Characterization of virus obtained from MDBK cells persistently infected with a variant of herpes simplex virus type 1 strain MP [HSV-1(MP)]

141,306-310

VIROLOGY

(1986)

Characterization of Virus Obtained from MDBK Cells Persistently Infected with a Variant of Herpes Simplex Virus Type 1 Strain MP [HSV-l(MP)] M. BARTOLETTI,* MAURO TOGNON,? ROBERTO MANSERvIGI,t AND ANNA MANNINI-PALENZONA**~

ANNA

*Institute

of Microbiology and Virobgg, and +Institutc of Microbidogy

University of Bdogna, I-40126 Bologna, Ita&, of Fevara,

I-44100 Ferraro,

Italy

Received August 3, 1984 accepted October 24, 1984 Virus clones which express glycoprotein gC (gC’) were obtained from two persistently infected (p.i.) MDBK cell lines which had been independently established by infection with HSV-l(MP)lOIll, a gC- syncytial (syn) variant of herpes simplex virus type 1 strain MP [HSV-l(MP)]. The gC+ revertants were syn in MDBK, HEp-2, and Vero cell lines and in primary human fibroblasts; this offers further evidence that glycoprotein gC does not inhibit cell fusion. The gC+ revertants represented from 70 to 100 percent of the virions present in the virus populations examined, thus suggesting a possible selective advantage of the gC+ revertants in this system of persistent infection. o 1985 Academic

Press, Inc.

tify using monoclonal antibodies the glycoproteins expressed by the virus obtained from persistent infection, (ii) examine the plaque morphology of the gC+ revertants, (iii) obtain information about the relative proportion of the gC? revertants in the viral populations obtained from p.i. cells. Two lines of independently established, p.i. MDBK cells (pi. cell line L and p.i. cell line H) were used in these studies. They had been obtained by infecting MDBK cells [at a multiplicity of infection (m.o.i.) of 0.1 and 10, respectively] with HSV-l(MP)10311, a gC- plaque-purified variant of HSV-l(MP). The variant is syn in HEp-2, Vero, and MDBK cells of low density; in MDBK cells of high density, however, it is cell aggregating (syn’) instead of syn (6). The p.i. cells were characterized by a cyclic pattern of monolayer destruction and reconstitution correlated with virus production (0.02 and 0.0003 PFU/cell in the phase of presence and absence of cytopathology, respectively); they were routinely maintained by trypsinizing the monolayers in the phase of absence of cytopathology. After passage 20, p.i. cell lesions became exclusively of the syncytial type, irrespective of the density of the monolayer in which they were

Evolution of the viral population during persistent infection has been amply documented, and, in some systems, there is evidence that a particular mutation may be essential for the maintenance of persistence (I, 2). In herpes simplex virus persistent infections, emergence of variants which differ from the parental virus with respect to plaque morphology, antigenicity, virulence, and thermosensitivity have been described (1). In the course of a study of MDBK cells persistently infected (p.i.) with a variant of herpes simplex virus type 1 strain MP [HSV-l(MP)], which is syncytial (syn) and does not express glycoprotein gC (gC-) (3), preliminary data indicated that the virus obtained from pi. cells had acquired the property of expressing gC (A. ManniniPalenzona and A. M. Bartoletti, International Workshop on Herpesviruses, Bologna, July 1981). gC+ revertants from HSVl(MP) have never been described (4), and the relationship between the absence of gC expression and the syn phenotype has not been completely defined (4, 5). It seemed, therefore, of interest to: (i) ideni Author dressed.

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localized. This had prompted us to compare the glycoprotein electrophoretic patterns of cells infected with parental virus HSV-l(MP)10311 and with virus obtained from p.i. cells. In fact a relationship exists between the type of cytopathology and the glycoproteins specified by the virus in the infected cells (3, 7, 8). Polyacrylamide gel electrophoresis (PAGE) of [14Clglucosamine-labeled cells had shown that a new band, whose apparent molecular weight corresponded to that of glycoprotein gC, appeared in both MDBK and HEp-2 cells infected with the virus obtained from passage 20 and 38 of the p.i. cell line L (A. Mannini-Palenzona and A. Bartoletti, International Workshop on Herpesviruses, Bologna, July 1981). To establish definitively that the virus obtained from p.i. cells had acquired the property of expressing gC, polypeptides from HEp-2 cells infected with plaque-

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purified virus clones (6) were immunoprecipitated both with anti-gC monoclonal antibodies and with a serum against all the major HSV-1 glycoproteins according to the technique described by Pereira et al. (9). The immunoprecipitates were then analyzed by PAGE (10). In Figs. 1 and 2 it is shown that virus clones obtained from passage 20 [20(l) and 20(6)] and 38 [38(2)] of the p.i. cell line L express glycoprotein gC and its precursors pg&, as well as all the major HSV-1 glycoprotein species; whereas parental virus HSVl(MP)10311 fails to accumulate glycoprotein gC and its precursor pg&=,. Prototype strains gC HSV-l(MP) and gC+ HSVl(F) (11) are also shown. In order to exclude a possible contamination of the p.i. cells with exogeneous virus, the DNAs of gC parental virus HSV-l(MP)10311 and gC+ virus clones 20(l), 20(6), and 38(2) were cleaved with

FIG. 1. Autoradiogram of electrophoretically separated polypeptides precipitated by anti-gC-1 (H-95) monoclonal antibody (a-gC) from detergent-solubilized extracts of HEp-2 cells infected with HSV-l(MP), HSV-l(F), parental virus HSV-l(MP)10311, and virus clones 20(l), 20(6), and 38(2). HEp-2 cell monolayers were labeled with FSJmethionine (10 &i/ml of Eagle’s MEM containing l/10 the normal concentration of methionine and supplemented with 1% fetal calf serum) from 4 to 18 hr postinfection with 10 PFU of virus. Extracts were prepared from the infected cells using a phosphate-buffered saline containing 1% Nonidet P-40 and 1% sodium deoxycholate (9). The extracts obtained after ultracentrifugation were reacted with the anti-gC antibody and the immunoprecipitates were analyzed by electrophoresis on a 8.5% SDS polyacrylamide gel (10). The hybridoma antibody chosen for this study was an anti-gC antibody designated U-95, produced by M. Para and obtained from P. Spear. Polypeptides present in samples of the extracts (Ext) are shown for comparison along with the immunoprecipitated polypeptides identified by closed triangles. The open triangles denote absence of detectable gC and pgC,,.

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FIG. 2. Autoradiogram of electrophoretically separated polypeptides precipitated by rabbit antiserum No. 3 (R # 3) from detergent-solubilized extracts of HEp-2 cells infected with HSVl(MP) HSV-l(F), parental virus HSV-l(MP)10311, and virus clones 20(l), 20(6), and 38(2). Infection and labeling of HEp-2 cell monolayers, and preparation of cell extracts were performed as described in the legend to Fig. 1. The extracts were reacted with antiserum R # 3, and the immunoprecipitates were analyzed by electrophoresis on a 8.5% SDS polyacrylamide gel (10). Antiserum R # 3 was prepared by immunization of a New Zealand white rabbit with HSV-l(13) (19) virion envelope proteins solubilized by Nonidet P-40 as previously described (20). Polypeptides present in samples of the extracts (Ext) are shown for comparison along with the immunoprecipitated polypeptides.

restriction endonuclease enzyme BamHI and subjected to electrophoresis in agarose gel according to the technique already described (12). In fact the DNAs of epidemiologically unrelated strains differ with respect to their specific restriction enzyme sites (1.3) and the electrophoretic pattern of HSV genome appears to be stable (14). Figure 3 shows that the electrophoretic profiles of all the DNAs were identical to the electrophoretic profile of HSV-l(MP). This finding excludes contamination of the p.i. cells with exogeneous virus. In fact the only other virus which could share the DNA pattern of HSV-l(MP) is HSV-l(mP), the virus from which HSV-l(MP) was derived (15). HSVl(mP) has never been used in this laboratory; furthermore, it does not cause cell fusion, while the virus clones which appear to be gC? revertants are syn (see below). The relationship between the syn phenotype and gC expression is still to be defined. Studies of recombinant viruses (4, 11, 16) have shown that gC does not inhibit fusion, as originally suggested (6);

however, many independent isolates of syn plaque morphology variants are gC (reported in (4)). It seemed therefore of interest to investigate the plaque morphology of our spontaneous gC+ revertants in different cell types. The gC+ revertants formed syncytia in HEp-2 and Vero cell lines, in primary human fibroblasts, and, at a difference with parental virus, in MDBK cells of both low and high cell density. In HEp-2 cells syncytia formed by gC+ revertants 20(6) and 38(2) were considerably smaller than syncytia formed by parental virus HSV-l(MP)10311 and gC? revertant 20(l) (data not shown). To explore the possibility that gC+ reversion might confer a selective advantage to the viruses in this system of persistence we began to examine the capacity of additional virus clones to express gC. Ten clones were obtained from both passages 20 and 38 of the p.i. cell line L and also from passage 22 of the p.i. cell line H. Analysis by PAGE of [14Clglucosaminelabeled infected HEp-2 cells (6) showed that 100 and ‘70 percent of the clones

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position within map coordinates 0.735 to 0.740. Our gC+ revertants were syn in HEp-2 and Vero cell lines, in MDBK cells of both low and high density, and in primary human fibroblasts. This constitutes further evidence that the syn phenotype of HSV-l(MP) cannot be due to the absence of gC expression and that the gC and the syn phenotypes of HSVl(MP) are segregable, in agreement with other reports (4, 11, 16, 17). These results also indicate that the possible selective advantage of the gC+ revertants in this system of persistent infection might be taken into consideration. In fact, the revertants were obtained from two independently established cell lines, persisted 2 through successive cell subcultivations, and appeared to represent from 70 to 100 percent of the virus population examined. Furthermore, gC+ revertants have never FIG. 3. DNA patterns of HSV-l(MP), HSV-l(F), been isolated from HSV-l(MP) (4), despite parental virus HSV-l(MP)10311, and virus clones the fact that this virus is extensively used 20(l), 20(6), and 33(2). Viral DNA was prepared in many laboratories (3, 5, 11, 16-18) for according to the method described by Wallboomers and Ter Shegget (21). The DNAs were digested with studies relating to HSV glycoproteins. The BarnHI restriction endonuclease (New England Bioapparent decrease in percent gC+ reverlabs, Beverly, Mass.), electrophoresed in 0.7% agarose tants found from passages 20 to 38 of the gel, and stained with ethidium bromide (12). The p.i. cell line L does not, in our opinion, fragments were designated according to the nomeninvalidate the hypothesis of a selective clature described in Tognon et al. (1.2). advantage of the gC’ virus. In fact it could be explained by the presence of a small fraction of gC virus which went obtained from the p.i. cell line L at pas- undetected at passage 20, and which flucsages 20 and 38, respectively, and 100 tuates during long-term cell cultivation. It would be interesting to explore a percent of the clones obtained from the p.i. cell line H at passage 22, were gC’ possible role for gC in persistent infections. In vitro studies have so far failed (data not shown). to assign a specific role to gC in the These results show that it is possible process of HSV lytic infection (4, 5). Howto isolate gC+ revertants from MDBK cells p.i. with a gC- syn variant of HSV- ever, it has recently been found that gC l(MP). Restriction endonuclease analysis acts as a receptor for the C3b complement of the revertant DNAs excluded a contam- component (IS), and, since most isolates ination from exogeneous source; there- from patients express gC, it has been fore this is the first report of gC+ rever- suggested that gC may be dispensable for the virus replication in vitro but essential tants obtained from HSV-l(MP). Recent for the survival of the virus in nature (4). studies indicate that the mutation responsible for the gC- phenotype of HSV-l(MP) may be located to a position within map ACKNOWLEDGMENTS coordinates 0.602 to 0.643 (~9, the region which contains the structural gene for gC These studies were aided by Grants from Minister0 (4,111. These studies also locate a mutation della Pubblica Istruzione 40 e 60%, and “Gruppo di responsible for the syn phenotype to a Virologia” del CNR.

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