Virus Research, 16 (1990) 195-210 Elsevier
195
VIRUS 00583
Monoclonal antibody to immediate early protein encoded by varicella-zoster virus gene 62
Bagher Forghani
‘, Ravi Mahalingam 2, Abbas Vafai 2, Jerry W. Hurst and Kent W. Dupuis ’
’
ofLaboratories,California State Department of Health Services, Berkeley, California, U.S.A. and 2 Department of NeuroIo~, University of Colorado, Health Sciences Center, Denver, Colorado, U.S.A.
’ Viral and Rickettsial Disease Laboratory Division
(Accepted 23 February 1990)
Monoclonal antibodies (mAbs) were prepared against varicella-zoster virus (VZV)-infected cell proteins, and 10 mAbs which reacted with nuclear antigens were selected. These mAbs recognized a major 175-180 kDa and three minor VZV-specific phosphoprotein species. Immunofluorescence staining of VZV-infected cells showed that the 175-180 kDa protein was synthesized within 6 h after infection. The synthesis of this protein was inhibited by cycloheximide (CH); however, reversal of CH treatment and addition of actinomycin D (ActD) resulted in the synthesis of the 175-180 kDa protein. To determine whether the 175-180 kDa protein seen in the infected cells is encoded by VZV immediate early (IE) gene 62, the predicted open reading frames of VZV genes 61 and 62 were cloned into pGEM transcription vectors. RNA was transcribed from each gene, translated in vitro and immunoprecipitated with a mAb which recognizes a major 175-180 kDa and three minor proteins. The reactivity of the in vitro translation products encoded by gene 62 with this mAb suggested that the 175-180 kDa protein is encoded by VZV IE gene 62.
VZV; Monoclonal antibody; Immediate early protein; Gene 62
Correspondence to: B. Forghani, Viral and Rickettsial Disease Laboratory, Division of Laboratories, California State Department of Health Services, 2151 Berkeley Way, Berkeley, CA 94704, U.S.A. 0168-1702/90/$03.50
0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
196
Introduction
Varicella-zoster virus (VZV), the causative agent of two distinct clinical syndromes, chicken-pox and shingles, is a member of the alphaherpesvirinae family (Roizman et al., 1981). DNA sequence analysis of VZV genome has identified 71 open reading frames (ORFs), 3 of which are repeated in the inverted repeat sequences (Davison and Scott, 1986). VZV, therefore, has the capacity to encode 68 proteins. However, due to the difficulty in obtaining high titered cell-free virus and the cell-associated nature of the virus, relatively few studies have been reported on the temporal expression of VZV genes. By contrast, extensive data are available for gene expression of other herpesviruses, especially herpes simplex virus (HSV). On the basis of the temporal order of gene expression, 3 classes of genes have been recognized in HSV (Honess and Roizman, 1974, 1975). They are designated immediate early (IE), early (E), and late (L) genes. The IE genes which are expressed immediately after infection consist of 5 genes (ICPO, ICP4, ICP22, ICP27 and ICP47). It has been shown that a functional ICP4 gene product is essential for the synthesis of mRNA from E and L genes, and for downregulation of transcription from its own gene (Watson and Clements, 1980; Deluca and Schaffer, 1985; Tedder and Pizer, 1988; O’Hara and Hayward, 1985). By DNA sequence analysis of the entire VZV genome (Davison and Scott, 1986), 3 VZV genes which display certain sequence similarities to HSV IE genes have been identified: gene 62 to ICP4; gene 63/70 to ICP22; and gene 4 to ICP27. Most notably, the predicted product of gene 62 shares a high degree of amino acid sequence similarity with the HSV ICP4 gene (Davison and McGeoch, 1986; Davison and Scott, 1986). Using polyclonal antisera to VZV, 4 IE polypeptides with apparent molecular weights of 185, 69, 43 and 34 have been reported (Lopetegui et al., 1983; Shiraki and Hyman, 1987). The 185 kDa protein species is phosphorylated and is the closest in size to the predicted 140 kDa polypeptide encoded by VZV gene 62 (Davison and Scott, 1986; Shiraki and Hyman, 1987). In addition, Felsner et al. (1988) have described a cell line expressing VZV gene 62 capable of complementing HSV IE gene ICPd It has been suggested that VZV 175-185 kDa phosphoprotein is the functional analog of HSV ICP4 protein (Felsner et al., 1988). We describe here the production of monoclonal antibodies (mAbs) to a VZV 175-180 kDa phosphoprotein. Indirect immunofluorescence (IIF) assays of cells infected with cell-free VZV indicated the reactivity of these mAbs with nuclear antigens within 6 h post infection (p.i.). The expression of this nuclear antigen was inhibited in the presence of metabolic inhibitors [cycloheximide (CH), actinomycin D (Act. D)]; however, nuclear antigens were detected following reversal of CHtreated VZV-infected cells. Furthermore, our results showed that these mAbs recognized the in vitro translation product encoded by VZV gene 62, suggesting that the 175-180 kDa protein is encoded by VZV gene 62.
197
Materials and Methods Cell culture and viruses Human fetal diploid lung (HFDL) cells were propagated in Eagle’s minimum essential medium (MEM) in Hanks’ balanced salt solution (BSS) with 10% fetal bovine serum and maintained in Eagle’s MEM in Earle’s BSS. The CaQu strain of VZV, an isolate of the Viral and Rickettsial Disease Laboratory (VRDL), California State Department of Health Services, was used in this study. Cell-free virus was prepared as described earlier (Schmidt and Lennette, 1976). Monolayers were infected with HSV-1 (McIntyre strain), HSV-2 (MS strain) and cytomegalovirus (CMV) (AD 169 strain). Monoclonal
antibodies
The complete procedure for the production of mAbs against VZV proteins has been described elsewhere (Forghani et al., 1982). Briefly, mAbs to IE protein of VZV were prepared by immunizing mice with whole cell lysates of VZV-infected HFDL cells. The first immunization was given intraperitoneally (i.p.) with Freund’s complete adjuvant. Hybridization of immune mouse spleen cells with mouse myeloma cells (SP2/0 line) was done by standard procedures using polyethylene glycol as fusing agent. The hybrid cells were screened for antibody secretion by IIF assay (Forghani et al., 1982). Monoclonal antibody 2XFl was selected for experiments described in this study. Ascites were prepared as described (Forghani et al., 1982). Briefly, each animal was primed with Pristane (2,6,10,14-tetramethyl pentadecane), injected with hybridoma i.p., and ascitic fluids were collected lo-12 days later. Zsotyping The isotype of each mAb was determined by the procedure described (Forghani et al., 1982). Briefly, tissue culture media from VZV positive hybridomas were concentrated 25-30 times by neutralized saturated ammonium sulfate and tested by agarose gel immunodiffusion against goat monospecific antisera to mouse IgGl, IgGZa, IgG2b, IgG3, IgA and IgM (Research Products International Corp., Elk Grove, IL). Radiolabeling
and radioimmunoprecipitation
assays (RZPA)
VZV-infected monolayers were prepared by mixing VZV-infected and uninfected HFDL cells at a ratio of 1: 3. At 24 to 48 h p.i., when cells showed a 60-70% cytopathic effect (CPE), the growth medium was replaced with phosphate-free medium containing 40 pCi/ml of [32Pi]orthophosphate or with methionine-free medium containing 20 pCi/ml [ 35S]methionine, or with 2 PCi of D-[‘4C]glucosamine, and labeled for 18-20 h. Radiolabeled viral antigens were prepared by
198
lysing the washed labeled cells with T&-saline buffer (0.05 M Tris-HCI, pH 7.4, 0.15 M NaCl) containing 1% deoxycholate, 1% NP-40 and 1% aprotinin as protease inhibitor, and clarifying at 40,000 x g for 1 h. As control, a labeled uninfected cell lysate was prepared in the same manner. RIPAs were performed by incubating labeled cell lysates with tissue culture fluids from hybridoma-secreted mAbs. The immune complexes were removed with protein A sepharose 4CL, disrupted with sodium dodecyl sulfate (SDS) and 2-mer~pt~th~ol, subjected to 10% polyacrylamide gel electrophoresis (PAGE), and finally autoradiographed, as described earlier (Forghani et al., 1984). Pulse-chase labeling
VZV-infected cells were labeled with 50 yCi/ml of ~35S]met~o~ne for 10, 20,40 and 60 min, and 6 h. After the pulse period, cells were washed 3 times, and the label was chased in fresh medium for 24 h. Procedures for SDS-PAGE-RIPA were similar to those described above.
Denatured uninfected and WY-infected cell lysates were separated by SDSPAGE (Laemmli, 1970). Preparation of VZV infected and uninfected cell lysates for SDS-PAGE was the same as described for RIPA except that they were not radiolabeled. The PAGE separated proteins were transferred to nitrocellulose filter paper (~hleicher h Schuell, Keene, NH) for 90 min by an el~troblotter (Hoeffer Scientific Instruments, San Francisco, CA) (Towbin et al., 1979; Burnette, 1981). The nitrocellulose filters were reacted with mAb for 1 h at 37 a C and overnight at 4” C, followed by 3 washings; they were then treated with horseradish peroxidaseconjugated rabbit anti-mouse IgG for 1 h at room temperature, and washed 3 times. The enzymatic reaction was visualized by addition of H202 as substrate and a~n~thyl carbazole as c~omogen donor (Forghani and Dennis, 1989). Non-denatured protein PAGE was run using buffer systems described by Laemmli (1970) but without addition of SDS to either gel or cell lysate. The separated proteins were transferred to nitrocellulose paper and the immunoenzymatic assays were done as described for denatured proteins. metabolic inhibitors
Monolayers of HFDL cells were grown on 4 sets of 8-chamber Lab-Tek slides (Miles Laboratories, Naperville, IL) and infected with cell-free virus. After 1 h adsorption at 37OC, the monolayers were washed 3 times with buffer to remove unadsorbed virus. Fresh medium containing 100 pg/ml CH was added to one set of slides to block the cellular and viral protein synthesis. These slides were incubated at 37 o C for 5 h, followed by removal of the medium and 3 washings. Fresh medium containing 10 pg/ml ActD was added to block transcription and allow the translation of preexisting mRNA. The cells were incubated for 14 h more at 37’C.
199
Controls were: (i) cell-free VZV-infected cells without drug treatment; and (ii) uninfected cells treated with and without metabolic inhibitors. Monolayers were rinsed briefly and, without drying, cells were fixed in acetone for 10 min at 6, 9, 12, 16 and 20 h p.i., which corresponds to 0, 3, 6, 9 and 14 h post removal of CH. The fixed cells were stored at 70 o C for future analysis. IIF staining
Cell culture fluids from cells secreting mAbs were added to each set of slides, incubated in a humidified chamber for 3 h, and washed 3 times. Fluorescein-conjugated goat anti-mouse IgG was added and the cells were incubated for 1 h at 37” C, washed 3 times, mounted and observed in a fluorescence microscope as described (Forghani et al., 1982). Monoclonal antibodies to VZV glycoproteins (gp) I, II, III and IV and normal mouse ascitic fluids were used as controls for specificity in this assay. Virus neutralization
assay
VZV neutralization assay was done using the VRDL standard plaque reduction assay with and without fresh guinea pig complement (Forghani et al., 1982). Cloning of VZV genes 61 and 62
The VZV BarnHI-B and J fragments (Fig. 1) contain the open reading frames (ORFs) for VZV genes 61 and 62 (Davison and Scott, 1986). The recombinant plasmid (pBR322) containing the BamHI-B and J fragments (Gilden et al., 1983) was cleaved with either AccI or iVde1 restriction enzymes. The 2.0~kilobase (kb) AccI fragment spanning VZV nucleotides 102715 to 104726 and containing the ORF of VZV gene 61 was electroeluted, blunt-ended with T4 DNA polymerase (Maniatis et al., 1982), and ligated into the SmaI site downstream from the T7 promoter of the pGEM7Zf transcription vector (Promega Biotec, Madison, WI) as described (Vafai et al., 1986). To clone gene 62, the 5.0 kb NdeI subfragment of BarnHI-B and J spanning VZV nucleotides 104420 to 109367 was ligated into the NdeI site of the polylinker of the pGEM5Zf transcription vector (Promega Biotec) downstream from the SP6 promoter. In vitro transcription,
translation and immunoprecipitation
The recombinant plasmids carrying either VZV gene 61 or gene 62 were cleaved with either PstI or Hind111 restriction enzymes downstream from the DNA insert, and the linearized DNA was used as a template for RNA transcription with T7 or SP6 RNA polymerase (Promega Biotec) respectively. RNA was transcribed from genes 61 and 62 and translated in vitro by the procedure described previously (Vafai et al., 1988). The in vitro translation (IVT) products were either analyzed by
200 32
P
MWM
69
46
Fig. 1. Fluorograph of SDS-PAGE electrophoretically separated phosphopeptides in immunoprecipitates of uninfected (lanes 1,4) and VZV-infected (lanes 2, 3) cell lysates labeled with [32Pi] and [ “Slmethionine reacted with mAb clone 2XFl. * Molecular weight markers (MWM) of “C-labeled proteins (myosin, phosphorylase B, bovine serum albumin, IgG heavy chain, ovalbumin).
SDS-PAGE or immunoprecipitated with mAb 2XFl. Before immunoprecipitation, the IVT products, suspended in 500 ~1 of lysis buffer (0.02 M sodium phosphate, pH 7.6, 0.1 M NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS) were incubated for 2 h at 4°C with 40 ~1 of a 10% formal&fixed suspension of protein A-containing Staphylococcus aweus Cowan I (Vafai et al., 1986). After centrifugation in a microfuge for 5 min at 4O C, VZV-specific proteins were immunoprecipitated at 4°C for 20 h in the presence of 100 ~1 of mAb 2XFl and 20 ~1 of goat anti-mouse IgG (Cappel Laboratories, Westchester, PA). Finally, 30 ~1 of a 10% formalin-fixed S. aweus suspension were added and, after 2 h at 4°C absorbed immune complexes were washed 3 times with lysis buffer and suspended in 20 ~1 of TNE buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA). After addition of 10 ~1 of 3 x sample buffer (150 mM Tris, pH 7.0, 6% SDS, 15% 2-mercaptoethanol, 0.03% bromophenol blue), the suspension was heated in boiling water for 4 min, cooled on ice, and analyzed by 8% polyacrylamide SDS-PAGE as described (Vafai et al., 1988).
201
Results Preparation
and selection of mAbs
Three attempts were made to hybridize immunized mouse spleen cells with murine SP2/0 myeloma cells. Ten independent mAbs were selected by IIF on 24-h VZV-infected monolayers. All 10 mAbs reacted only with the nucleus and nuclear membrane of the infected cells, and not with those of uninfected cells. To ensure the purity of each hybridoma, each clone was subcloned 3 times. These mAbs did not show any cross-reactivity with HSV-1, HSV-2 or CMV infected cells by IIF. When cells were infected with 25 recent VZV isolates (obtained from the VRDL diagnostic file) and tested blindly by IIF, all showed ‘nuclear staining reactivities similar to those seen with the CaQu laboratory strain. The following experiments were performed to further characterize our panel of mAbs: (i) Agarose gel immunodiffusion on concentrated cell culture media showed that all 10 mAbs from 3 separate hybridizations were of IgGl subclass of murine immunoglobulin. (ii) Cell culture media and ascitic fluids of 10 mAbs were tested for virus neutralizing activity with and without complement. As expected, no plaque reduction was observed either with cell culture media or with ascites, indicating that these mAbs do not neutralize VZV infectivity. (iii) Each ascitic fluid was titrated, tested by IIF, and found to have a titer of at least 1: 100,000. The reactions were specific only for VZV-infected cells and no cross-reactivity was observed with HSVand CMV-infected, or uninfected HFDL cells. Identification
of polypeptides
recognized by each mAb
To identify the VZV-specific proteins which are recognizedby the mAbs, the cell culture medium of each hybridoma was reacted with virus-infected cell lysates which had been labeled with [32Pi]orthophosphate, [3SS]methionine and [‘4C]glucosamine. The results shown in Fig. 1 indicate a prominent 175-180 kDa peptide and three minor polypeptide species in both [ 35S]methionine- and [ 32Pi]orthophosphatelabeled cell lysates with molecular weights ranging from 105 to 160 (Fig. 1, lanes 2 and 3). These results indicate that the 175-180 kDa polypeptide was highly phosphorylated and the others were at least partially phosphorylated. No precipitation bands were observed with [‘4C]glucosamine-labeled cell lysate, indicating that this protein was not glycosylated. When additional experiments were conducted with radioisotope-labeled uninfected cell lysates, no protein bands of similar size were seen (Fig. 1, Lanes 1 and 4). These experiments were repeated with immune mouse and normal ascites. Again, no precipitation was seen with normal mouse ascites; however, all 10 immune ascites immunoprecipitated protein bands similar to those detected by tissue culture media from hybridoma cells (data not shown). These results are in accord with two earlier publications identifying a 185 kDa major IE protein of VZV (Lopetegui et al., 1983; Shiraki and Hyman, 1987) using polyclonal antisera prepared in monkey and guinea pig respectively.
202
Pulse-chase
experiments
To determine the relationship between the major 175-180 kDa phosphoprotein and the three minor phosphoproteins, VZV-infected cells were labeled at various times and used in immunoprecipitation assays. The results of these experiments are shown in Fig. 2. During 10 min pulse (lane 2), two faint precipitation bands (175-180 kDa and 105-110 kDa) were detected. During additional 20, 40 and 60 min pulse labeling (lanes 3, 4 and 5) the intensity of all bands increased and two additional precipitation bands were visible. During 6 h pulse labeling (lane 6) the intensity of all bands increased slightly and no additional virus-specific bands were observed. When the 10 min pulse label was chased for 24 h, again only one major and three minor phosphoproteins were seen (lane 9). No polypeptides of similar size were detected by immunoprecipitation of uninfected cell lysates which were pulselabeled for either 10 min or 6 h and chased for 14 h (Fig. 2, lanes 1, 7 and 8). These data suggest that these three minor protein species are either a degradation product of the 175-180 kDa protein or are another VZV-specific protein which non-specifically reacts with mAb 2XFl.
I
23456709
Fig. 2. Autoradiographic image of pulse-chase labeled VZV IE protein. The optimal pulse-labeling time intervals for pulse-chase experiments were determined by labeling VZV-infected cell cultures showing a CPE of 60-708 with 50 rCi/ml [35S]methionine. At the IO-min pulse the labeled medium was removed, and the monolayer was washed 3 times, replenished with fresh medium without the label, and chased for 24 h. Arrows indicate proteins precipitated at the lo-min pulse with mAb clone 2XF1, which intensified at subsequent time intervals, and the chase. Lanes 2, 3,4 and 5: 10, 20, 40 and 60 min pulse; lane 6: 6 h pulse; lane 9: 24 h chase of VZV-infected cell lysate. Lanes 1 and 7: 10 min and 6 h pulse; lane 8: 24 h chase of uninfected cell lysate.
203
Immunoblotting
Experiments were conducted to react the cell culture media of the individual hybridomas with VZV denatured proteins which were separated by SDS-PAGE and electrophoretically transferred onto nitrocellulose filters. only the cell culture medium and ascitic fluid of clone 2XFl reacted with 175-180 kDa and three minor polypeptide species; the other 9 mAbs were non-reactive (Fig. 3). We considered the possibility that the lack of reactivity of these mAbs could be due to denaturation of the antigenic sites (epitopes) during SDS-PAGE separation. However, similar (negative) results were obtained when several renaturation processes (Dunn, 1986; Mandrel1 and Zollinger, 1984) were attempted (data not shown). In addition, no reactivity was observed with any of the 9 mAbs when a non-denaturing gel (Laemmli, 1970) was employed for separation of VZV protein and transferto nitrocellulose filter (data not shown). Again, only clone 2XFl was reactive.
Fig. 3. Electrophoretic transfer to nitrocelhdose paper of denatured uninfected and VZV-infected cell lysates separated by SDS-PAGE and reacted with mAb clone 2XFl. Lane 1: uninfected cell lysate. Lane 2: VZV-infected cell lysate.
204
CH
Fig. 4. Immunofluorescent staining of acetone-fixed WV-infected cells. HFDL cell monolayers were prepared on Lab-Tek slides infected with cell-free virus. One hour p.i. the inoculum was removed, fresh medium containing CH was added and incubated for 5 h. Then, the CH medium was removed, medium containing ActD was added and incubated for the next 14 h. The fixed monolayers were stained by IIF using mAb clone 2XFl and FITC-conjugated rabbit anti-mouse IgG. Lane U: untreated; lane CH: cycloheximide treated. Row 1: uninfected cell monolayer; row 2: infected cells at 6 h p.i.; row 3: at 9 h p.i.; row 4: at 12 h p.i.; row 5: at 16 h p.i.; row 6: at 20 h p.i.
205
Temporal expression and localization To examine the temporal expression and cellular distribution of the 175-180 kDa IE protein at various times p.i., we used the 10 mAbs in IIF assays. Four sets of HFDL cell monolayers were prepared: (i) untreated VZV infected cells; (ii) VZV infected cells treated with CH and ActD; (iii) untreated uninfected cells; and (iv) uninfected cells treated with CH and ActD. In the set of untreated VZV-infected cells, by 4 h p.i., less than 0.1% of single cells showed a very faint nuclear staining with dusty appearance (data not shown), and by 6 h p.i., a generalized granular nuclear and nuclear membrane stain were visualized (Fig. 4). By 9 h and 12 h p.i., the intensity of nuclear staining had increased, with areas of bright fluorescence. By 16 h p.i., infectious foci of 4-5 cells with various degrees of nuclear staining were visible. By 20 h p.i., the foci had enlarged to 8-10 cells with intense nuclei. Surprisingly, the type of staining in the nucleus of certain cells had also changed dramatically, showing intense patchy areas resembling polymorphonuclear leukocytes. On VZV-infected monolayers treated with CH, no fluorescence staining was observed at 0 h or 1 h after removal of the drug. However, by 3 h post removal of the CH, corresponding to 9 h p.i., single cells were showing nuclear and nuclear membrane staining resembling that seen at 6 h p.i. of untreated infected cells. By 9, 12 and 16 h post removal of CH, corresponding to 12, 16 and 20 h p.i., the nuclear staining intensified and became more granular. The uninfected cells with and without drug treatment, as well as the VZV-infected, CH-treated cells without reversal of CH treatment, showed no staining. In addition, no cytoplasmic or nuclear staining was observed at 4, 6, 9 and 12 h p.i. with mAbs to VZV gp1, gpI1 gpII1 and gpIV and normal mouse ascitic fluids used as controls. Reactivity of mAb 2XFl with translation products encoded by VZV gene 62 To test the possibility that the 175-180 kDa protein, detected by mAb 2XF1, is the product of VZV IE gene 62, the predicted ORFs of VZV genes 61 and 62 (Davison and Scott, 1986) were subcloned from the BarnHI-B and J DNA fragments (Fig. 5) into in vitro transcription vectors (pGEM5Zf and pGEM7Zf) as described in Materials and Methods. The RNA transcribed from these genes was translated in vitro, and the in vitro translation (IVT) products were immunoprecipitated with mAb 2XFl and analyzed by SDS-PAGE. Fig. 6A shows the primary translation products encoded by VZV genes 61 (62 kDa) and 62 (175 kDa). Immunoprecipitation of the IVT products with mAb 2XFl revealed a strong reactivity of the products encoded by gene 62, but not gene 61, suggesting that this mAb recognizes epitope(s) on VZV gene 62 (Fig. 6B). Three additional protein bands, smaller than the 175 kDa species, were also detected by SDS-PAGE analysis after immunoprecipitation of the IVT products encoded by gene 62. These protein bands may represent the degradation of the IVT products, or could be due to the translation products of the truncated RNA species generated during the in vitro
206 TR L
UL
'RL
‘R s
us
TRs
Fig. 5. Construction of recombinant plasmids carrying VZV genes 61 and 62. Recombinant plasmid pBR322 containing the BarnHI-B and J fragments, located within the unique long (UL), inverted repeat long (IR,) and inverted repeat short (IRS) sequences, was cleaved with either Accl (A) or Ndel (N) restriction enzymes, and the 2.0 kb and 5.0 kb restriction fragments containing VZV open reading frames 61 and 62, respectively, were cloned in pGEM vectors downstream from ‘I7 or SP6 promoters as described in Materials and Methods. B: BumHI site; S: Smal site.
transcription of gene 62. It is also possible that these bands may result from random initiation of different AUG codons present on gene 62 RNA.
Discussion Studies described in this report demonstrate that our panel of mAbs, generated by 3 independent hybridization attempts, recognized a phosphorylated IE protein of VZV. Using 32P- and 35S-labeled infected VZV cell lysates, a major 175-180 kDa and three minor phosphopolypeptides were immunoprecipitated with hybridoma cell media and immune mouse ascitic fluids. The lack of immunoprecipitation of 14C-labeled infected cell lysates with the 175-180 kDa protein species suggested that this phosphoprotein is not glycosylated. DNA sequence analysis of VZV genome (Davison and Scott, 1986) has predicted a 140 kDa protein encoded by gene 62 which displays a degree of amino acid sequence similarity to HSV IE gene ICP4 (McGeoch et al., 1988). Also, two earlier studies using polyclonal antisera to VZV identified an IE phosphoprotein with an apparent size of 185 kDa (Lopetegui et al., 1983; Shiraki and Hyman, 1987); our data are consistent with theirs. Despite amino acid sequence similarities between VZV gene 62 and HSV-1 ICP4, no antigenic cross-reactivity was observed with HSV-1, HSV-2 and CMV using anti-175-180 kDa mAbs.
207
,61
ivt
imp
Fig. 6. Expression and immunoprecipitation of WV genes 61 and 62 products. The recombinant plasmid containing either VZV gene 61 or 62 was cleaved with either PsrI (gene 61) or Hind111 (gene 62). ‘Ihe linearized DNA was used as a template for transcription. RNA was transcribed, translated in vitro, and the in vitro translation (WI) products were analyzed by SDS-IO% PAGE. The IVT products were also immunoprecipitated (imp) with mAb 2XFl and analyzed by SDS-8% PAGE. The apparent molecular weights (in kilodaltons) of the major polypeptides encoded by genes 61 and 62 are shown. No RNA is shown; RNA was excluded from translation reaction.
Experiments were conducted to determine the reactivity of our mAbs with denatured VZV proteins. SDS-PAGE-separated VZV proteins were electrotransferred to nitrocellulose filters and reacted with the mAbs. Although ail 10 mAbs were murine subclass IgGl, only one clone, 2XF1, was reactive to a 175-180 kDa and three minor polypeptides by Western blot analysis; aII the others were non-reactive. By Western blot analysis, using an antiserum prepared in rabbit against a synthetic peptide derived from VZV gene 62 DNA sequences, Felsner et al (1988) have identified a polypeptide with an apparent molecular weight of 175. Our Western blot data were consistent with these results and showed that mAb 2XFl recognized a 175-180 kDa polypeptide species.
208
As described under Results, we performed experiments to determine whether the non-reactivity of the other 9 mAbs in immunoblot assays could be due to denaturation or damage of the antibody-binding sites (epitopes) on the antigen by SDS-PAGE separation. Since our results were negative, we are now considering the possibility that the non-reactivity may be due to recognition of different antigenic domain(s) by these mAbs which are easily damaged. Competitive binding assays will be required to determine the likelihood of this possibility. In addition to the 175-180 kDa major phosphopolypeptide, our panel of mAbs recognized three minor phosphopolypeptides in VZV-infected cells. To explain the relationship between these phosphopolypeptides, the following experiments were done: (i) We considered the possibility that our hybridomas were not pure, although they were generated by 3 independent hybridization attempts. To ensure the purity of each hybridoma, we subcloned each hybridoma 3 times; the SDS-PAGE-RIPA results were identical in each step of subcloning (data not shown). (ii) Results of pulse-chase experiments indicated that by 10 min pulse two phosphoproteins (105-110 kDa and 175-180 kDa) were detected, and two additional bands were visible by 20,40, and 60 min and 6 h pulse periods. Again, by 10 min pulse and 24 h chase all four phosphoproteins were detected. These results suggest that these proteins may be either a cleavage product of the 175-180 kDa protein or an additional VZV-specific protein recognized by these mAbs. Detection of the smaller protein species could also be due to the translation of internal AUG codons. However, recent studies on IE mRNA of equine herpesvirus type 1 (EHV-1) have determined that it encodes four closely related IEPs in in vivo and in vitro translation (Cat&man et al., 1988; Robertson et al., 1988). It is also possible that, similar to IE mRNA of EHV-1, VZV gene 62 encodes a family of antigenically related phosphoproteins. It is striking to note that the genomic structure of VZV and EHV-1 both have two isomeric arrangements (Takahashi, 1983) and perhaps their IEPs have the same function. CH, an inhibitor of protein synthesis, and ActD, an inhibitor of RNA synthesis, have been used extensively to determine early protein synthesis of herpesviruses, including VZV (Shin&i and Hyman, 1987). We have applied these two metabolic inhibitors to examine the immunological reactivity of our MAbs to IE VZV-specific proteins. In the absence of these drugs a faint nuclear stain was observed at 4 h p.i., and a bright granular nuclear stain at 6 h p.i.; the staining intensified with additional incubation time. However, when virus infection was carried out in the presence of the drugs, the first nuclear stain was observed by 3 h after CH-reversal of the infected cells, corresponding to 9 h pi. These data demonstrate that the polypeptide recognized by the mAbs is an IE VZV protein (175-180 kDa) being synthesized within 6 h following infection. The synthesis of this protein was inhibited by CH treatment; however, treatment of the infected cells with CH resulted in the accumulation of RNA encoding the 175-180 kDa protein which was translated following the reversal of CH-treated VZV-infected cells. Our results indicated that the primary translation products encoded by VZV gene 62, but not gene 61, reacted with mAb 2XF1, further substantiating the immunological reactivity of this mAb to a major VZV IE protein.
209
In this paper we have described the first successful attempt at preparation and partial characterization of mAbs directed against the products of VZV IE gene 62. These mAbs will help to further characterize the functional properties of the 175-180 kDa protein and the three minor proteins encoded by gene 62 and their role in the expression of other viral genes during VZV replication in the infected cells.
References Burnette, W.N. (1981) “Western blotting”; electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Analyt. B&hem. 112, 195-203. Caughman, G.B., Robertson, A.T., Gray, W.L., Sullivan, D.A. and G’CaIlaghan, D.J. (1988) Characterization of equine herpesvirus type 1 immediate early proteins. Virology 163, 563-571. Davison, A.J. and McGeoch, D.J. (1986) Evolutionary comparisons of the S segments in the genomes of herpes simplex virus type 1 and varicella-zoster virus. J. Gen. Virol. 67, 597-611. Davison, A.J. and Scott, J.E. (1986) The complete DNA sequence of varicella-zoster virus. J. Gen. Virol. 67,1759-1816. Deluca, N.A. and Schaffer, P.A. (1985) Isolation and characterization of deletion mutants of herpes simplex virus type 1 in the gene encoding immediate early regulatory protein ICW. J. Virol. 56, 558-570. Dunn, SD. (1986) Effects of the modification of transfer buffer composition and the renaturation of proteins in gels on the recognition of proteins on Western blots by monoclonal antibodies. Analyt. B&hem. 157,144-153. Felsner, J.M., Kintchington, P.R., Inchauspe, G., Straus, S.E. and Ostrove, J.M. (1988) Cell lines containing varicella-zoster virus open reading frame 62 and expressing the “IE” 175 protein complement ICPF4 mutants of herpes simplex virus type 1. J. Virol. 62, 2076-2082. Forghani, B. and Dennis, J. (1989) Immunoenzymatic staining. In: N.J. Schmidt and R.W. Emmons (Eds.), Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections, 4th edit., pp. 135-156. American Public Health Association, Inc., Washington, D.C. Forghani, B., Dupuis, K.W. and Schmidt, N.J. (1984) Varicella-zoster viral glycoproteins analyzed with monoclonal antibodies. J. Virol. 52, 55-62. Forghani, B., Schmidt, N.J., Myoraku, C.K. and Gallo, D. (1982) Serological reactivity of some moncclonal antibodies to varicella-zoster virus. Arch. Virol. 73, 311-317. Gilden, D.H., Vafai, A., Shtram, Y., Becker, Y., Devlin, M. and Wellish, M. (1983) Varicella-zoster virus DNA in human sensory ganglia. Nature (London) 306,478-480. Honess, R.W. and Roizman, B. (1974) Regulation of herpesvirus macromolecular synthesis. 1. Cascade regulation of the synthesis of three groups of viral proteins. J. Virol. 14, 18-19. Honess, R.W. and Roizman, B. (1975) Regulation of herpesvirus macromolecular synthesis: sequential transition of polypeptide synthesis requires functional polypeptides. Proc. Natl. Acad. Sci. USA 72, 1276-1280. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 277, 680-685. Lopetegui, P., Matsunaga, Y., Okuno, T., Ogino, T. and Yamanishi, K. (1983) Expression of varicellazoster virus-related antigens in biochemically transformed cells. J. Gen. Virol. 64, 1181-1186. Mandrell, R.E. and Zollinger, W.D. (1984) Use of a zwitterionic detergent for restoration of the antibody-binding capacity of electroblotted meningococcal outer membrane proteins. J. Immun. Methods 67, 1-11. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular cloning: a Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
210
McGeoch, D.J., Dahymaple, M.A., Davison, A.J., Dolan, A., Frame, M.C., McNab, D., Perry, L.J., Scott, J.E. and Taylor, P. (1988) The complete DNA sequence of the long unique region in the genome of herpes simplex virus type 1. J. Gen. Virol. 69, 1531-1574. O’Hare, P. and Hayward, G.S. (1985) Three tram-acting regulatory proteins of herpes simplex virus modulate immediate-early gene expression in a pathway involving positive and negative feedback regulation. J. Virol. 56, 723-733. Robertson, A.T., Cat&man, G.B., Gray, W.L., Bauman, R.P., Staczek, J. and O’Callaghan, D.J. (1988) Analysis of the in vitro translation products of the equine herpesvirus type 1 immediate early mRNA. Virology 166, 451-462. Roizman, B., Carmichael, L.E., Deinhardt, F., de-The, G., Nahmias, A.J., Plowright, W., Rapp, F., Takahashi, M. and Wolf, K. (1981) Herpesviridae. Intervirology 16, 201-217. Schmidt, N.J. and Lennette, E.H. (1976) Improved yields of cell-free varicella-zoster virus. Infect. Immun. 14,709-715. Shiraki, K. and Hyman, R.W. (1987) The immediate early proteins of varicella-zoster virus. Virology 156, 423-426. Takahashi, M. (1983) Chicken pox. In: M.A. Lauffer and K. Maramorosch (Eds.), Advances in Virus Research, Vol. 28, Academic Press, New York, N.Y. Tedder, D.G. and Pizer, L.I. (1988) Role for DNA-protein interaction in activation of the herpes simplex virus glycoprotein D gene. J. Virol. 62, 4661-4672. Towbin, H., Staehlin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Prcc. Natl. Acad. Sci. USA, 76, 4350-4354. Vafai, A., Wellish, M. and Gilden, D.H. (1986) Polypeptides encoded by varicella-zoster virus unique short sequences. Virus Res. 5, 67-76. Vafai, A., Wroblewska, Z., Mahalingam, R., Cabirac, G., Wellish, M., Cisco, M. and Gilden, D.H. (1988) Recognition of similar epitopes on varicella-roster virus gp I and gpIV by monoclonal antibodies. J. Virol. 62, 2544-2551. Watson, R.J. and Clements, J.B. (1980) A herpes simplex virus type 1 function continuously required for early and late virus RNA synthesis. Nature (London) 285, 320-330. (Received 22 August 1989; revision received 15 February 1990)
195
VIRUS 00583
Monoclonal antibody to immediate early protein encoded by varicella-zoster virus gene 62
Bagher Forghani
‘, Ravi Mahalingam 2, Abbas Vafai 2, Jerry W. Hurst and Kent W. Dupuis ’
’
ofLaboratories,California State Department of Health Services, Berkeley, California, U.S.A. and 2 Department of NeuroIo~, University of Colorado, Health Sciences Center, Denver, Colorado, U.S.A.
’ Viral and Rickettsial Disease Laboratory Division
(Accepted 23 February 1990)
Monoclonal antibodies (mAbs) were prepared against varicella-zoster virus (VZV)-infected cell proteins, and 10 mAbs which reacted with nuclear antigens were selected. These mAbs recognized a major 175-180 kDa and three minor VZV-specific phosphoprotein species. Immunofluorescence staining of VZV-infected cells showed that the 175-180 kDa protein was synthesized within 6 h after infection. The synthesis of this protein was inhibited by cycloheximide (CH); however, reversal of CH treatment and addition of actinomycin D (ActD) resulted in the synthesis of the 175-180 kDa protein. To determine whether the 175-180 kDa protein seen in the infected cells is encoded by VZV immediate early (IE) gene 62, the predicted open reading frames of VZV genes 61 and 62 were cloned into pGEM transcription vectors. RNA was transcribed from each gene, translated in vitro and immunoprecipitated with a mAb which recognizes a major 175-180 kDa and three minor proteins. The reactivity of the in vitro translation products encoded by gene 62 with this mAb suggested that the 175-180 kDa protein is encoded by VZV IE gene 62.
VZV; Monoclonal antibody; Immediate early protein; Gene 62
Correspondence to: B. Forghani, Viral and Rickettsial Disease Laboratory, Division of Laboratories, California State Department of Health Services, 2151 Berkeley Way, Berkeley, CA 94704, U.S.A. 0168-1702/90/$03.50
0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
196
Introduction
Varicella-zoster virus (VZV), the causative agent of two distinct clinical syndromes, chicken-pox and shingles, is a member of the alphaherpesvirinae family (Roizman et al., 1981). DNA sequence analysis of VZV genome has identified 71 open reading frames (ORFs), 3 of which are repeated in the inverted repeat sequences (Davison and Scott, 1986). VZV, therefore, has the capacity to encode 68 proteins. However, due to the difficulty in obtaining high titered cell-free virus and the cell-associated nature of the virus, relatively few studies have been reported on the temporal expression of VZV genes. By contrast, extensive data are available for gene expression of other herpesviruses, especially herpes simplex virus (HSV). On the basis of the temporal order of gene expression, 3 classes of genes have been recognized in HSV (Honess and Roizman, 1974, 1975). They are designated immediate early (IE), early (E), and late (L) genes. The IE genes which are expressed immediately after infection consist of 5 genes (ICPO, ICP4, ICP22, ICP27 and ICP47). It has been shown that a functional ICP4 gene product is essential for the synthesis of mRNA from E and L genes, and for downregulation of transcription from its own gene (Watson and Clements, 1980; Deluca and Schaffer, 1985; Tedder and Pizer, 1988; O’Hara and Hayward, 1985). By DNA sequence analysis of the entire VZV genome (Davison and Scott, 1986), 3 VZV genes which display certain sequence similarities to HSV IE genes have been identified: gene 62 to ICP4; gene 63/70 to ICP22; and gene 4 to ICP27. Most notably, the predicted product of gene 62 shares a high degree of amino acid sequence similarity with the HSV ICP4 gene (Davison and McGeoch, 1986; Davison and Scott, 1986). Using polyclonal antisera to VZV, 4 IE polypeptides with apparent molecular weights of 185, 69, 43 and 34 have been reported (Lopetegui et al., 1983; Shiraki and Hyman, 1987). The 185 kDa protein species is phosphorylated and is the closest in size to the predicted 140 kDa polypeptide encoded by VZV gene 62 (Davison and Scott, 1986; Shiraki and Hyman, 1987). In addition, Felsner et al. (1988) have described a cell line expressing VZV gene 62 capable of complementing HSV IE gene ICPd It has been suggested that VZV 175-185 kDa phosphoprotein is the functional analog of HSV ICP4 protein (Felsner et al., 1988). We describe here the production of monoclonal antibodies (mAbs) to a VZV 175-180 kDa phosphoprotein. Indirect immunofluorescence (IIF) assays of cells infected with cell-free VZV indicated the reactivity of these mAbs with nuclear antigens within 6 h post infection (p.i.). The expression of this nuclear antigen was inhibited in the presence of metabolic inhibitors [cycloheximide (CH), actinomycin D (Act. D)]; however, nuclear antigens were detected following reversal of CHtreated VZV-infected cells. Furthermore, our results showed that these mAbs recognized the in vitro translation product encoded by VZV gene 62, suggesting that the 175-180 kDa protein is encoded by VZV gene 62.
197
Materials and Methods Cell culture and viruses Human fetal diploid lung (HFDL) cells were propagated in Eagle’s minimum essential medium (MEM) in Hanks’ balanced salt solution (BSS) with 10% fetal bovine serum and maintained in Eagle’s MEM in Earle’s BSS. The CaQu strain of VZV, an isolate of the Viral and Rickettsial Disease Laboratory (VRDL), California State Department of Health Services, was used in this study. Cell-free virus was prepared as described earlier (Schmidt and Lennette, 1976). Monolayers were infected with HSV-1 (McIntyre strain), HSV-2 (MS strain) and cytomegalovirus (CMV) (AD 169 strain). Monoclonal
antibodies
The complete procedure for the production of mAbs against VZV proteins has been described elsewhere (Forghani et al., 1982). Briefly, mAbs to IE protein of VZV were prepared by immunizing mice with whole cell lysates of VZV-infected HFDL cells. The first immunization was given intraperitoneally (i.p.) with Freund’s complete adjuvant. Hybridization of immune mouse spleen cells with mouse myeloma cells (SP2/0 line) was done by standard procedures using polyethylene glycol as fusing agent. The hybrid cells were screened for antibody secretion by IIF assay (Forghani et al., 1982). Monoclonal antibody 2XFl was selected for experiments described in this study. Ascites were prepared as described (Forghani et al., 1982). Briefly, each animal was primed with Pristane (2,6,10,14-tetramethyl pentadecane), injected with hybridoma i.p., and ascitic fluids were collected lo-12 days later. Zsotyping The isotype of each mAb was determined by the procedure described (Forghani et al., 1982). Briefly, tissue culture media from VZV positive hybridomas were concentrated 25-30 times by neutralized saturated ammonium sulfate and tested by agarose gel immunodiffusion against goat monospecific antisera to mouse IgGl, IgGZa, IgG2b, IgG3, IgA and IgM (Research Products International Corp., Elk Grove, IL). Radiolabeling
and radioimmunoprecipitation
assays (RZPA)
VZV-infected monolayers were prepared by mixing VZV-infected and uninfected HFDL cells at a ratio of 1: 3. At 24 to 48 h p.i., when cells showed a 60-70% cytopathic effect (CPE), the growth medium was replaced with phosphate-free medium containing 40 pCi/ml of [32Pi]orthophosphate or with methionine-free medium containing 20 pCi/ml [ 35S]methionine, or with 2 PCi of D-[‘4C]glucosamine, and labeled for 18-20 h. Radiolabeled viral antigens were prepared by
198
lysing the washed labeled cells with T&-saline buffer (0.05 M Tris-HCI, pH 7.4, 0.15 M NaCl) containing 1% deoxycholate, 1% NP-40 and 1% aprotinin as protease inhibitor, and clarifying at 40,000 x g for 1 h. As control, a labeled uninfected cell lysate was prepared in the same manner. RIPAs were performed by incubating labeled cell lysates with tissue culture fluids from hybridoma-secreted mAbs. The immune complexes were removed with protein A sepharose 4CL, disrupted with sodium dodecyl sulfate (SDS) and 2-mer~pt~th~ol, subjected to 10% polyacrylamide gel electrophoresis (PAGE), and finally autoradiographed, as described earlier (Forghani et al., 1984). Pulse-chase labeling
VZV-infected cells were labeled with 50 yCi/ml of ~35S]met~o~ne for 10, 20,40 and 60 min, and 6 h. After the pulse period, cells were washed 3 times, and the label was chased in fresh medium for 24 h. Procedures for SDS-PAGE-RIPA were similar to those described above.
Denatured uninfected and WY-infected cell lysates were separated by SDSPAGE (Laemmli, 1970). Preparation of VZV infected and uninfected cell lysates for SDS-PAGE was the same as described for RIPA except that they were not radiolabeled. The PAGE separated proteins were transferred to nitrocellulose filter paper (~hleicher h Schuell, Keene, NH) for 90 min by an el~troblotter (Hoeffer Scientific Instruments, San Francisco, CA) (Towbin et al., 1979; Burnette, 1981). The nitrocellulose filters were reacted with mAb for 1 h at 37 a C and overnight at 4” C, followed by 3 washings; they were then treated with horseradish peroxidaseconjugated rabbit anti-mouse IgG for 1 h at room temperature, and washed 3 times. The enzymatic reaction was visualized by addition of H202 as substrate and a~n~thyl carbazole as c~omogen donor (Forghani and Dennis, 1989). Non-denatured protein PAGE was run using buffer systems described by Laemmli (1970) but without addition of SDS to either gel or cell lysate. The separated proteins were transferred to nitrocellulose paper and the immunoenzymatic assays were done as described for denatured proteins. metabolic inhibitors
Monolayers of HFDL cells were grown on 4 sets of 8-chamber Lab-Tek slides (Miles Laboratories, Naperville, IL) and infected with cell-free virus. After 1 h adsorption at 37OC, the monolayers were washed 3 times with buffer to remove unadsorbed virus. Fresh medium containing 100 pg/ml CH was added to one set of slides to block the cellular and viral protein synthesis. These slides were incubated at 37 o C for 5 h, followed by removal of the medium and 3 washings. Fresh medium containing 10 pg/ml ActD was added to block transcription and allow the translation of preexisting mRNA. The cells were incubated for 14 h more at 37’C.
199
Controls were: (i) cell-free VZV-infected cells without drug treatment; and (ii) uninfected cells treated with and without metabolic inhibitors. Monolayers were rinsed briefly and, without drying, cells were fixed in acetone for 10 min at 6, 9, 12, 16 and 20 h p.i., which corresponds to 0, 3, 6, 9 and 14 h post removal of CH. The fixed cells were stored at 70 o C for future analysis. IIF staining
Cell culture fluids from cells secreting mAbs were added to each set of slides, incubated in a humidified chamber for 3 h, and washed 3 times. Fluorescein-conjugated goat anti-mouse IgG was added and the cells were incubated for 1 h at 37” C, washed 3 times, mounted and observed in a fluorescence microscope as described (Forghani et al., 1982). Monoclonal antibodies to VZV glycoproteins (gp) I, II, III and IV and normal mouse ascitic fluids were used as controls for specificity in this assay. Virus neutralization
assay
VZV neutralization assay was done using the VRDL standard plaque reduction assay with and without fresh guinea pig complement (Forghani et al., 1982). Cloning of VZV genes 61 and 62
The VZV BarnHI-B and J fragments (Fig. 1) contain the open reading frames (ORFs) for VZV genes 61 and 62 (Davison and Scott, 1986). The recombinant plasmid (pBR322) containing the BamHI-B and J fragments (Gilden et al., 1983) was cleaved with either AccI or iVde1 restriction enzymes. The 2.0~kilobase (kb) AccI fragment spanning VZV nucleotides 102715 to 104726 and containing the ORF of VZV gene 61 was electroeluted, blunt-ended with T4 DNA polymerase (Maniatis et al., 1982), and ligated into the SmaI site downstream from the T7 promoter of the pGEM7Zf transcription vector (Promega Biotec, Madison, WI) as described (Vafai et al., 1986). To clone gene 62, the 5.0 kb NdeI subfragment of BarnHI-B and J spanning VZV nucleotides 104420 to 109367 was ligated into the NdeI site of the polylinker of the pGEM5Zf transcription vector (Promega Biotec) downstream from the SP6 promoter. In vitro transcription,
translation and immunoprecipitation
The recombinant plasmids carrying either VZV gene 61 or gene 62 were cleaved with either PstI or Hind111 restriction enzymes downstream from the DNA insert, and the linearized DNA was used as a template for RNA transcription with T7 or SP6 RNA polymerase (Promega Biotec) respectively. RNA was transcribed from genes 61 and 62 and translated in vitro by the procedure described previously (Vafai et al., 1988). The in vitro translation (IVT) products were either analyzed by
200 32
P
MWM
69
46
Fig. 1. Fluorograph of SDS-PAGE electrophoretically separated phosphopeptides in immunoprecipitates of uninfected (lanes 1,4) and VZV-infected (lanes 2, 3) cell lysates labeled with [32Pi] and [ “Slmethionine reacted with mAb clone 2XFl. * Molecular weight markers (MWM) of “C-labeled proteins (myosin, phosphorylase B, bovine serum albumin, IgG heavy chain, ovalbumin).
SDS-PAGE or immunoprecipitated with mAb 2XFl. Before immunoprecipitation, the IVT products, suspended in 500 ~1 of lysis buffer (0.02 M sodium phosphate, pH 7.6, 0.1 M NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS) were incubated for 2 h at 4°C with 40 ~1 of a 10% formal&fixed suspension of protein A-containing Staphylococcus aweus Cowan I (Vafai et al., 1986). After centrifugation in a microfuge for 5 min at 4O C, VZV-specific proteins were immunoprecipitated at 4°C for 20 h in the presence of 100 ~1 of mAb 2XFl and 20 ~1 of goat anti-mouse IgG (Cappel Laboratories, Westchester, PA). Finally, 30 ~1 of a 10% formalin-fixed S. aweus suspension were added and, after 2 h at 4°C absorbed immune complexes were washed 3 times with lysis buffer and suspended in 20 ~1 of TNE buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA). After addition of 10 ~1 of 3 x sample buffer (150 mM Tris, pH 7.0, 6% SDS, 15% 2-mercaptoethanol, 0.03% bromophenol blue), the suspension was heated in boiling water for 4 min, cooled on ice, and analyzed by 8% polyacrylamide SDS-PAGE as described (Vafai et al., 1988).
201
Results Preparation
and selection of mAbs
Three attempts were made to hybridize immunized mouse spleen cells with murine SP2/0 myeloma cells. Ten independent mAbs were selected by IIF on 24-h VZV-infected monolayers. All 10 mAbs reacted only with the nucleus and nuclear membrane of the infected cells, and not with those of uninfected cells. To ensure the purity of each hybridoma, each clone was subcloned 3 times. These mAbs did not show any cross-reactivity with HSV-1, HSV-2 or CMV infected cells by IIF. When cells were infected with 25 recent VZV isolates (obtained from the VRDL diagnostic file) and tested blindly by IIF, all showed ‘nuclear staining reactivities similar to those seen with the CaQu laboratory strain. The following experiments were performed to further characterize our panel of mAbs: (i) Agarose gel immunodiffusion on concentrated cell culture media showed that all 10 mAbs from 3 separate hybridizations were of IgGl subclass of murine immunoglobulin. (ii) Cell culture media and ascitic fluids of 10 mAbs were tested for virus neutralizing activity with and without complement. As expected, no plaque reduction was observed either with cell culture media or with ascites, indicating that these mAbs do not neutralize VZV infectivity. (iii) Each ascitic fluid was titrated, tested by IIF, and found to have a titer of at least 1: 100,000. The reactions were specific only for VZV-infected cells and no cross-reactivity was observed with HSVand CMV-infected, or uninfected HFDL cells. Identification
of polypeptides
recognized by each mAb
To identify the VZV-specific proteins which are recognizedby the mAbs, the cell culture medium of each hybridoma was reacted with virus-infected cell lysates which had been labeled with [32Pi]orthophosphate, [3SS]methionine and [‘4C]glucosamine. The results shown in Fig. 1 indicate a prominent 175-180 kDa peptide and three minor polypeptide species in both [ 35S]methionine- and [ 32Pi]orthophosphatelabeled cell lysates with molecular weights ranging from 105 to 160 (Fig. 1, lanes 2 and 3). These results indicate that the 175-180 kDa polypeptide was highly phosphorylated and the others were at least partially phosphorylated. No precipitation bands were observed with [‘4C]glucosamine-labeled cell lysate, indicating that this protein was not glycosylated. When additional experiments were conducted with radioisotope-labeled uninfected cell lysates, no protein bands of similar size were seen (Fig. 1, Lanes 1 and 4). These experiments were repeated with immune mouse and normal ascites. Again, no precipitation was seen with normal mouse ascites; however, all 10 immune ascites immunoprecipitated protein bands similar to those detected by tissue culture media from hybridoma cells (data not shown). These results are in accord with two earlier publications identifying a 185 kDa major IE protein of VZV (Lopetegui et al., 1983; Shiraki and Hyman, 1987) using polyclonal antisera prepared in monkey and guinea pig respectively.
202
Pulse-chase
experiments
To determine the relationship between the major 175-180 kDa phosphoprotein and the three minor phosphoproteins, VZV-infected cells were labeled at various times and used in immunoprecipitation assays. The results of these experiments are shown in Fig. 2. During 10 min pulse (lane 2), two faint precipitation bands (175-180 kDa and 105-110 kDa) were detected. During additional 20, 40 and 60 min pulse labeling (lanes 3, 4 and 5) the intensity of all bands increased and two additional precipitation bands were visible. During 6 h pulse labeling (lane 6) the intensity of all bands increased slightly and no additional virus-specific bands were observed. When the 10 min pulse label was chased for 24 h, again only one major and three minor phosphoproteins were seen (lane 9). No polypeptides of similar size were detected by immunoprecipitation of uninfected cell lysates which were pulselabeled for either 10 min or 6 h and chased for 14 h (Fig. 2, lanes 1, 7 and 8). These data suggest that these three minor protein species are either a degradation product of the 175-180 kDa protein or are another VZV-specific protein which non-specifically reacts with mAb 2XFl.
I
23456709
Fig. 2. Autoradiographic image of pulse-chase labeled VZV IE protein. The optimal pulse-labeling time intervals for pulse-chase experiments were determined by labeling VZV-infected cell cultures showing a CPE of 60-708 with 50 rCi/ml [35S]methionine. At the IO-min pulse the labeled medium was removed, and the monolayer was washed 3 times, replenished with fresh medium without the label, and chased for 24 h. Arrows indicate proteins precipitated at the lo-min pulse with mAb clone 2XF1, which intensified at subsequent time intervals, and the chase. Lanes 2, 3,4 and 5: 10, 20, 40 and 60 min pulse; lane 6: 6 h pulse; lane 9: 24 h chase of VZV-infected cell lysate. Lanes 1 and 7: 10 min and 6 h pulse; lane 8: 24 h chase of uninfected cell lysate.
203
Immunoblotting
Experiments were conducted to react the cell culture media of the individual hybridomas with VZV denatured proteins which were separated by SDS-PAGE and electrophoretically transferred onto nitrocellulose filters. only the cell culture medium and ascitic fluid of clone 2XFl reacted with 175-180 kDa and three minor polypeptide species; the other 9 mAbs were non-reactive (Fig. 3). We considered the possibility that the lack of reactivity of these mAbs could be due to denaturation of the antigenic sites (epitopes) during SDS-PAGE separation. However, similar (negative) results were obtained when several renaturation processes (Dunn, 1986; Mandrel1 and Zollinger, 1984) were attempted (data not shown). In addition, no reactivity was observed with any of the 9 mAbs when a non-denaturing gel (Laemmli, 1970) was employed for separation of VZV protein and transferto nitrocellulose filter (data not shown). Again, only clone 2XFl was reactive.
Fig. 3. Electrophoretic transfer to nitrocelhdose paper of denatured uninfected and VZV-infected cell lysates separated by SDS-PAGE and reacted with mAb clone 2XFl. Lane 1: uninfected cell lysate. Lane 2: VZV-infected cell lysate.
204
CH
Fig. 4. Immunofluorescent staining of acetone-fixed WV-infected cells. HFDL cell monolayers were prepared on Lab-Tek slides infected with cell-free virus. One hour p.i. the inoculum was removed, fresh medium containing CH was added and incubated for 5 h. Then, the CH medium was removed, medium containing ActD was added and incubated for the next 14 h. The fixed monolayers were stained by IIF using mAb clone 2XFl and FITC-conjugated rabbit anti-mouse IgG. Lane U: untreated; lane CH: cycloheximide treated. Row 1: uninfected cell monolayer; row 2: infected cells at 6 h p.i.; row 3: at 9 h p.i.; row 4: at 12 h p.i.; row 5: at 16 h p.i.; row 6: at 20 h p.i.
205
Temporal expression and localization To examine the temporal expression and cellular distribution of the 175-180 kDa IE protein at various times p.i., we used the 10 mAbs in IIF assays. Four sets of HFDL cell monolayers were prepared: (i) untreated VZV infected cells; (ii) VZV infected cells treated with CH and ActD; (iii) untreated uninfected cells; and (iv) uninfected cells treated with CH and ActD. In the set of untreated VZV-infected cells, by 4 h p.i., less than 0.1% of single cells showed a very faint nuclear staining with dusty appearance (data not shown), and by 6 h p.i., a generalized granular nuclear and nuclear membrane stain were visualized (Fig. 4). By 9 h and 12 h p.i., the intensity of nuclear staining had increased, with areas of bright fluorescence. By 16 h p.i., infectious foci of 4-5 cells with various degrees of nuclear staining were visible. By 20 h p.i., the foci had enlarged to 8-10 cells with intense nuclei. Surprisingly, the type of staining in the nucleus of certain cells had also changed dramatically, showing intense patchy areas resembling polymorphonuclear leukocytes. On VZV-infected monolayers treated with CH, no fluorescence staining was observed at 0 h or 1 h after removal of the drug. However, by 3 h post removal of the CH, corresponding to 9 h p.i., single cells were showing nuclear and nuclear membrane staining resembling that seen at 6 h p.i. of untreated infected cells. By 9, 12 and 16 h post removal of CH, corresponding to 12, 16 and 20 h p.i., the nuclear staining intensified and became more granular. The uninfected cells with and without drug treatment, as well as the VZV-infected, CH-treated cells without reversal of CH treatment, showed no staining. In addition, no cytoplasmic or nuclear staining was observed at 4, 6, 9 and 12 h p.i. with mAbs to VZV gp1, gpI1 gpII1 and gpIV and normal mouse ascitic fluids used as controls. Reactivity of mAb 2XFl with translation products encoded by VZV gene 62 To test the possibility that the 175-180 kDa protein, detected by mAb 2XF1, is the product of VZV IE gene 62, the predicted ORFs of VZV genes 61 and 62 (Davison and Scott, 1986) were subcloned from the BarnHI-B and J DNA fragments (Fig. 5) into in vitro transcription vectors (pGEM5Zf and pGEM7Zf) as described in Materials and Methods. The RNA transcribed from these genes was translated in vitro, and the in vitro translation (IVT) products were immunoprecipitated with mAb 2XFl and analyzed by SDS-PAGE. Fig. 6A shows the primary translation products encoded by VZV genes 61 (62 kDa) and 62 (175 kDa). Immunoprecipitation of the IVT products with mAb 2XFl revealed a strong reactivity of the products encoded by gene 62, but not gene 61, suggesting that this mAb recognizes epitope(s) on VZV gene 62 (Fig. 6B). Three additional protein bands, smaller than the 175 kDa species, were also detected by SDS-PAGE analysis after immunoprecipitation of the IVT products encoded by gene 62. These protein bands may represent the degradation of the IVT products, or could be due to the translation products of the truncated RNA species generated during the in vitro
206 TR L
UL
'RL
‘R s
us
TRs
Fig. 5. Construction of recombinant plasmids carrying VZV genes 61 and 62. Recombinant plasmid pBR322 containing the BarnHI-B and J fragments, located within the unique long (UL), inverted repeat long (IR,) and inverted repeat short (IRS) sequences, was cleaved with either Accl (A) or Ndel (N) restriction enzymes, and the 2.0 kb and 5.0 kb restriction fragments containing VZV open reading frames 61 and 62, respectively, were cloned in pGEM vectors downstream from ‘I7 or SP6 promoters as described in Materials and Methods. B: BumHI site; S: Smal site.
transcription of gene 62. It is also possible that these bands may result from random initiation of different AUG codons present on gene 62 RNA.
Discussion Studies described in this report demonstrate that our panel of mAbs, generated by 3 independent hybridization attempts, recognized a phosphorylated IE protein of VZV. Using 32P- and 35S-labeled infected VZV cell lysates, a major 175-180 kDa and three minor phosphopolypeptides were immunoprecipitated with hybridoma cell media and immune mouse ascitic fluids. The lack of immunoprecipitation of 14C-labeled infected cell lysates with the 175-180 kDa protein species suggested that this phosphoprotein is not glycosylated. DNA sequence analysis of VZV genome (Davison and Scott, 1986) has predicted a 140 kDa protein encoded by gene 62 which displays a degree of amino acid sequence similarity to HSV IE gene ICP4 (McGeoch et al., 1988). Also, two earlier studies using polyclonal antisera to VZV identified an IE phosphoprotein with an apparent size of 185 kDa (Lopetegui et al., 1983; Shiraki and Hyman, 1987); our data are consistent with theirs. Despite amino acid sequence similarities between VZV gene 62 and HSV-1 ICP4, no antigenic cross-reactivity was observed with HSV-1, HSV-2 and CMV using anti-175-180 kDa mAbs.
207
,61
ivt
imp
Fig. 6. Expression and immunoprecipitation of WV genes 61 and 62 products. The recombinant plasmid containing either VZV gene 61 or 62 was cleaved with either PsrI (gene 61) or Hind111 (gene 62). ‘Ihe linearized DNA was used as a template for transcription. RNA was transcribed, translated in vitro, and the in vitro translation (WI) products were analyzed by SDS-IO% PAGE. The IVT products were also immunoprecipitated (imp) with mAb 2XFl and analyzed by SDS-8% PAGE. The apparent molecular weights (in kilodaltons) of the major polypeptides encoded by genes 61 and 62 are shown. No RNA is shown; RNA was excluded from translation reaction.
Experiments were conducted to determine the reactivity of our mAbs with denatured VZV proteins. SDS-PAGE-separated VZV proteins were electrotransferred to nitrocellulose filters and reacted with the mAbs. Although ail 10 mAbs were murine subclass IgGl, only one clone, 2XF1, was reactive to a 175-180 kDa and three minor polypeptides by Western blot analysis; aII the others were non-reactive. By Western blot analysis, using an antiserum prepared in rabbit against a synthetic peptide derived from VZV gene 62 DNA sequences, Felsner et al (1988) have identified a polypeptide with an apparent molecular weight of 175. Our Western blot data were consistent with these results and showed that mAb 2XFl recognized a 175-180 kDa polypeptide species.
208
As described under Results, we performed experiments to determine whether the non-reactivity of the other 9 mAbs in immunoblot assays could be due to denaturation or damage of the antibody-binding sites (epitopes) on the antigen by SDS-PAGE separation. Since our results were negative, we are now considering the possibility that the non-reactivity may be due to recognition of different antigenic domain(s) by these mAbs which are easily damaged. Competitive binding assays will be required to determine the likelihood of this possibility. In addition to the 175-180 kDa major phosphopolypeptide, our panel of mAbs recognized three minor phosphopolypeptides in VZV-infected cells. To explain the relationship between these phosphopolypeptides, the following experiments were done: (i) We considered the possibility that our hybridomas were not pure, although they were generated by 3 independent hybridization attempts. To ensure the purity of each hybridoma, we subcloned each hybridoma 3 times; the SDS-PAGE-RIPA results were identical in each step of subcloning (data not shown). (ii) Results of pulse-chase experiments indicated that by 10 min pulse two phosphoproteins (105-110 kDa and 175-180 kDa) were detected, and two additional bands were visible by 20,40, and 60 min and 6 h pulse periods. Again, by 10 min pulse and 24 h chase all four phosphoproteins were detected. These results suggest that these proteins may be either a cleavage product of the 175-180 kDa protein or an additional VZV-specific protein recognized by these mAbs. Detection of the smaller protein species could also be due to the translation of internal AUG codons. However, recent studies on IE mRNA of equine herpesvirus type 1 (EHV-1) have determined that it encodes four closely related IEPs in in vivo and in vitro translation (Cat&man et al., 1988; Robertson et al., 1988). It is also possible that, similar to IE mRNA of EHV-1, VZV gene 62 encodes a family of antigenically related phosphoproteins. It is striking to note that the genomic structure of VZV and EHV-1 both have two isomeric arrangements (Takahashi, 1983) and perhaps their IEPs have the same function. CH, an inhibitor of protein synthesis, and ActD, an inhibitor of RNA synthesis, have been used extensively to determine early protein synthesis of herpesviruses, including VZV (Shin&i and Hyman, 1987). We have applied these two metabolic inhibitors to examine the immunological reactivity of our MAbs to IE VZV-specific proteins. In the absence of these drugs a faint nuclear stain was observed at 4 h p.i., and a bright granular nuclear stain at 6 h p.i.; the staining intensified with additional incubation time. However, when virus infection was carried out in the presence of the drugs, the first nuclear stain was observed by 3 h after CH-reversal of the infected cells, corresponding to 9 h pi. These data demonstrate that the polypeptide recognized by the mAbs is an IE VZV protein (175-180 kDa) being synthesized within 6 h following infection. The synthesis of this protein was inhibited by CH treatment; however, treatment of the infected cells with CH resulted in the accumulation of RNA encoding the 175-180 kDa protein which was translated following the reversal of CH-treated VZV-infected cells. Our results indicated that the primary translation products encoded by VZV gene 62, but not gene 61, reacted with mAb 2XF1, further substantiating the immunological reactivity of this mAb to a major VZV IE protein.
209
In this paper we have described the first successful attempt at preparation and partial characterization of mAbs directed against the products of VZV IE gene 62. These mAbs will help to further characterize the functional properties of the 175-180 kDa protein and the three minor proteins encoded by gene 62 and their role in the expression of other viral genes during VZV replication in the infected cells.
References Burnette, W.N. (1981) “Western blotting”; electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Analyt. B&hem. 112, 195-203. Caughman, G.B., Robertson, A.T., Gray, W.L., Sullivan, D.A. and G’CaIlaghan, D.J. (1988) Characterization of equine herpesvirus type 1 immediate early proteins. Virology 163, 563-571. Davison, A.J. and McGeoch, D.J. (1986) Evolutionary comparisons of the S segments in the genomes of herpes simplex virus type 1 and varicella-zoster virus. J. Gen. Virol. 67, 597-611. Davison, A.J. and Scott, J.E. (1986) The complete DNA sequence of varicella-zoster virus. J. Gen. Virol. 67,1759-1816. Deluca, N.A. and Schaffer, P.A. (1985) Isolation and characterization of deletion mutants of herpes simplex virus type 1 in the gene encoding immediate early regulatory protein ICW. J. Virol. 56, 558-570. Dunn, SD. (1986) Effects of the modification of transfer buffer composition and the renaturation of proteins in gels on the recognition of proteins on Western blots by monoclonal antibodies. Analyt. B&hem. 157,144-153. Felsner, J.M., Kintchington, P.R., Inchauspe, G., Straus, S.E. and Ostrove, J.M. (1988) Cell lines containing varicella-zoster virus open reading frame 62 and expressing the “IE” 175 protein complement ICPF4 mutants of herpes simplex virus type 1. J. Virol. 62, 2076-2082. Forghani, B. and Dennis, J. (1989) Immunoenzymatic staining. In: N.J. Schmidt and R.W. Emmons (Eds.), Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections, 4th edit., pp. 135-156. American Public Health Association, Inc., Washington, D.C. Forghani, B., Dupuis, K.W. and Schmidt, N.J. (1984) Varicella-zoster viral glycoproteins analyzed with monoclonal antibodies. J. Virol. 52, 55-62. Forghani, B., Schmidt, N.J., Myoraku, C.K. and Gallo, D. (1982) Serological reactivity of some moncclonal antibodies to varicella-zoster virus. Arch. Virol. 73, 311-317. Gilden, D.H., Vafai, A., Shtram, Y., Becker, Y., Devlin, M. and Wellish, M. (1983) Varicella-zoster virus DNA in human sensory ganglia. Nature (London) 306,478-480. Honess, R.W. and Roizman, B. (1974) Regulation of herpesvirus macromolecular synthesis. 1. Cascade regulation of the synthesis of three groups of viral proteins. J. Virol. 14, 18-19. Honess, R.W. and Roizman, B. (1975) Regulation of herpesvirus macromolecular synthesis: sequential transition of polypeptide synthesis requires functional polypeptides. Proc. Natl. Acad. Sci. USA 72, 1276-1280. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 277, 680-685. Lopetegui, P., Matsunaga, Y., Okuno, T., Ogino, T. and Yamanishi, K. (1983) Expression of varicellazoster virus-related antigens in biochemically transformed cells. J. Gen. Virol. 64, 1181-1186. Mandrell, R.E. and Zollinger, W.D. (1984) Use of a zwitterionic detergent for restoration of the antibody-binding capacity of electroblotted meningococcal outer membrane proteins. J. Immun. Methods 67, 1-11. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular cloning: a Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
210
McGeoch, D.J., Dahymaple, M.A., Davison, A.J., Dolan, A., Frame, M.C., McNab, D., Perry, L.J., Scott, J.E. and Taylor, P. (1988) The complete DNA sequence of the long unique region in the genome of herpes simplex virus type 1. J. Gen. Virol. 69, 1531-1574. O’Hare, P. and Hayward, G.S. (1985) Three tram-acting regulatory proteins of herpes simplex virus modulate immediate-early gene expression in a pathway involving positive and negative feedback regulation. J. Virol. 56, 723-733. Robertson, A.T., Cat&man, G.B., Gray, W.L., Bauman, R.P., Staczek, J. and O’Callaghan, D.J. (1988) Analysis of the in vitro translation products of the equine herpesvirus type 1 immediate early mRNA. Virology 166, 451-462. Roizman, B., Carmichael, L.E., Deinhardt, F., de-The, G., Nahmias, A.J., Plowright, W., Rapp, F., Takahashi, M. and Wolf, K. (1981) Herpesviridae. Intervirology 16, 201-217. Schmidt, N.J. and Lennette, E.H. (1976) Improved yields of cell-free varicella-zoster virus. Infect. Immun. 14,709-715. Shiraki, K. and Hyman, R.W. (1987) The immediate early proteins of varicella-zoster virus. Virology 156, 423-426. Takahashi, M. (1983) Chicken pox. In: M.A. Lauffer and K. Maramorosch (Eds.), Advances in Virus Research, Vol. 28, Academic Press, New York, N.Y. Tedder, D.G. and Pizer, L.I. (1988) Role for DNA-protein interaction in activation of the herpes simplex virus glycoprotein D gene. J. Virol. 62, 4661-4672. Towbin, H., Staehlin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Prcc. Natl. Acad. Sci. USA, 76, 4350-4354. Vafai, A., Wellish, M. and Gilden, D.H. (1986) Polypeptides encoded by varicella-zoster virus unique short sequences. Virus Res. 5, 67-76. Vafai, A., Wroblewska, Z., Mahalingam, R., Cabirac, G., Wellish, M., Cisco, M. and Gilden, D.H. (1988) Recognition of similar epitopes on varicella-roster virus gp I and gpIV by monoclonal antibodies. J. Virol. 62, 2544-2551. Watson, R.J. and Clements, J.B. (1980) A herpes simplex virus type 1 function continuously required for early and late virus RNA synthesis. Nature (London) 285, 320-330. (Received 22 August 1989; revision received 15 February 1990)
