MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK
Correspondence
Nigel Stow
n.stow{at}vir.gla.ac.uk
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The requirements for HCMV DNA replication were identified through the use of transient transfection assays in permissive HF cells (Anders et al., 1992; Pari & Anders, 1993
; Anders & McCue, 1996
). These assays defined an origin of lytic DNA synthesis (oriLyt) and 11 protein-encoding loci. The required proteins comprise six that constitute the replication fork machinery, plus a set of auxiliary proteins involved in their expression and possible origin-specific functions (Anders & McCue, 1996
; Iskenderian et al., 1996
; Fortunato & Spector, 1999
). OriLyt-dependent DNA synthesis was subsequently demonstrated in transfection assays with non-permissive Vero cells, although in this case expression of the viral proteins was driven by strong constitutive promoters (Sarisky & Hayward, 1996
). These results reveal that there is no intrinsic block to the functioning of the HCMV replicative machinery in Vero cells.
Several different laboratory strains of HCMV are in common use and differences between strains have been reported both in tissue culture and the SCID-hu mouse model system (Brown et al., 1995; Sinzger et al., 1999
, 2000
). The first complete genome sequence was determined for HCMV strain AD169 (Chee et al., 1990
), and the majority of the plasmids utilized in investigations of viral origin-dependent DNA replication contain inserts of strain AD169 DNA (Anders et al., 1992
; Pari & Anders, 1993
; Sarisky & Hayward, 1996
). It was reported that up to 10 % of Vero cells infected with strain AD169 expressed IE1 protein, a proportion slightly higher than that previously observed with strain Towne virus (Bystrevskaya et al., 1997
; LaFemina & Hayward, 1988
).
In view of the above observations it was interesting to determine whether the limited expression of IE genes in infected Vero and 293 cells might be sufficient to activate the lytic cycle and allow viral DNA synthesis. We therefore employed a sensitive DNA blot hybridization approach to analyse replication of genomic DNA and a transfected oriLyt-containing plasmid in HF, Vero and 293 cells infected or superinfected with HCMV strain AD169.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Plasmids.
The HCMV oriLyt-containing plasmid, pSP50 (Anders et al., 1992) was kindly provided by David Anders. Plasmid pADori was constructed by cloning an AatII plus BstEII oriLyt-containing fragment spanning nucleotides 9132194168 of HCMV strain AD169 (Chee et al., 1990
; accession no. X17403), into pGEM7Zf(-). Plasmid pADori
XhoI contains a 1160 bp deletion between the two XhoI sites in pADori which removes sequences essential for origin function (Zhu et al., 1998
).
Infection with HCMV.
Monolayers of cells in 35 mm diameter Petri dishes were infected with 3 p.f.u. per cell HCMV strain AD169. After 1 h at 37 °C the inoculum was removed and the cells washed with acid glycine to inactivate residual virus (Stow, 2001), prior to continuation of incubation in EFC10 or EFC5.
Transfections.
Cell monolayers in 35 mm diameter Petri dishes were transfected using the calcium phosphate precipitation technique followed by brief exposure at 4 h post-transfection to 25 % DMSO (Stow & Wilkie, 1976). Each plate of cells received 0·5 ml of precipitate containing 12 µg calf thymus carrier DNA and 1·6 µg origin-containing plasmid. Six hours post-transfection the cells were infected with HCMV as described above except that the acid glycine treatment was omitted. Transfection efficiencies were routinely monitored using the plasmid pElacZ, which comprises the Escherichia coli
-galactosidase gene inserted into the expression vector pCMV10. Uninfected plates were fixed, stained for
-galactosidase expression and positive cells counted in representative microscope fields (Stow et al., 1993
).
DNA preparation and analysis.
Monolayers of cells were harvested at the indicated times post-infection (p.i.) and total cellular DNA was prepared and analysed as previously described (Hodge & Stow, 2001). One-tenth of the DNA recovered from each plate was digested with EcoRI (for the analysis of HCMV genome replication) or EcoRI plus DpnI (for the analysis of oriLyt-containing plasmid replication). DNA fragments were resolved by electrophoresis through a 0·8 % agarose gel, transferred to nitrocellulose and detected by blot hybridization. 32P-labelled pSP50 and pAT153 DNAs were used to detect viral and plasmid fragments, respectively. The plasmid vector pAT153 includes the majority of the vector sequences present in the HCMV oriLyt-containing plasmids. Following hybridization, the washed filters were exposed to a phosphorimager screen and data collected and analysed with the personal Molecular Imager and Quantity One software (Bio-Rad).
Immunofluorescence.
Cells were seeded onto glass coverslips in Linbro wells (1·5x105 cells per 13 mm diameter coverslip) 1 day prior to infection with 3 p.f.u. per cell HCMV. At 48 h p.i. the cells were fixed and permeabilized as previously described (Abbotts et al., 2000). The coverslips were reacted with FITC-conjugated mouse monoclonal antibody CCH-2 against HCMV UL44 (Dako) for 1 h at room temperature, extensively washed, treated with 0·5 µg Hoechst 33258 ml-1 in PBS for 10 min to enable visualization of cell nuclei, washed again and mounted. Slides were examined under a UV fluorescence microscope (Nikon Microphot SA) and the images captured on a digital camera.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Amplification of HCMV oriLyt-containing plasmids in HFFF, Vero and 293 cells
The ability of the HCMV DNA replication machinery to function in the three cell types was also assessed by superinfecting cells that had been transfected with oriLyt-containing plasmids. This approach has the advantage that unreplicated input plasmid DNA can be distinguished from molecules which have been replicated because of its susceptibility to digestion with DpnI. The plasmids used are shown in Fig. 2. Plasmid pSP50 contains the complete core and auxiliary regions required for maximum origin activity, whilst pADori more closely resembles a minimum functional origin. Replicate monolayers transfected with pADori were also mock-infected or following superinfection were incubated in the presence of 200 µg phosphonoacetic acid (PAA) ml-1, which specifically blocks the activity of the viral DNA polymerase (Huang, 1975
).
|
|
Over a large number of experiments we observed that, in marked contrast to genomic DNA, the accumulation of oriLyt-containing plasmids was of the same order in non-permissive Vero and 293 cells as in permissive HFFFs. This is probably because the significantly smaller proportion of infected Vero and 293 cells which express DNA replication proteins is counterbalanced by the much greater efficiency with which these cells can be transfected. Comparisons of transfection efficiencies throughout the course of these studies using a beta-galactosidase expressing plasmid, pElacZ, yielded values in the range 1·510 % and 520 % positive Vero and 293 cells, respectively. In contrast, the proportion of positive HFFF cells was generally approximately 0·1 %.
Expression of the UL44 protein
The above experiments provide evidence that proteins required for HCMV DNA synthesis are expressed after infection of non-permissive Vero and 293 cells. Immunofluorescence experiments were performed to assess the proportion of cells that expressed one particular DNA replication protein, the DNA polymerase processivity subunit, UL44.
Monolayers of HFFF, Vero and 293 cells grown on glass coverslips were infected with 3 p.f.u. HCMV per cell and examined for UL44 expression 48 h p.i. by UV fluorescence microscopy. The number of cells per field was determined by counting Hoechst-stained nuclei. Fig. 4 shows that, as expected, almost all HFFF cells expressed UL44. In contrast, UL44 expression was detectable in only a very small proportion of infected Vero or 293 cells. In repeat experiments the fraction of Vero and 293 cells which expressed UL44 ranged between 0·11 % and 0·52 %, respectively. Although UL44 was expressed in many fewer Vero and 293 than HFFF cells, the fluorescence intensities suggested that similar amounts were present in positive cells of the three different cell types. The expression of the early HCMV DNA replication protein UL44 in only a fraction of Vero cells agrees with an earlier report for viral early antigen in these cells (Einhorn et al., 1982
).
|
LaFemina & Hayward (1986, 1988
) previously demonstrated that HCMV infection of non-permissive cells may be blocked at two distinct stages. In several rodent and simian fibroblastic cell types the IE1 protein is abundantly expressed but viral DNA synthesis is undetectable, suggesting that in such cells the viral IE (or possibly early) proteins are functionally defective. In contrast, in cell lines such as Vero and 293, in which only a small fraction of infected cells express IE1 protein, the block was suggested to operate at the level of transcription initiation or mRNA stability. This block to expression from the major IE promoter was not evident in the context of transfected plasmid DNAs, again suggestive of some form of discrimination between plasmid and genomic DNA. More recently, it has been observed that fibroblast-adapted laboratory strains of HCMV exhibit 100- to 1000-fold lower infectivity in human endothelial cells than low passage isolates grown in these cells and that this appears to correlate with defects in transport of capsids towards the nucleus and nuclear import of the viral genome (Slobbe van Drunen et al., 1998
; Sinzger et al., 2000
). Although the proportion of pre-labelled input viral DNA recovered from Vero cell nuclei was somewhat less than from the nuclei of permissive cells, the reduction did not appear sufficient to account for the low level expression of IE1 protein (LaFemina & Hayward, 1988
). Nevertheless it remains possible that inefficient delivery of genomes to the nucleus may contribute to the observed block in protein expression and DNA replication, particularly in 293 cells in which nuclear DNA uptake has not been examined.
HCMV growth in HFFF, Vero and 293 cells
Since we had detected both viral origin-dependent DNA synthesis and expression of the UL44 DNA replication protein in HCMV-infected Vero and 293 cells, it was of interest to determine whether infectious progeny were generated. Monolayers of HFFF, Vero and 293 cells were infected with 3 p.f.u. per cell HCMV and after adsorption were washed with acid glycine to minimize the level of residual infectious input virus. Replicate plates were harvested 1 h and 4 days post-adsorption, and titrated on monolayers of HFFF cells. Table 1 shows that little or no infectious virus was detectable in the 1 h samples. Efficient virus replication occurred in HFFF cells, but no infectious progeny was produced in either Vero or 293 cells. The almost 105-fold reduction in virus yield from Vero and 293 cells is significantly greater than the reduction in either the proportion of cells expressing UL44, or in HCMV DNA accumulation in 293 cells, suggesting that there may be a further block to infection, possibly at the level of late gene expression or virion assembly in 293 (and probably also Vero) cells.
|
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anders, D. G. & McCue, L. A. (1996). The human cytomegalovirus genes and proteins required for DNA synthesis. Intervirology 39, 378388.[Medline]
Anders, D. G., Kacica, M. A., Pari, G. & Punturieri, S. M. (1992). Boundaries and structure of human cytomegalovirus oriLyt, a complex origin for lytic-phase DNA replication. J Virol 66, 33733384.[Abstract]
Brown, J. M., Kaneshima, H. & Mocarski, E. S. (1995). Dramatic interstrain differences in the replication of human cytomegalovirus in SCID-Hu mice. J Infect Dis 171, 15991603.[Medline]
Bystrevskaya, V. B., Lobova, T. V., Smirnov, V. N., Makarova, N. E. & Kushch, A. A. (1997). Centrosome injury in cells infected with human cytomegalovirus. J Struct Biol 120, 5260.[CrossRef][Medline]
Chee, M. S., Bankier, A. T., Beck, S. & 12 other authors (1990). Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD169. Curr Top Microbiol Immunol 154, 125169.[Medline]
Einhorn, L., Gadler, H. & Wahren, B. (1982). Adsorption of purified human cytomegalovirus and induction of early antigens in different cells. J Med Virol 10, 225234.[Medline]
Everett, R. D. (1984). Trans-activation of transcription by herpes virus products: requirement for two HSV-1 immediate-early polypeptides for maximum activity. EMBO J 3, 31353141.[Abstract]
Fortunato, E. A. & Spector, D. H. (1999). Regulation of human cytomegalovirus gene expression. Adv Virus Res 54, 61128.[Medline]
García-Ramírez, J. J., Ruchti, F., Huang, H., Simmen, K., Angulo, A. & Ghazal, P. (2001). Dominance of virus over host factors in cross-species activation of human cytomegalovirus early gene expression. J Virol 75, 2635.
Hodge, P. D. & Stow, N. D. (2001). Effects of mutations within the herpes simplex virus type 1 DNA encapsidation signal on packaging efficiency. J Virol 75, 89778986.
Huang, E. S. (1975). Human cytomegalovirus. IV. Specific inhibition of virus-induced DNA polymerase activity and viral DNA replication by phosphonoacetic acid. J Virol 16, 15601565.[Medline]
Iskenderian, A. C., Huang, L., Reilly, A., Stenberg, R. M. & Anders, D. G. (1996). Four of eleven loci required for transient complementation of human cytomegalovirus DNA replication cooperate to activate expression of replication genes. J Virol 70, 383392.[Abstract]
LaFemina, R. & Hayward, G. S. (1986). Constitutive and retinoic acid-inducible expression of cytomegalovirus immediate-early genes in human teratocarcinoma cells. J Virol 58, 434440.[Medline]
LaFemina, R. L. & Hayward, G. S. (1988). Differences in cell type-specific blocks to immediate early gene expression and DNA replication of human, simian and murine cytomegalovirus. J Gen Virol 69, 355374.[Abstract]
Mocarski, E. S. & Tan Courcelle, C. (2001). Cytomegaloviruses and their replication. In Fields Virology, 4th edn, pp. 26292673. Edited by D. M. Knipe & P. M. Howley. Philadelphia: Lippincott Williams & Wilkins.
Pari, G. S. & Anders, D. G. (1993). Eleven loci encoding trans-acting factors are required for transient complementation of human cytomegalovirus oriLyt-dependent DNA replication. J Virol 67, 71267131.
Pari, G. S., Kacica, M. A. & Anders, D. G. (1993). Open reading frames UL44, IRS1/TRS1, and UL3638 are required for transient complementation of human cytomegalovirus oriLyt-dependent DNA synthesis. J Virol 67, 25752582.[Abstract]
Pizzorno, M. C., O'Hare, P., Sha, L., LaFemina, R. L. & Hayward, G. S. (1988). Trans-activation and autoregulation of gene expression by immediate-early region 2 gene products of human cytomegalovirus. Journal of Virology 62, 11671179.[Medline]
Sarisky, R. T. & Hayward, G. S. (1996). Evidence that the UL84 gene product of human cytomegalovirus is essential for promoting oriLyt-dependent DNA replication and formation of replication compartments in cotransfection assays. J Virol 70, 73987413.[Abstract]
Sinzger, C., Schmidt, K., Knapp, J., Kahl, M., Beck, R., Waldman, J., Hebart, H., Einsele, H. & Jahn, G. (1999). Modification of human cytomegalovirus tropism through propagation in vitro is associated with changes in the viral genome. J Gen Virol 80, 28672877.
Sinzger, C., Kahl, M., Laib, K., Klingel, K., Rieger, P., Plachter, B. & Jahn, G. (2000). Tropism of human cytomegalovirus for endothelial cells is determined by a post-entry step dependent on efficient translocation to the nucleus. J Gen Virol 81, 30213035.
Slobbe van Drunen, M. E., Hendrickx, A. T., Vossen, R. C., Speel, E. J., van dam Mieras, M. C. & Bruggeman, C. A. (1998). Nuclear import as a barrier to infection of human umbilical vein endothelial cells by human cytomegalovirus strain AD169. Virus Res 56, 149156.[CrossRef][Medline]
St Jeor, S. C., Albrecht, T. B., Funk, F. D. & Rapp, F. (1974). Stimulation of cellular DNA synthesis by human cytomegalovirus. J Virol 13, 353362.
Stow, N. D. (2001). Packaging of genomic and amplicon DNA by the herpes simplex virus type 1 UL25 null mutant, KUL25 NS. J Virol 75, 1075510765.
Stow, N. D. & Wilkie, N. M. (1976). An improved technique for obtaining enhanced infectivity with herpes simplex virus type 1 DNA. J Gen Virol 33, 447458.[Abstract]
Stow, N. D., Hammarsten, O., Arbuckle, M. I. & Elias, P. (1993). Inhibition of herpes simplex virus type 1 DNA replication by mutant forms of the origin-binding protein. Virology 196, 413418.[CrossRef][Medline]
Zhu, Y., Huang, L. & Anders, D. G. (1998). Human cytomegalovirus oriLyt sequence requirements. J Virol 72, 49894996.
Received 4 September 2002;
accepted 15 November 2002.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |