Resistance of herpes simplex virus to 9-{1[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl}guanine: Physical mapping of drug

Transcription

1 Proc. Natl. Acad. Sci. USA Vol. 81, pp , March 1984 Microbiology Resistance of herpes simplex virus to 9-{1[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl}guanine: Physical mapping of drug synergism within the viral DNA polymerase locus (antiviral resistance/active center of herpes DNA polymerase) C. S. CRUMPACKER*t, P. N. KOWALSKY*t, S. A. OLIVER*t, L. E. SCHNIPPER*t, AND A. K. FIELD *Charles A. Dana Research Institute and the Harvard Thorndike Laboratory, Beth Israel Hospital, and the Divisions of tinfectious Disease and toncology, Department of Medicine, Beth Israel Hospital and Harvard Medical School, Boston, MA 02215; and Department of Virus and Cell Biology, Merck Sharp & Dohme Research Laboratories, West Point, PA Communicated by Harold S. Ginsberg, October 27, 1983 ABSTRACT A herpes simplex virus type 2 () mutant TS6 (strain HG52) induces a heat-labile viral DNA polymerase at the nonpermissive temperature and is markedly resistant to 9-{[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl}- guanine [2'-nor-2'-deoxyguanosine; 2'NDG]. This antiviral drug requires HSV thymidine kinase for phosphorylation to an active inhibitor (2'NDG-triphosphate), and thymidine kinasedeficient mutants of HSV exhibit varying degrees of resistance to 2'NDG, with the HSV type 1 () B2006 mutant (Kit) being markedly resistant. The ts6 mutation and the 2'ndg"-1 mutation within the viral DNA polymerase locus have been physically mapped by marker rescue and generation of HSV- 1/ intertypic recombinants. The physical map limits for the ts6 mutation and 2'ndg"-1 mutation are closely linked within a 2.2-kilobase-pair region of DNA sequences and are physically separate from the paarl and acvr-1 mutations. Resistance to 2'NDG by ts6 can be overcome in the presence of combinations of 2'NDG and phosphonoacetic acid, indicating drug synergism within the viral DNA polymerase locus. These physical mapping studies expand the limits of DNA sequences defining an active center in the viral polymerase to 3.5 kilobase pairs, indicating that regions spanning the entire polymerase polypeptide may contribute to a specialized surface able to interact with nucleotides of different structure. The new antiherpes nucleoside analog 9-{[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl}guanine (2'-nor-2'-deoxyguanosine; 2'NDG) is an efficient inhibitor of herpes simplex virus types 1 and 2 ( and ), human cytomegalovirus, and Epstein-Barr virus replication (1-4). It is a preferential substrate for the thymidine kinase (TK), is readily phosphorylated to the triphosphate, and is a potent inhibitor of viral DNA polymerase (4). It inhibits herpes virus replication in cell culture and has therapeutic efficacy against herpes virus infections in mice. 2'NDG is a structural analog of acyclovir [9-(2-hydroxyethoxymethyl)guanine; ACV]. Restriction endonuclease analysis of the genomes of plaque-purified intertypic recombinants of and HSV- 2, generated by marker rescue, has permitted physical mapping of temperature-sensitive mutations and drug resistance mutations associated with HSV DNA polymerase activity. These included tsc4, tsc7, tsd9 (, strain KOS), tsh (, strain 17), and ts6 (, strain HG52), which are all associated with a thermolabile DNA polymerase activity (5-9). Drug resistance mutations conferring resistance to phosphonoacetic acid (PAA), ACV, and adenine arabinonucleoside (araa) have also been shown to be defined by a 2.6- kilobase-pair (kbp) region of DNA sequences within the viral DNA polymerase locus (5, 10, 11). The recombi- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact. nants containing the drug resistance mutations induce an altered viral DNA polymerase enzyme exhibiting altered enzyme kinetics and a higher Ki in the presence of PAA, dideoxynucleotide triphosphates, ACV-TP, ara-atp, and (E)- 5-(2-bromovinyl)-2'-deoxyuridine triphosphate (BrVdUrd- TP) (ref. 12; unpublished data). Purified viral polymerase from other drug-resistant mutants have also exhibited altered enzyme kinetics in the presence of ACV-TP (13) and ara-atp (14). The physical mapping of drug resistance mutations that result in altered viral DNA polymerase activity in the presence of these nucleotide triphosphates was used to propose an active center on the viral polymerase enzyme. This is a region of 1.3 kbp in the DNA polymerase locus or a maximal region of 2.6 kbp if the region of uncertainty where crossover events occur is included (12). In this report we describe a mutant of ts6 (strain HG52) that contains a mutation in the structural gene of viral DNA polymerase and is markedly resistant to 2'NDG. The 2'ndgR-1 mutation has been physically mapped to a 2.2-kbp region of DNA sequences within the DNA polymerase locus and is physically separate from the DNA sequences containing the resistance mutations for ACV and PAA. The resistance of ts6 to 2'NDG can be overcome in the presence of 2'NDG and PAA, demonstrating drug synergy within the viral DNA polymerase locus. These physical mapping studies expand the limits of DNA sequences defining the viral DNA polymerase active center to 3.5 kbp. These observations suggest that through tertiary folding almost the entire DNA polymerase polypeptide may contribute to a specialized surface that is able to interact with nucleotides of different structure. MATERIALS AND METHODS Cells. A continuous line of African green monkey kidney cells (Vero) was grown in Eagle's minimum essential medium supplemented with 10% calf serum. Cell monolayers (2.4 x 106 cells) in 60-mm (diameter) plastic Petri dishes (Flow Laboratories) were used throughout. Viruses. The wild-type strains of (KOS and 17) and (HG52) are PAAS and ACV'. The temperature-sensitive (TS) mutant of ts6 (strain HG52) has been described (15, 16). The PAA-resistant mutants of strain 17 (ts+, PAAR_1) and strain KOS (ts+, PAAR-5) were obtained from John Subak-Sharpe and Priscilla Schaffer, respectively. The TK-deficient mutant (dpyk7) of Abbreviations: 2'NDG, 9-{[2-hydroxy-1-(hydroxymethyl)ethoxy]- methyl}guanine or 2'-nor-2'-deoxyguanosine; ACV, 9-(2-hydroxyethoxymethyl)guanine or acyclovir; PAA, phosphonoacetic acid; araa, adenine arabinonucleoside; BrVdUrd, (E)-5-(2-bromovinyl)- 2'-deoxyuridine; kbp, kilobase pair(s); and, herpes simplex virus types 1 and 2, respectively; TK, thymidine kinase. 1556

2 Microbiology: Crumpacker et al (strain 17) had been isolated by growth of wild-type virus in BrdUrd (5,ug/ml) (17) and the mutation was mapped by marker rescue to the viral TK locus (18). The ACVR-6 mutant of was obtained by passing a clinical isolate of in the presence of ACV (0.5,ug/ml) for six passages to select a tk- mutant that expressed only 8% of the TK activity of the wild-type virus (11). The markedly TK-deficient mutant B2006 of the Kit strain was obtained from David Knipe and its properties have been described (19). Drugs. 2'NDG was supplied by Richard Tolman (Merck Sharp & Dohme). A stock solution of 5 mm 2'NDG was prepared in distilled H20 and stored at -20'C. ACV was supplied by Burroughs Wellcome (Research Triangle Park, NC) and a 10 mm stock solution was prepared in distilled H20 and stored at -200C. PAA as the disodium salt was the gift of Abbott. Intertypic Marker Rescue and Analysis of Genome Structure of Recombinants of and. The technique of intertypic marker rescue of ts mutations has been described in detail (5), and the genome structures and phenotypic characteristics of the R6 recombinants have been published (5). In brief, dishes of baby hamster kidney cells were coinfected at the nonpermissive temperature (38.5 C), per dish, with 0.4,g of intact DNA from the ts6 (HG52) mutant and 0.4 pg of unseparated, restriction endonuclease-cleaved fragments of DNA from strain 17 (ts+, paar1). DNA transfection was by use of the modified calcium phosphate technique (20). The ts+ virus progeny from such crosses were plaque-purified three times at 38.5 C, and the parental origin of restriction endonuclease sites in the DNA was determined (5). Resistance to 100 pg of PAA was present in the DNA crosses as a nonselected marker. The ts+ recombinants were previously tested for resistance to PAA by deter- Proc. NatL Acad. Sci. USA 81 (1984) 1557 mining the relative efficiency of plaguing in the presence and absence of drug and for resistance to ACV by determining the ID50 (,M) of drug (10). The physical maps of strain 17 and strain HG52 for the restriction endonucleases Xpa I (1), HindIII (2), EcoRI (3), Bgl 11 (4), Hpa I (5), Kpn I (6), and BamHI (7) in the region of map units on the HSV genome have been determined (5, 18). All of the restriction sites are summarized at the top of Fig. 1. The numbers correspond to the numbers assigned to each restriction endonuclease, and the sites are aligned with the corresponding one for each map. The restriction sites and the corresponding fragments are shown above the line for and below the line for HSV- 2. One map unit is equivalent to 106 daltons (1.6 kbp), and the genome orientation is as described (18). The order and size of the restriction endonuclease fragments for each type have been defined (21, 22). Inhibition of HSV Plaque Formation by 2'NDG. The antiviral activity of 2'NDG was assayed by a plaque-reduction method performed in the presence of increasing concentrations of drug in Vero cells. Inhibition of plaque formation by 2'NDG and PAA together was determined by keeping one drug constant (PAA) and increasing the other over a broad range of concentrations. Both drugs were present in the overlay medium. The ID50 (pg/mi) of each drug combination or single drug relative to control wells without drug was calculated directly from the plot relating surviving plaques in the presence of increasing concentrations of drug on semilogarithmic paper. The relation of the percentage of survivors vs. the logarithm of the drug concentrations is a linear function in the region of 50% reduction, and the best linear plot was obtained by the method of a least squares fit (23). To determine synergism, the ID50s obtained in the presence ~~~ R6-26 R6-30 R6-34 R X ID 50 (Mg/m 2'NDG ACV ts6 PAA -I HSV-I (strain 17 ) WT (stain HG52)WT FIG. 1. Genome structure determined by restriction endonuclease analysis (5, 10) and sensitivities to 2'NDG and ACV of R6 recombinants and parental strains of HSV. Endonuclease cleavage sites for the enzymes given in the Materials and Methods are shown above the line for and below the line for ; solid regions indicate DNA sequences, clear regions indicate DNA sequences, and hatching indicates regions of uncertainty. Data on 50o inhibition with ACV are from ref. 10.

3 1558 Microbiology: Crumpacker etalp Table 1. Sensitivity of mutants of HSV to 2'NDG, ACV, and PAA ID50, pg/mi Type 2'NDG ACV PAA KOS WT PAAR5 (KOS) Strain 17 WT PAAR_1 (strain 17) dpyk-7 (strain 17) ACVR-6 (clinical isolate) B2006 (Kit) HG52 (WT) ts6 (HG52) The 50% inhibitory dose (ID50) is the concentration of drug that inhibited viral plaque formation by 50% on Vero cells. Each value is the mean of three independent assays and in each case the SD was <10% of the mean. of various combinations of 2'NDG and PAA are plotted on an isobologram. The isobologram using ID50 concentration of two antiviral drugs against a single virus is defined to illustrate synergism for the combination when 50% inhibition occurs with only one-fourth the ID50 concentration of each drug alone (24). RESULTS Resistance of ts6 and TK- Mutants to 2'NDG. The strains PAAR_1 (strain 17) and PAAR_5 (strain KOS) were derived by passage in the presence of PAA (100 Mg/ml) and exhibit resistance to PAA, ACV, and araa (6, 10, 11, 13). These two mutants are sensitive to plaque reduction by 2'NDG and the ID50 is similar to that of the strain 17 and strain KOS viruses (Table 1). The ts6 (strain HG52) mutant is sensitive to inhibition by PAA and ACV but it is 10-fold more resistant to 2'NDG than the parent (HG52) (ID50 = 15.5,g/ml). The ts6 mutant induces normal levels of viral TK and alkaline DNase and these loci can be excluded as playing a role in resistance of (ts6) to 2'NDG (12, 25). These studies indicate that resistance to 2'NDG is mediated by the viral DNA polymerase locus, but by a different region of DNA sequences than those containing resistance mutations for paar_1 and acvr1. Bgl-ll i-d Bgl-ll o-c Bam Hi Resistance to 2'NDG can also be mediated by the viral TK locus. The dpyk-7 mutant of (strain 17) induces only a small amount of viral TK activity, is markedly resistant to ACV, and exhibits only one-half as much sensitivity to 2'NDG compared to strain 17 (Table 1). However, the ADVR-6 mutant, a TK- mutant derived by serial passage in ACV and inducing only 8% of the viral TK of the initial WT virus, exhibits marked resistance to both ACV and 2'NDG. Mutant B2006 (Kit) expresses no TK activity, fails to induce the 43-kilodalton viral TK protein, and is the most markedly resistant mutant to ACV and 2'NDG [ID50 = 55,g/ml]. Thus, resistance to 2'NDG is mediated by both the viral DNA polymerase and TK loci of HSV. A mutant expressing diminished TK activity, dpyk-7 (strain 17), is markedly resistant to ACV but only one-half as sensitive to 2'NDG, suggesting a difference in the manner in which these two drugs are processed by the viral TK enzyme. Physical Map Limits of the 2'ndg"-l Mutation. To physically define the map limits for the 2'ndgR-1 mutation within the viral DNA polymerase locus, / intertypic recombinants previously employed to demonstrate the close linkage of the acvr-1, paarj, and araar_1 mutations were tested for plaque inhibition in the presence of 2'NDG. These recombinants contain a small insertion of DNA sequences from mutant PAAR_1 of (strain 17) in an otherwise genome (ts6, strain HG52). The insertion of DNA sequences corrects the ts6 defect, enabling the recombinants to grow at the nonpermissive temperature of 38.5 C (5). The genome structure of these recombinants in the region of map units on the HSV genome, the restriction endonuclease enzymes used in the analysis, and the ID50 for each recombinant and the parental strains to 2'NDG and ACV are depicted in Fig. 1. The R6-26 recombinant contains an DNA insertion extending from the Bgl II i-d site to the BamHI h'-j' site (maps units, , including the region of uncertainty where crossover events occur). This is the smallest region of DNA sequences that all of the R6 recombinants have in common and this region of DNA sequences defines the maximal map units containing the ts6 mutation. The R6-30 recombinant contains an DNA insertion extending from the BamHI g-v site to the BamHI h'-j' site (map units, ); the R6-34 recombinant contains an DNA insertion extending from the Bgl II i-d site to the EcoRI m-o site (map units, ); and the R6-19 recombinant contains h'-j' Kpn x-c I1 Proc. NatL Acad Sci. USA 81 (1984) HSV- 1 Eco RI m-n I 'ndg r_, ara-a r_ 1, acv r_ 1, paa r-1 evima/ 'S"""0X' 5 bvdu r -1 Kbp FIG. 2. Physical map limits of drug resistance mutations within the DNA polymerase locus of HSV. Restriction endonuclease sites and fractional location on the genome are shown. Hatched areas indicate regions of uncertainty where crossover events can occur. Maximal limits for the 2 ndgr-1 mutation are obtained from this study and are compared with previously derived limits for paar-1 (5, 6), acvr-1 (10), araar_1, and bvdu -1 (mutation for resistance to BrVdUrd) (11). Limits shown for paar_1, acvr-1, and araar_1 do not imply order but only that resistance mutations for these three drugs are closely linked within a 2.6-kbp region of HSV DNA. I

4 Microbiology: Crumpacker et al 0 z CMj 20r 8 12 PAA [Mug/mi] FIG. 3. Isobologram using the 50% plaque-reduction concentration (ID50) of 2'NDG and PAA (pg/ml) to illustrate the synergistic effect of the combination of two drugs on ts6. The ID50 for each combination was determined by plaque-inhibition assay in Vero cells and a plot of survivors vs. the logarithm of the drug concentration for 2'NDG with PAA held constant over several concentrations. an DNA insertion extending from the Kpn I j-m site to the Kpn I x-c site (map units, ). All of these recombinants have corrected the ts6 mutation and therefore grow at 38.50C. All are markedly sensitive to 2'NDG (ID50s = ,ug/ml) but have varying phenotypes with respect to PAA, ACV, araa, and BrVdUrd resistance. Recombinant R6-34 is resistant to PAA, ACV, araa, and BrVdUrd; R6-19 is resistant to PAA, ACV, and araa; and R6-26 and R6-30 are sensitive to PAA, ACV, araa, and BrVdUrd (10, 11). The physical map limits of DNA sequence containing the resistance mutation for 2'NDG overlap with the DNA region containing the resistance mutations for ACV, PAA, and araa (Fig. 2). However, the resistance mutations for these three drugs and for BrVdUrd can be transferred separately from the 2'ndgR_1 mutation. This indicates that the resistance mutations occur in separate parts of the DNA polymerase locus, even though the mapping limits contain regions of overlap. Synergism of 2'NDG and PAA on ts6. The ts6 mutant exhibits marked resistance to 2'NDG (ID50 = 15.5,ug/ml) but this resistance can be overcome in the presence of small amounts of PAA. The isobologram of the ID50s for ts6 in the presence of both drugs indicates that the decrease in amount of 2'NDG required to inhibit plaque formation by 50% in the presence of PAA is greater than would be expected for an additive relationship (Fig. 3). In the presence of 1 Ag of PAA per ml, only 4.4 pag of 2'NDG per ml is required to inhibit plaque formation by 50% compared to 18.0 ug/ml with 2' NDG alone. For the purposes of this study, synergism is defined to occur when the concentration of drug that produces 50% inhibition is reduced to one-fourth of the drug concentration required when acting alone (24). By this definition 2'NDG and PAA are able to act synergistically to overcome the resistance to 2'NDG exhibited by ts6. The resistance mutations to these two drugs occur in physically distinct regions of the viral DNA polymerase locus. This indicates that these two drugs can act synergistically to inhibit viral DNA polymerase, probably by their interaction with distinct regions of the viral polymerase enzyme. DISCUSSION The ts6 mutation had been physically mapped to the viral DNA polymerase locus and this mutation has been associated with a thermolabile HSV DNA polymerase activity (5, 7). In this report we establish that the physical location of the ts6 mutation is either identical or closely linked to the Proc. NatL Acad. Sci. USA 81 (1984) 1559 resistance mutation for 2'NDG. The ts6 mutant is markedly resistant to 2'NDG, and correction of the ts6 mutation by insertion of a 2.2-kbp region of DNA sequences extending from the Bgi II i-d site to the BamHI h'-j' site (map units, ) also results in recombinants that are sensitive to 2'NDG. The mapping limits for the 2'ndgR_1 mutation are identical to those obtained for the ts6 mutation. An alternative explanation that a "second site" rearrangement of sequences results in intergenic or intragenic suppression of the mutant phenotype is unlikely because of the wide range in the length of the rescuing DNA sequence. This argues against crossovers that result in intragenic suppression. The assertion that the ts6 mutation and the 2'ndgR_1 mutation are identical or very closely linked within the DNA polymerase locus is analogous to the assertion for the tsd9 (strain KOS) and the paar mutation (26, 27). The HSV- 1 tsd9 mutant has a mutation in the structural gene for DNA polymerase, is unable to replicate viral DNA at 38.50C, and is resistant to PAA (8, 26, 27). A revertant that is able to grow at the nonpermissive temperature also becomes sensitive to PAA (26, 27). Physical mapping of the tsd9 mutation 14 / intertypic recombi- by marker rescue in nants indicates that the physical map limits for the tsd9 and the paar_1 mutations are contained in a 2.6-kbp region of DNA sequences extending from the Bgl II o-c site to the Kpn I x-c site (map units, ) (6). This region of DNA sequences also contains acvr_1 and araar_1 mutations (10, 11) and overlaps with the region of DNA defining the 2'ndgR-1 mutation, but the phenotype for resistance to 2'NDG can be physically separated from resistance to the other three drugs. All of these drug resistance mutations taken together are defined by a 3.5-kbp region of DNA within the DNA polymerase locus. Synergy between 2'NDG (referred to as BIOLF-62) and PAA or phosphonoformate has been demonstrated for HSV- 1 and (28). A combination of 2'NDG and ACV did not exhibit synergy in this system. In this report, we have shown that 2'NDG and PAA can interact in a synergistic manner to overcome the resistance of ts6 to inhibition by 2'NDG alone. Synergistic antiviral activity has been observed for araa and PAA acting in combination to inhibit herpes virus replication (29). In addition to the physical mapping studies indicating that the paar_1 and 2'ndgR_1 mutations are defined by different regions of the viral DNA polymerase locus, the demonstration of synergism between PAA and 2'NDG strongly suggests that these drugs interact with different parts of the viral DNA polymerase. An alternative explanation could be that PAA stimulates error-prone viral DNA synthesis, which is more susceptible to internucleotide incorporation or chain termination by 2'NDG-TP. These studies extend the region of DNA sequences in that contain mutations conferring resistance to nucleoside analogs (Fig. 2). The previously described 2.9-kbp region containing the bvdur-1 mutation ( BamHI h'-j' site to EcoRI m-o site; map units, ) and the 2.6-kbp region containing the paar-1, acvr_1, and araar_1 mutations ( BgI II o-c site to Kpn I x-c site; map units, ) are now joined by a 2.2-kbp region containing the 2'ndgR_1 mutation ( Bgl II i-d site to BamHI h'-j' site; map units, ). This extends the region of DNA sequence containing mutations that confer resistance to nucleoside analogs leftward to the HSV- 1 Bgl II i-d site (map units, 39.6) and rightward to the EcoRI m-o site (map units, 42.8). The right-hand limit can be reduced further to the Kpn I x-c site (map units, 41.8) by considering the limits previously used to define an active center on the viral DNA polymerase and where the limits for the paar_1, acvr_1, araar_1, and bvdur-1 mutations overlap (map units, ) (11, 12).

5 1560 Microbiology: Crumpacker et al. The region of DNA extending from the Bgl II i-d site to the Kpn x-c site (map units, ) comprises a 3.5-kbp region of DNA containing all of the drug resistance mutations mapping within the DNA polymerase locus and is in close agreement with the region of DNA sequences determining the potential mrna for the viral DNA polymerase. This is a 4.2- to 4.3-kbp transcript, whose 5' end begins near the BamHI v-r site, spans the Bgl II i-d site, and extends to a 3' end between the BamHI k'-w site and the Kpn I x-c site (L. Holland and M. Levine, personal communication). The fact that resistance mutations can be shown to physically map along the entire length of DNA transcribing this potential mrna suggests that an active center or several active centers of polymerase function are determined by tertiary folding of the entire DNA polymerase polypeptide. A detailed picture of this tertiary folding must await the amino acid sequence analysis of the viral DNA polymerase enzyme or be deduced from the nucleotide base sequence analysis of the viral DNA polymerase gene. We thank Drs. John Subak-Sharpe, Priscilla Schaffer, David Knipe, and Ian Hay for kindly providing stocks of mutant viruses and also thank Drs. Lou Holland and Myron Levine for permission to cite their work prior to publication. We gratefully acknowledge the assistance of Ms. Eileen Poe in the preparation of this manuscript. This work was supported by Contract AI from the Antiviral Substances Program of the National Institute of Allergy and Infectious Disease and National Dental Institute, Grant NIA-1- P01-AG0059 from National Institute of Aging, and a grant from Merck Sharpe & Dohme Research Laboratories. 1. Smith, K. O., Galloway, K. S., Dennell, W. C., Ogilvia, K. K. & Radatus, B. K. (1982) Antimicrob. Agents Chemother. 22, Ashton, W. T., Karkas, J. D., Field, A. K. & Tolman, R. L. (1982) Biochem. Biophys. Res. Commun. 108, Cheng, Y. C., Huang, E. S., Lin, J. C., Mar, E. C., Pagano, J. S., Dutschman, G. E. & Grill, S. P. (1983) Proc. Natl. Acad. Sci. USA 80, Field, A. K., Davies, M. E., DeWitt, C., Perry, H. C., Liou, R., Germershausen, J., Karkas, J. D., Ashton, W. T., Johnson, D. B. R. & Tolman, R. L. (1983) Proc. Natl. Acad. Sci. USA 80, Proc. NatL Acad ScL USA 81 (1984) 5. Chartrand, P., Stow, N. P., Timbury, M. C. & Wilkie, N. M. (1979) J. Virol. 31, Chartrand, P., Crumpacker, C. S., Schaffer, P. A. & Wilkie, N. M. (1980) Virology 103, Hay, J. & Subak-Sharpe, J. H. (1976) J. Gen. Virol. 31, Aron, G. M., Purifoy, D. J. M. & Schaffer, P. A. (1975) J. Virol. 16, Purifoy, D. J. M. & Powell, K. C. (1981) J. Gen. Virol. 54, Crumpacker, C. S., Chartrand, P., Subak-Sharpe, J. H. & Wilkie, N. M. (1980) Virology 105, Crumpacker, C. S., Schnipper, L. E., Kowalsky, P. N. & Sherman, D. M. (1982) J. Infect. Dis. 146, Knopf, K. W., Kaufman, E. R. & Crumpacker, C. S. (1981) J. Virol. 39, Coen, D. M., Furman, P. A., Gelep, P. T. & Schaffer, P. A. (1982) J. Virol. 41, Furman, P. A., Coen, D. M., St. Clair, M. H. & Schaffer, P. A. (1981) J. Virol. 40, Timbury, M. C. (1971) J. Gen. Virol. 13, Timbury, M. C. & Calder, L. (1976) J. Gen. Virol. 30, Jamieson, A. T., Gentry, G. A. & Subak-Sharpe, J. H. (1974) J. Gen. Virol. 24, Stow, N. D. & Wilkie, N. M. (1978) Virology 90, Dubbs, D. R. & Kit, S. (1965) Virology 25, Graham, F. L. & van der Eb, A. I. (1973) Virology 52, Wilkie, N. M. (1976) J. Virol. 20, Cortini, R. & Wilkie, N. M. (1978) J. Gen. Virol. 39, Crumpacker, C. S., Schnipper, L. E., Zaia, J. A. & Levin, M. J. (1979) Antimicrob. Agents Chemother. 15, Jawetz, E. & Gunnison, J. H. (1953) Pharmacol. Rev. 5, Crumpacker, C. S., Schnipper, L. E., Chartrand, P. & Knopf, K. W. (1981) in Antiviral Chemotherapy: Design of Inhibitors of Viral Functions, ed. Gauri, K. K. (Academic, New York), pp Joffre, J. T., Schaffer, P. A. & Parris, D. S. (1977) J. Virol. 23, Purifoy, D. J. M. & Powell, K. L. (1977) J. Virol. 24, Smith, K. O., Galloway, K. S., Ogilvie, K. K. & Cheriyan, U. 0. (1982) Antimicrob. Agents Chemother. 22, Shannon, W. M. & Schabel, R. M. (1980) Pharmacol. Ther. Part B,