Growth and Persistence of Polyoma Early Region ... - Journal of Virology

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May 5, 1981 - Department ofMicrobiology, Guy's Hospital Medical School, London Bridge SEI 9RT, England. Received 22 January 1981/Accepted 5 May ...
Vol. 39, No. 3

JOURNAL OF VIROLOGY, Sept. 1981, p. 958-962 0022-538X/81/090958-05$02.00/o

Growth and Persistence of Polyoma Early Region Deletion Mutants in Mice D. J. McCANCE Department of Microbiology, Guy's Hospital Medical School, London Bridge SEI 9RT, England

Received 22 January 1981/Accepted 5 May 1981

Replication of early region deletion mutations in polyoma in the kidneys of mice was in most cases reduced by 10- to 10,000-fold, as compared with wild-type polyoma, and the mutants failed to produce the persistent infection observed with wild-type polyoma. Polyoma virus has a small circular genome apparently organized to obtain maximum utility from a miniimum of length. This is achieved by overlapping genes which are expressed through differential processing of RNA transcripts (5). A number of viable deletion mutations mapping within different parts of the polyoma virus early region have been isolated. The mutants grow well in tissue culture cells without helper viruses and have been crucial in the elucidation of the genetic organization of the viruses (1, 2, 4). Because these mutants are viable in tissue culture, this presents something of a paradox: if polyoma virus has been selected through evolution for minimum genome size, why does some of the DNA sequence, including parts of certain coding regions, appear dispensible? The obvious answer is that the virus evolved in mice and not in cell culture. I have examined whether polyoma virus mutants containing viable deletion mutants are able, like wild-type strain A2 polyoma virus (3), to grow and persist in their host, the mouse. Figure 1 shows the deletable regions of the genome compared with the regions which determine the three early mRNA's and their translation products, small, middle, and large T antigens. The deletions can be arranged into three groups depending on the area of the early region deleted. Viable deletions have been isolated from the early region of the genome clockwise from the origin of replication but before the common translational initiation codon for the three T antigens, between map units 71.6 and 73.3 (1, 4, 6). This region of the genome, or part of it, determines the 5' ends of the wild-type early mRNA's (5); therefore, this region may be important in the specification and regulation of early mRNA synthesis. The mutants containing deletions in this region grow well in tissue culture (1, 4, 6), although the two studied in this paper, d117 and d175, were chosen because they have large deletions and a reduced capacity, as compared with wild-type polyoma virus, to grow 958

in vitro (1). The replication of dll7 and d175 in the kidneys of mice was assayed over 5 weeks postinfection, and Table 1 shows virus titers in the kidneys when neonatal mice were inoculated with 107 50% tissue culture infective doses (TCID5o) of polyoma virus. Maximum kidney titers for both mutants were reduced by 1 to 3 logs at days 7 and 10 postinfection, as compared with the wild type. The high titer of d117 at day 4 postinfection which was comparable to that of the wild type may have been due to the high input of virus, because when mice were inoculated with 104 TCID5o, kidney titers at day 4 were 106.7, 106.7, and 107 TCID50 per g of tissue for wild-type virus and 104, 105-25, and 104.8 TCID50 per g of tissue for d117. In in vitro work, dll7 was found to have a defect in viral DNA replication, causing a twofold reduction in DNA over a 36-h infection (1). The high initial inoculum of 107 TCID5o may have produced in vivo a situation in which many cells were infected in the first round of replication, producing a similar burst size, and only later in infection was a difference in replicative ability detected. At day 14 postinfection with the low inoculum, titers for both viruses reached the same levels as in mice inoculated with the high inoculum. At day 21 postinfection, there was no significant difference between the virus titers in the kidneys of mice inoculated with d117 or wild-type virus. This may have been due to the high kidney titer (1088 TCID50) of one of the three mice when the other litter mates had titers of 1068 TCID50. Mutant dl75 had a much lower replication potential in vivo than wild-type virus. This mutant produces low amounts of large T antigen (1), which initiates viral DNA synthesis and regulates early mRNA transcription, and therefore shows reduced capacity to replicate efficiently. The host range mutants, 18-5 and 21.2 (Fig. 1; M. Friend and M. Griffiths, personal communication), have mutations in the sequence spliced out of the mRNA for large T but retained in the

VOL. 39, 1981

NOTES small-T

A

'U

=

An

A-

mRNA

An

mRNA

An

mRNA

middle-T

large -T

I-

-

721

Deletion mutants

716

733

_

.

_

-

dl 75 82-9

79 5

1di 17

73-1

82-9±1%

21-2 18-5

_

959

88 2

dl8

91 6

A16

89-7±0 6%

945

916

dl 23

FIG. 1. Diagrammatic representation of the major polyoma virus early mRNA's of productively infected cells and the deletions within this early region. The early region of the genome is shown in linear fashion, with both standard map units (3) and nucleotide number (11) indicated. Above the linear genome are the three early mRNA's. The areas enclosed in boxes are the regions of mRNA's coding for the three early proteins, small, middle, and large T. The intron regions are indicated (=.=t). Below the genome the map units of the deletion mutants are shown (1, 4; M. Fried, personal communication). Dotted lines represent the possible extent of deletions not more precisely mapped. An, poly(A) tails attached to 3' ends of cytoplasmic mRNA's.

TABLE 1. Polyoma virus titers in the kidneys of neonatal mice infected intraperitoneallya Days postinfection

Polyoma virus titersb

Wild-type polyoma (A2 strain)

d117

d175

ND ND 0.1) 8.8, 8.4, 8.4 6.8, 6.5 (P < 0.1) 6.8, 5.5 (P < 0.1) 6.1, 6.5, 7.8 (P < 0.1) 8.0, 8.8, 8.8 ND 6.5, 5.8, 6.5 6.25, 8.5, >8 a Each neonatal mouse was inoculated within 24 h of birth with 107 TCID5o of virus. b The data are expressed as the logarithm of the endpoint dilution of kidney homogenates which were plated on secondary mouse embryo fibroblasts in multiwell dishes and which exhibited a viral cytopathic effect in 50% of the wells 14 days postinfection. Dilutions of kidney homogenates tested were in the range of 10-2 to 10-10. Data are expressed as TCID50 per gram of kidney. Each datum represents the titer for the kidneys of one mouse. ND, Not done. 1 4 7 10 14 21 28 35

mRNA for small and middle T. These mutations result in a normal large T protein but truncated versions of small and middle T proteins, because the deletions cause frameshifts leading to premature termination. These and other host range mutants (2) produce ca. 10 to 100 times less virus per cycle than does the wild type when grown in vitro on continuous lines such as 3T3 cells but are indistinguishable from wild-type virus when primary cell cultures are used (2). Nevertheless, as shown in Table 2, both these mutants grow very poorly in mice, with maximum titers 4 to 5 logs below those for wild-type virus. The third group of mutants have mutations in

the region of the viral genome which codes for both the middle and large T antigens (4). Although the coding regions for the two proteins overlap, different codon reading frames are used to produce distinct polypeptide chains. In vitro replication of d18 is 1 log lower than that of wildtype virus, but the transforming ability of d18 is similar to that of wild-type virus, whereas d123 grows as well as wild-type virus in cell culture, but its transforming ability is impaired (4). Mutant A16 grows as well as wild-type virus in cell culture, and its transforming ability is normal (M. Fried, personal communication). In kidneys of mice, d18 and d123 titers were 2 to 4 logs lower

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than wild-type virus titers (Table 3), whereas A16 grew at least as well as wild-type virus and maintained higher titers during the 4 weeks of this experiment. It would seem that the structure of middle or large T antigen or both is more adversely affected by the deletions in mutants d18 and dl23 than by those in A16. Five (not including host range mutants) of the seven viable mutant viruses were isolated from kidneys of infected animals and grown in tissue culture, after which a restriction enzyme analysis was carried out. All restriction enzyme patterns of viruses isolated from kidneys were those of the inoculated mutant showing that no helper virus was involved. The antibody response by the mice to the polyoma viruses was measured by the hemagglutination inhibition test (7). Antibody titers against all the mutants containing deletions were similar to those against wild-type virus, with titers rising to 10,240 hemagglutination inhibiTABLE 2. Polyoma virus titers in the kidneys of neonatal mice infected intraperitoneallya Polyoma virus titersb Days postin- Wild-type fection polyoma 18-5 21-2 (A2 strain) 1 mM Trishydrochloride [pH 7.51-5 mM NaOALc-1.0 mM EDTA). The DNA was nicked by partialIdepurina(14). This is not Thssno totype to~f nir-ProttpBKviu tion (13), and the material was transferr Md cellulose filters by the Southern blot techinique (12). Filters were hybridized at 68°C with nick -translated (9) 32P-labeled polyoma DNA (specific a ctivity, 2 x to 10 pg of high-molecular-weight DNA (1 genome 108 cpm per pg of DNA) for 48 h and exjposed after equivalent) from an uninfected mouse and restricted EcoRI-rewashing to Fuji X-ray film for 7 days. (ra) Lanes 1 with EcoRL (b) Lanes 1 and 2 containDNA from through 3 contain EcoRI-restricted high -molecular- stricted high-molecular-weight kidney in lanes I and 3 weight kidney DNA from mice inoculated with mu- mice inoculated with mutant A16 as described above, tant A16, and lanes 4 through 6 containt EcoRI-re- of (a). Lane 4 is a reconstruction as stricted high-molecular-weight kidney DNA from and lane m is a form I marker (supercoiled polyoma mice inoculated with mutant dM8. Lane r is a recon- DNA). The arrows indicate the positions of form I, struction in which lOpg ofpolyoma DNA was added II, and III configurations ofpolyoma DNA.

dral

surprising

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because polyoma virus and strains of BK virus do differ most significantly in the early region of the genome (with BK virus having no middle T), and it appears that BK virus can dispense with small T for replication in vitro and persistence in natural hosts. The fact that titers of polyoma virus mutants were reduced in vivo and that no persistence was detected (except for A16) suggests that these viruses would not normally arise and persist in a population of mice. I thank the following for their generous gifts of mutants containing deletions: B. E. Griffin (mutants d123 and d18) M. Fried (18-5, 21-2, and A16), and W. R. Folk (d117 and d175). I also thank R. Kamen for helpful discussion and Valerie Finucane for excellent technical assistance.

LITERATURE CITED 1. Bendig, M. M., T. Thomas, and W. R. Folk. 1980. Regulatory mutants of polyoma virus defective in DNA replication and synthesis of early proteins. Cell 20:401409. 2. Goldman, E., and T. L. Benjamin. 1975. Analysis of host range of non-transforming polyoma virus mutants. Virology 66:372-384. 3. Griffin, B. E., M. Fried, and A. Cowie. 1974. Polyoma DNA: a physical map. Proc. Natl. Acad. Sci. U.S.A. 71: 2077-2081. 4. Griffin, B. E., and C. Maddock. 1979. New classes of viable deletion mutants in the early region of polyoma virus. J. Virol. 31:645-656. 5. Kamen, R., J. Favaloro, J. Parker, R. Treisman, L.

6. 7. 8. 9.

10. 11.

12.

13.

14.

Lania, M. Fried, and A. Mellor. 1980. Comparison of polyoma virus transcription in productively infected mouse cells and transformed rodent cell lines. Cold Spring Harbor Symp. Quant. Biol. 44:63-75. Magnusson, G., and P. Berg. 1979. Construction and analysis of viable deletion mutants of polyoma virus. J. Virol. 32:523-529. McCance, D. J., and C. A. Mims. 1977. Transplacental transmission of polyoma virus in mice. Infect. Immun. 18:196-202. McCance, D. J., and C. A. Mims. 1979. Reactivation of polyoma virus in kidneys of persistently infected mice during pregnancy. Infect. Immun. 25:998-1002. Rigby, P. W. S., M. Dieckman, C. Rhodes, and P. Berg. 1977. Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113:237-251. Rowe, W. P., J. W. Hartley, J. D. Estes, and R. J. Huebner. 1960. Growth curves of polyoma in mice and hamsters. Natl. Cancer Inst. Monogr. 4:189-209. Soeda, E., J. R. Arrand, N. Sinolar, J. E. Walsh, and B. E. Griffin. 1980. Coding potential and regulatory signals of the polyoma virus genome. Nature (London) 283:445-453. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517. Wahl, G. M., M. Stern, and G. R. Stark. 1979. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl paper and rapid hybridization using dextran sulfate. Proc. Natl. Acad. Sci. U.S.A. 76: 3683-3687. Yang, R. C. A., and R. Wu. 1979. BK virus DNA: complete nucleotide sequence of a human tumour virus. Science 206:456-462.