3 % of the original volume of medium. Yields of infectious virus were determined by titration on S22 cells, and particle numbers were determined by electron microscopy, using latex beads of a known concentration. To prepare DNA, the pellets of virus particles were resuspended in 0·25 ml per flask 10 mM Tris/HCl (pH 7·5), 1 mM EDTA (TE), lysed by addition of EDTA to 10 mM and SDS to 0·5 % and digested with proteinase K (1 mg ml-1) for 30 min at 50 °C. The samples were then gently extracted once each with phenol and chloroform and dialysed extensively against TE. The concentration of viral DNA was determined by Southern blot hybridization (see Results).
DNA transfections.
Monolayers of S22 cells in 35 mm dishes were transfected with viral DNAs using either calcium phosphate co-precipitation in conjunction with DMSO treatment (Stow & Wilkie, 1976) or Lipofectamine with PLUS reagent (Invitrogen) as recommended by the manufacturer. The cells were incubated for 3 days at 37 °C and plaques counted.
Virus yield assay.
Monolayers of Vero cells in 35 mm Petri dishes were infected with the specified viruses. One hour after addition of virus, the inoculum was removed and the monolayers were washed with acid glycine as described previously (Stow, 2001). Incubation was continued for a further 15 h. The cells were harvested by scraping into the growth medium, sonicated and the yield of virus was determined by titration on monolayers of S22 cells.
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RESULTS |
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Single-step growth curves demonstrated that, in BHK and Vero cells, the yield of ambUL12 was consistently 200- to 1000-fold lower than wt and ambUL12R (data not shown). The above reduction in encapsidated DNA is therefore insufficient to account for this decrease in yield. Shao et al. (1993) reported that AN-1 also demonstrated a marked reduction in egress of capsids from the nucleus. Analysis of DNase-resistant DNA from the nuclear and cytoplasmic fractions of Vero and BHK cells infected with ambUL12 or wt HSV-1 showed that ambUL12 was not greatly impaired in this process, and that DNase-resistant DNA was readily detected in cytoplasmic fractions of ambUL12-infected cells (Fig. 1b
). When the ratios of DNase-resistant DNA in the nuclear and cytoplasmic fractions were compared, ambUL12 was found to exhibit a small defect (within the range 1- to 3·6-fold) in nuclear egress compared with wt HSV-1 in both Vero and BHK cells. Some variability in the proportion of packaged DNA found in the cytoplasm was observed between experiments, possibly reflecting the difficulty of obtaining clean fractions. Nevertheless, electron microscopic examination of thin sections of infected cells demonstrated the presence of cytoplasmic enveloped ambUL12 particles, albeit in reduced numbers compared with wt HSV-1 and 12R (data not shown).
Time-course of DNA synthesis and packaging in ambUL12-infected cells
To determine whether the loss of the UL12 function had a similar effect throughout infection, a time-course of DNA replication and packaging in BHK cells infected with ambUL12 or wt HSV-1 was performed (Fig. 2). The amounts of total and DNase-resistant DNA were quantified and the proportion of the total DNA recovered in the DNase-resistant DNA fraction was calculated for each time point. The amount of ambUL12 DNA replicated was approximately 3-fold lower than wt throughout infection, and DNA packaging was also impaired at all times examined. This result suggests that the viral alkaline nuclease has a direct or indirect involvement in both DNA replication and packaging throughout infection, including early times when relatively little DNA has been synthesized and the replication compartments are small.
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Fig. 3 shows that linear HSV genomes were detected in total cellular DNA (Fig. 3a
) and DNase-treated whole cell, cytoplasmic and nuclear samples (Fig. 3b, c and d
, respectively) from both ambUL12- and 12R-infected cells, whereas the material that failed to enter the gel (well DNA) was degraded by DNase treatment. Quantification revealed that, for both viruses, approximately half the linear genomes present in the total DNA sample were recovered in the whole cell packaged DNA, indicating that the stability of packaging of ambUL12 and 12R genomes and their susceptibility to DNase were similar. However, the amount of linear ambUL12 DNA present in these fractions was reduced about 20-fold compared with 12R DNA, consistent with the reduction in packaging described above. In agreement with Fig. 1(b)
, linear ambUL12 molecules were detected in both the nuclear and cytoplasmic fractions.
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Analysis of ambUL12 particles released from BHK and Vero cells
In view of the above results, we tested whether infection of non-permissive cells with ambUL12 might generate significant numbers of virus particles unable to initiate infection in complementing S22 cells. Since stocks of wt HSV-1 derived from extracellular virus routinely exhibit the greatest infectivity (i.e. lowest particle/p.f.u. ratios), virus progeny released from BHK or Vero cells infected with ambUL12 or 12R were titrated on monolayers of complementing S22 cells, and the numbers of released virus particles were determined by electron microscopic examination of negatively stained samples. For both viruses, the majority of these particles were enveloped. The titres, particle concentrations and calculated particle/p.f.u. ratios are shown in Table 1.
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Characterization of DNA from ambUL12 particles
To determine whether the higher particle/p.f.u. ratios of ambUL12 progeny were a consequence of the packaging of poorer quality genomes, we examined the DNA isolated from extracellular virus particles from infected BHK cells.
The DNA was extracted using a gentle procedure to avoid damage through ethanol precipitation and resuspension (see Methods). To determine the concentration of viral DNA, samples were cleaved with EcoRI and analysed by using agarose gels alongside known amounts of EcoRI-cleaved pGX153. Fragment EcoRI N, from near the middle of UL, was detected by hybridization to labelled pGX153 DNA, and the signal intensities for the viral and plasmid-derived bands were measured on a phosphorimager. The same known amounts of ambUL12 and 12R DNAs were then compared in gels and subsequent transfection assays.
Fig. 4(a) shows a Southern blot analysis of EcoRI and BamHI digests probed with BamHI P and BamHI K, respectively. The genome locations and fragments detected are shown in Fig. 4(c)
. The patterns obtained for the two DNAs with the BamHI P probe were very similar, although the ambUL12 sample appeared slightly more diffuse in the region of the gel containing the co-migrating EcoRI F and G fragments. A much greater difference was apparent when BamHI digests of the same DNAs were probed with BamHI K. Although the pattern and intensities of the bands were very similar, an additional distinct smear was present in the lane containing ambUL12 DNA. Phosphorimager analysis showed that, whereas the total radioactivity hybridized to the two DNAs was very similar for the BamHI P probe, approximately twice as many counts hybridized to ambUL12 as to 12R DNA when BamHI K was the probe.
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These results indicate that packaged ambUL12 DNA prepared by two different methods is structurally distinct from 12R DNA and that different regions of the genome appear to be differently affected. It is possible that the same structural abnormality is responsible for both the apparent size heterogeneity of ambUL12 genomes seen by PFGE (Fig. 3) and the unusual patterns of hybridization seen in Fig. 4
.
Infectivity of ambUL12 DNA
Although DNA from ambUL12 particles exhibited structural abnormalities compared with 12R DNA, this did not necessarily account for the higher particle/p.f.u. ratios of the mutant virus. The infectivity of the two DNAs was therefore compared on S22 cells using both calcium phosphate- and liposome-mediated transfection procedures (Table 2).
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Dominant inhibitory effects of ambUL12 DNA and particles
Since the particle/p.f.u. ratios for ambUL12 grown in BHK cells exceeded those of 12R by only 28- to 44-fold (Table 1), it was surprising that no ambUL12 plaques were observed under conditions when 12R produced >100 plaques. We therefore tested whether ambUL12 might exert a dominant inhibitory effect on the infectivity of co-transfected wt HSV-1 DNA. The results of such an experiment, in which DNAs were introduced into the cells using liposomes, are shown in Table 3
. The addition of ambUL12 DNA completely abolished plaque formation by wt HSV-1 DNA, whereas an approximately additive effect was observed with 12R DNA. An essentially identical result was obtained using the calcium phosphate procedure, except that fewer plaques were produced by 12R and wt HSV-1 DNAs (data not shown).
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DISCUSSION |
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Virus particles released from non-permissive cells infected with UL12 null mutants have not previously been characterized. Our results reveal several interesting features about these particles: (i) large numbers of such particles are generated, but they exhibit higher particle/p.f.u. ratios than wt HSV-1 or a rescued virus; (ii) the genomes packaged into the particles are structurally aberrant and non-infectious in transfection assays; and (iii) both the virus particles and the DNA isolated from them can exert a dominant inhibitory effect on the replication of wt HSV-1. DNA isolated from ambUL12 particles in the cytoplasmic fraction of infected cells exhibited aberrant genomes and a lack of infectivity essentially identical to the released genomes (data not shown), indicating that no selectivity exists in the nature of particles released from the cell.
Comparisons with a rescued virus, 12R, demonstrated that the defects exhibited by ambUL12 were a consequence of the original mutation introduced into the UL12 gene. Interestingly, however, ambUL12 stocks derived from S22 cells exhibited significantly higher particle/p.f.u. ratios than wt HSV-1 grown on these cells, and structural abnormalities were detectable in the packaged genomes by blot hybridization (data not shown). These latter observations are consistent with previous reports that complementing cell lines, including S22, fail to support the growth of UL12 null mutants to wt levels (Shao et al., 1993; Weller et al., 1990
).
The mechanistic activities of the HSV-1 alkaline nuclease during infection have not been fully elucidated, and it remains difficult to explain our observations in terms of the absence of its function. The protein has been implicated in two possible recombinational activities, namely the removal of branched structures from concatemeric viral DNA (Martinez et al., 1996a) and the initiation of single strand exchange (Reuven et al., 2003
). Replicative intermediates that accumulate in the absence of the nuclease are structurally more complex than those in wt HSV-1-infected cells, and probably contain a greater frequency of branched structures (Martinez et al., 1996a
). It can be envisaged that the reduction in DNA synthesis reported here, and by others (Shao et al., 1993
; Martinez et al., 1996b
), might result from such branches imposing topological constraints on replicating molecules and impeding the progress of the DNA synthetic machinery. An alternative explanation, that UL12 plays a role in a switch to a recombination-driven late mode of DNA synthesis, as occurs in bacteriophage T4 (Mosig, 1987
), seems less likely, since the effects of the absence of the alkaline nuclease were seen as early as 6 h p.i. (Fig. 2
).
The presence of a greater number of branches in replicative intermediates also provides a plausible explanation for (i) the reduced efficiency of packaging that we observed in ambUL12-infected cells, and (ii) the packaged molecules appearing to be structurally aberrant. The structural abnormalities were observed in both intracellular DNase-resistant DNA and DNA extracted from extracellular capsids, and are therefore unlikely to be artefacts of the DNA preparation. The smearing and increased hybridization to certain restriction enzyme fragments of viral DNA (Fig. 4) may reflect the presence of residual single- or double-stranded branched structures that would normally be removed by UL12 prior to encapsidation. It is interesting to note that the degree of smearing and enhanced hybridization was much greater for fragments containing the a sequence and portions of the RL and RS regions (BamHI K, Q and S) than for fragments from UL (EcoRI F, G and N). This possibly correlates with previous observations that the a sequence and repeat regions represent recombinational hot spots in the HSV-1 genome associated with segment inversion (for a review see Umene, 1999
). It remains to be determined whether the presence of branched structures might also explain the diffuse migration of packaged linear ambUL12 genomes seen by PFGE (Fig. 3
). Work is in progress to characterize further the structural abnormalities of the packaged ambUL12 genomes.
It is clear from our characterization of DNA from extracellular ambUL12 particles that capsids containing structurally abnormal DNA can exit from the nucleus and become enveloped. DNA transfection experiments indicated that the increased particle/p.f.u. ratios observed for the cell-released ambUL12 were likely to be primarily a consequence of the aberrant nature of the packaged DNA. Exactly why these genomes are reduced in their ability to initiate plaque formation is not known, but could reflect an incomplete gene content, or cis-acting effects that might preclude genome replication or the normal expression of one or more essential genes. The presence of multiple branches and associated strand breaks in an infecting genome, if left unrepaired, might be anticipated to impede normal movement of replication or transcription complexes, and lead to an increased frequency of double-strand breaks. The residual infectivity, on complementing S22 cells, of ambUL12 particles released from non-complementing cells possibly indicates that a small proportion of the packaged genomes contain relatively few, if any, of the abnormal structures, or that the alkaline exonuclease expressed by S22 cells can function in their repair prior to replication.
Rather surprisingly, ambUL12 DNA and particles from non-permissive cells also interfered with the infectivity of wt HSV-1. In DNA transfections, a complete inhibition of plaque formation by wt HSV-1 DNA was observed, whilst the inhibitory effect of virus particles, although dose dependent, was not as great (Tables 3 and 4). We speculate that, in cells receiving both wt and mutant viral genomes (delivered as either DNA or virus particles), recombination events might introduce aberrant structures into the wt genomes, thereby reducing their ability to give rise to infectious progeny. The effect is possibly more dramatic in the transfection experiments because the successfully transfected cells receive many more viral genomes than those infected with virus particles, thereby increasing the likelihood of recombination. Recombination between normal and aberrant genomes may also explain why UL12 null mutants tend to form tiny plaques in non-complementing cells (Martinez et al., 1996b
; Shao et al., 1993
; Weller et al., 1990
). In this situation, the small proportion of viable progeny released from the originally infected cell might become recombinationally inactivated in the presence of an excess of abnormal genomes upon infection of surrounding cells.
Taken together, our data and previous reports (Martinez et al., 1996b; Shao et al., 1993
; Weller et al., 1990
) demonstrate that the failure to express a functional HSV-1 viral alkaline nuclease can have multiple effects on viral DNA synthesis and packaging, release of capsids from the nucleus, infectivity of progeny virus and structure of encapsidated DNA. Although it is tempting to conclude that the alkaline nuclease is a multifunctional protein, it remains possible that all these effects result from the loss of a single biochemical function such as the ability to process correctly the branched structures in replicative intermediates. Further research on the role of the alkaline nuclease in recombination events should hopefully clarify this situation.
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ACKNOWLEDGEMENTS |
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Received 16 September 2003;
accepted 26 November 2003.