Servizio di Virologia, Istituto di Ricovero e Cura a Carattere Scientifico Policlinico San Matteo, 27100 Pavia, Italy1
Author for correspondence: Giuseppe Gerna. Fax +39 0382 502599. e-mail g.gerna{at}smatteo.pv.it
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Abstract |
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Introduction |
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Although PMNL carry infectious virus, they do not seem to be productively infected in vivo (Gerna et al., 1992a , 2000
; Grefte et al., 1994
). However, results obtained using an in vitro model for the study of the PMNLEC/HELF (human embryonic lung fibroblasts) interaction suggest that PMNL may contribute to the haematogenous dissemination of HCMV in immunocompromised patients (Grundy et al., 1998
; Revello et al., 1998b
).
In the present study, we have plaque-purified isolates that do not have the capacity of the parental strains to be transferred to PMNL (PMNL tropism) or to grow in EC (EC tropism). These virus variants, referred to as transfer-deficient variants, are genetically related to the parental strains and behave in a way similar to laboratory-adapted HCMV strains in terms of PMNL and EC tropism. Transfer-deficient variants are useful tools for studying HCMVPMNL interactions and may help to identify the viral gene(s) that is responsible for PMNL and EC tropism.
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Methods |
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Coculture of PMNL with HCMV-infected cell cultures.
Concentrated PMNL preparations were cocultured with either HELF or HUVEC 96 h post-infection (p.i.) with either parental HCMV strains or their plaque isolates (Gerna et al., 1998b ; Revello et al., 1998b
). Coculture times were 3 h for HUVEC and 1824 h for HELF, unless otherwise indicated. Following coculture, PMNL were separated from the infected cells that had detached from the growth surface. To achieve this, cell suspensions were placed in the upper compartment of a cell culture insert, which consists of a 6·5 mm diameter transwell filter (5 µm pore size, Costar), for 3 h at 37 °C in 5% CO2; the lower compartment contained 10-8 M N-formyl-met-leu-phe-ala (Sigma) (Gerna et al., 1998b
; Revello et al., 1998b
). This procedure causes PMNL to migrate to the lower compartment and activates a respiratory burst. The level of PMNL purification achieved is comparable to that of fluorescence-activated cell sorting (Revello et al., 1998b
).
Assay for PMNL tropism.
Plaque isolates were screened for PMNL tropism by screening for the presence of HCMV pp65 in PMNL. Each virus plaque was picked and propagated in HELF until 100% CPE was reached (parental strains were also assayed when 100% CPE was reached). Each parental strain or plaque isolate was then tested for PMNL tropism according to the procedure described by Gerna et al. (1992b , 1998b
). Plaque isolates that maintained the same PMNL tropism as that of the original clinical isolate were referred to as parental strains, whereas plaque isolates that lacked this tropism were designated transfer-deficient variants. Virus stocks were prepared in HELF followed by two additional plaque isolation steps for each of the four parental strains and each of the transfer-deficient variants.
Assay for EC tropism.
The parental strain and transfer-deficient variants of VR6110 were assayed for EC tropism as follows. HELF were infected with the VR6110 variants at an m.o.i. of 15. After 7 days of incubation at 37 °C, infected HELF (80100% CPE) were trypsinized and inoculated at a ratio of 1:3 (infected HELF:uninfected HUVEC) onto confluent monolayers of uninfected HUVEC grown in 24-well plates; repeated attempts to infect HUVEC with HELF-derived sonicated cell-free virus suspensions were unsuccessful. After 7 days of incubation, CPE was observed in wells inoculated with either the parental strain or the transfer-deficient variant. Infected HUVEC were then trypsinized and mixed at a ratio of 1:2 with uninfected HUVEC at passage 2. Cells were reseeded and cultured for an additional 7 days. This procedure was repeated weekly until passage 6, after which cells were sonicated and cell-free HCMV was collected. The relative proportion of HELF in the coculture progressively decreased until, finally, no more were present (by passage 6). After each passage of virus growth in HUVEC, cell monolayers were incubated with monoclonal antibodies against either immediate-early (IE) or late viral proteins and stained, as described previously (Gerna et al., 1990 ). The three additional clinical isolates were assayed according to the same protocol.
Growth of VR6110 or its transfer-deficient variant in HELF.
To compare the growth of the VR6110 parent and transfer-deficient variant strains, HELF were infected with the VR6110 strains at an m.o.i. of 35. The infected cell culture medium and samples of HELF collected on days 38 p.i. were then used to determine the relative proportions of cell-free and cell-associated virus. The Towne strain was titrated for comparison.
HCMV load in infected HELF and in PMNL cocultures.
HELF were synchronously infected with VR6110 (either the parental strain or the transfer-deficient variant) at an m.o.i. of 15. Infected HELF were then harvested 120 h p.i. (100% CPE) and samples of 1x105 cells were used for nucleic acid quantification. The copy number of the viral DNA, IE mRNA and pp67 mRNA in infected HELF was determined by dividing the total copy number of viral nucleic acid by 1x105. PMNL cocultured for 24 h with infected HELF (96120 h p.i.) were collected and aliquoted to determine (i) the number of pp65- and p72-positive cells, (ii) the number of PMNL carrying infectious virus and (iii) the number of PMNL positive for viral DNA or mRNAs. To quantify PMNL positive for viral nucleic acids, serial PMNL mixtures each containing a progressively decreasing number of cocultured PMNL in a progressively increasing number of PMNL from a healthy donor were prepared and tested for viral DNA by quantitative PCR (1x105 cell per sample) and for viral mRNAs by nucleic acid sequence-based amplification (NASBA; Organon Teknika, Boxtel, The Netherlands).
Virus assays.
To determine and quantify the different virus parameters in PMNL, the number of pp65- and p72-positive PMNL was determined on PMNL (1x105) cytospin preparations that were fixed and stained according to a procedure reported previously (Gerna et al., 1992b , 1998b
). Infectious virus carried by PMNL after cocultivation was quantified by inoculating 1x105 PMNL onto HELF monolayers grown in shell vials and counting the number of p72-positive nuclei (stained 1624 h p.i. with an anti-p72 monoclonal antibody) (Gerna et al., 1990
). HCMV DNA was quantified in PMNL (1x105) samples by quantitative PCR using a primer pair specific for exon 4 of the major IE gene, as reported previously (Gerna et al., 1994
, 1998a
). HCMV IE and pp67 mRNA were quantified by NASBA (Middeldorp et al., 1999
). The reliability of these methods for both pp67 (Blok et al., 1998a
, b
; Gerna et al., 1999
) and IE mRNA (Blok et al., 1998a
, 1999
) determination has been reported previously.
Restriction fragment length polymorphism (RFLP) analysis.
Four genomic regions of the parental strain and two of its plaque isolates (one with transfer capacity comparable to that of the parental strain and the other with transfer-deficient capacity) of VR6110 as well as AD169, Toledo and Towne strains were amplified by PCR using the following pairs of primers: Bw1 and Bw2 (nt 7656277992), Pol1 and Pol2 (nt 7790578023), Pol3 and Pol4 (nt 7799679918) and Bw3 and Bw4 (nt 7986380844) for the UL54 ORF; UL97.1 and UL97.6 (nt 141288142917) for the UL97 ORF; IE1.1 and IE1.2 (nt 173619174206) for the UL123 ORF; and TLF15 and TLR22 (nt 47518491), TLF25 and TLR30 (nt 847611355) and TLF31 and TLR36 (nt 1056814041) for the ULb' fragment (Fig. 1). PCR products were then cleaved using two to four of the frequently used endonucleases HaeIII, MspI, HinP1I, AluI and BstUI (New England Biolabs). RFLP patterns were compared by agarose gel electrophoresis (Fig. 1
).
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Southern blot analysis of the VR6110 parental strain and its transfer-deficient variant.
The genomes of VR6110 and its transfer-deficient variant as well as that of a VR6110 transfer-positive plaque isolate were digested with EcoRI, HindIII and BamHI, blotted onto nylon membranes (Boehringer Mannheim) and hybridized using a set of cosmid probes from the VR6110 genome library spanning almost (9498%) the entire virus genome. The Towne and Toledo strains were also digested and probed as above.
Time-course of VR6110 infection in permissive cells by infection of PMNL following coculture at different times p.i.
Uninfected HUVEC and HUVEC infected with VR6110 parental strain at an m.o.i. of 35 were cocultured with PMNL for 3 h at time 0 (prior to infection) and thereafter at 24, 48, 72, 96, 120 and 144 h p.i. In similar experiments, uninfected HELF and HELF infected with either the VR6110 parental strain or its transfer-deficient variant at an m.o.i. of 13 were cocultured with PMNL for 24 h at the same times p.i. Following PMNL purification by migration and repeated washings, infectious virus and viral antigens as well as viral nucleic acids were quantified at different times, as reported above.
To investigate the role of late virus products in PMNL tropism, PMNL were cocultured for 3 h with HUVEC synchronously infected with VR6110 parental strain. Coculture was carried out 96 h p.i. in either the absence or the presence of phosphonoformic acid (PFA), which was added at a concentration of 400 µM to HUVEC at the time of infection.
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Results |
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We also investigated the transfer ability of three additional clinical isolates. All of the 15 plaque isolates obtained directly from the urine sample of a congenitally infected baby (VR6952) were found to be capable of transfer. However, after subsequent passage in HELF, the first transfer-deficient variant was found at passage 20. As for the other two clinical isolates, the first transfer-deficient variants were detected as single plaque isolates at passage 33 for VR6340 and at passage 37 for VR1814. Thus, transfer-deficient variants could be recovered from each of the four clinical isolates tested between passages 10 and 40. VR6952 became transfer-deficient at passage 50 in HELF, whereas VR6340 became transfer-deficient at passage 59. VR1814 apparently retained its transfer capacity at the same level as in the initial passages (5000 pp65-positive PMNL per 1x105 PMNL) up to and after 60 passages, even though a small number of plaque isolates with reduced transfer capacity were present.
Quantification of PMNL tropism
PMNL tropism of the clinical isolates as well as their plaque isolates was quantified according to the mean number of pp65-positive PMNL per 1x105 PMNL examined (Table 1) and were graded as negative (no positive cells detected), low (110 positive cells), intermediate (10100 positive cells) or high (>100 positive cells). The VR6952 parental strain and its plaque isolates showed high levels of pp65-positive PMNL both in the original clinical sample as well as at passage 10 (1020 plaques were examined each time). Subsequently, starting from passage 20, the number of plaque isolates with transfer-deficient capacity progressively increased until all of the plaque isolates at passage 50 were transfer-deficient. VR6340 showed a similar trend. On the other hand, the transfer capacity of VR1814 was consistently positive at a high level both for the clinical isolate and for the plaque isolates tested within passage 50, except for a single plaque isolate that showed a transfer-deficient phenotype at passage 37. At passage 60, two plaque isolates with intermediate or low transfer capacity were identified, while the relevant clinical isolate (VR1814) still maintained a high transfer capacity. Two further plaque purification steps of plaque isolates without the transfer capacity consistently yielded plaque isolates with a transfer-deficient phenotype.
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Genetic relatedness of the VR6110 parental strain and its transfer-deficient variant
The genetic relatedness of VR6110 parental strain and its transfer-deficient variants to the AD169, Toledo and Towne strains was analysed by RFLP (Fig. 4A) and Southern blot (Fig. 4B
, C
). These studies were carried out to verify that the VR6110 transfer-deficient variants were actually derived from the same parental strain. RFLP analysis results with respect to the clinical isolate VR6110 showed that the transfer-positive plaque isolate was different in 0 of 26 RFLP profiles, while the transfer-deficient plaque isolate was different in 2 of 26 (7·7%) RFLP profiles following HaeIII/MspI digestion of the TLF31TLR36 fragment of the ULb' ORF (Fig. 4A
). In contrast, the AD169, Toledo and Towne strains were different from VR6110 in 10 of 19 (52·6%), 15 of 26 (57·6%) and 14 of 21 (66·6%) RFLP profiles, respectively. Analysis of the two different RFLP profiles from both the VR6110 parental strain and the transfer-deficient plaque isolates showed the presence of a 2·0 kb deletion in a restricted ULb' region (TLF31TLR36, nt 1056814041; see Fig. 1
) of the transfer-deficient variant identified at passage 13. However, this deletion (spanning the UL130 and UL132 genes) was not found in the transfer-deficient variant identified at passage 67 (data not reported), indicating that it was not responsible for the transfer-deficient phenotype and that independent mutations could occur in the ULb' region of the transfer-deficient variants.
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Time-course of VR6110 transfer from HUVEC to PMNL
We investigated the time taken for VR6110 and its virus products to travel from infected HUVEC to uninfected PMNL following a 3 h coculture (Fig. 5A, B
). As expected, it was found that infectious virus, viral DNA, pp67 mRNA and pp65 were transferred to PMNL in the late phases of the virus replication cycle only, i.e. at 7296 h p.i. or later. However, IE mRNA and p72, which were abundant in infected HUVEC during the first 2448 h p.i., were only detected in PMNL when these cells were cocultured 7296 h p.i. or later.
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Discussion |
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The capacity of the 50 clinical isolates tested to date to transfer infectious virus to PMNL (Gerna et al., 2000 ) indicates that all HCMV strains that infect humans are likely to possess this property in vivo. However, all of the laboratory-adapted strains, such as AD169, Davis, Towne and even Toledo (considered the prototype wild-type strain), have been shown repeatedly to lack this property (Revello et al., 1998b
; Gerna et al., 2000
). It is reasonable to assume that these strains did have a transfer-positive phenotype initially, but that it was lost during extensive propagation in human fibroblasts. Based on this assumption, we decided to examine VR6110 (the clinical isolate previously shown to exhibit PMNL tropism) following extensive propagation and sequential plaque isolation in order to verify whether (i) transfer capacity could be lost after serial passaging and (ii) transfer-deficient variants could be recovered during propagation in HELF. The first VR6110 transfer-deficient variant was recovered at passage 13. By passage 67, the number of transfer-deficient variants increased, while the number of pp65-positive PMNL progressively decreased. The same trend was observed for the other three HCMV clinical isolates (recovered from urine, milk and cervix) examined and showed a lack of transfer-deficient variants at either the time of virus recovery or within the first 20 passages in HELF. So far, no transfer-deficient variants have been identified after extensive propagation of the four clinical isolates in HUVEC (data not reported).
The major biological difference detected between the VR6110 parental strain (transfer phenotype) and the transfer-deficient variants is the different ability to grow and propagate in HUVEC. Initially, the cell-associated parental strain was needed to propagate the virus in HUVEC, but after passage 6, cell-free virus could be passaged efficiently. Conversely, HUVEC were not permissive for the transfer-deficient variant. Thus, the initial cell-to-cell spread of the parental strain in HUVEC appears to correlate with the spread of virus from permissive cells to PMNL. While HCMV growth in HUVEC is restricted to the PMNL-tropic parental strains, both parental and transfer-deficient variant strains grow readily in HELF; only parental strains grown at low passage numbers are consistently PMNL-tropic. Transfer-deficient variants do, however, show a selective advantage in HELF. Thus, EC and PMNL tropism are linked, but HELF and PMNL tropism are dissociated and independent. Interestingly, both EC and PMNL tropism are lost after the extensive propagation of HCMV in HELF, suggesting that the genes involved in both functions are associated. It has been reported previously that EC-tropic variants may be lost following extensive passage of clinical isolates in fibroblasts (MacCormack & Grundy, 1999 ; Sinzger et al., 1999
; Waldman et al., 1989
) and that this loss is probably due to genetic mutations rather than to different functions of the viral gene products in different cell types (Sinzger et al., 1999
). However, loss of PMNL tropism in HELF was never reported.
As for the kinetics of virus replication in HELF, the cell-associated virus yield reached its peak 68 days p.i. for the parental strain, whereas the cell-associated virus yield for the transfer-deficient variant peaked 6 days p.i. and then started to drop by about 1 log per day. Interestingly, the Towne strain behaved similarly to the transfer-deficient variant. In PMNL cocultured with HELF infected with the parental strain, pp65 and p72 antigens and infectious virus were detected. However, the number of PMNL positive for viral DNA and mRNAs was markedly reduced in PMNL cocultured with HELF infected with the transfer-deficient variant (uptake by endocytosis).
Initially, finding that 15 or 13 kb genome fragments were missing in the laboratory-adapted AD169 and Towne strains (Cha et al., 1996 ) suggested that the gene responsible for PMNL tropism was located in this region of ULb'. However, when the Toledo strain (currently considered the wild-type prototype) was also found to lack the transfer phenotype, even in the presence of the entire ULb' region (although in the opposite orientation with respect to all clinical isolates tested), the previous assumption became less appropriate. The subsequent identification of transfer-deficient variants from VR6110 (as well as from each of the other clinical isolates tested) confirmed that the loss of transfer capacity was not directly related to the loss of a major fragment of the ULb' region of the HCMV genome. In the present study, comparative analysis of the VR6110 parental strain and its transfer-deficient variants by RFLP and Southern blot confirmed the origin of the two transfer-deficient variants from the common parental HCMV strain. In particular, the presence of large differences in the genomes was excluded following Southern blot analysis of the parental isolate and the plaque-purified variants, while they are commonly detected in epidemiologically unrelated strains (Chandler & McDougall, 1986
). Additionally, the conserved RFLP patterns of multiple genome regions suggest the absence of frequent nucleotide mutations, which is in contrast to reported findings for unrelated clinical isolates (Chou, 1990
; Baldanti et al., 1998
). In this paper, the ability of both Southern blot and RFLP techniques to detect genetic differences between unrelated HCMV isolates was readily confirmed by comparative analysis between the clinical isolates and the reference strains. Thus, the deletion detected in the transfer-deficient variant at passage 13 but not at passage 67 does not seem to be related to the loss of transfer capacity. However, this minor genetic alteration suggests a pressure for the selection of multiple transfer-deficient variants from the same genetic background.
We have suggested previously that the transfer of virus and virus products from permissive HUVEC to PMNL may be mediated by transitory microfusion events occurring between two contiguous cells that have come in close contact through interactions between adhesion molecules and integrins (Gerna et al., 2000 ). Following contact between PMNL and infected HUVEC, fusion is probably mediated by a viral protein which, as suggested by our findings, could be a late viral protein produced only by the parental strain. In this study, the requirement for a late viral protein to transfer IE virus products (mRNA and p72) to PMNL was demonstrated by both the time-course of HCMV transfer to PMNL (after 3 h of coculture) at sequential times after infection of HUVEC and the blockage of transfer from infected HUVEC at 96 h p.i. in the presence of PFA. In the absence of this protein(s), virus cannot be transferred to other cells and, thus, virus spread and dissemination is stopped. In our model, it is not surprising that HCMV synthesizes a fusogenic protein late in the replication cycle. In fact, PMNL-mediated dissemination of HCMV requires the presence of PMNL-derived infectious virus (Grundy et al., 1998
; Penfold et al., 1999
). The viral gene responsible for the production of this hypothetical protein is now under investigation.
The identification of transfer-deficient variants, which seems to be a general finding following in vitro propagation of HCMV clinical isolates in HELF, may represent an important step in the development of an HCMV vaccine. In addition, identification of the viral gene(s) involved in virus dissemination could indicate an important target for antiviral therapy.
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Acknowledgments |
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References |
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Received 24 November 2000;
accepted 16 February 2001.