VIRUS GENES 6:2, 107-118, 1992 © Kluwer Academic Publishers, Manufactured in The Netherlands

Simian Virus 40 Mutants with Amino-Acid Substitutions Near the Amino Terminus of Large T Antigen KEITH W.C. PEDEN ~'3 AND JAMES M. PIPAS 2

1Howard Hughes Medical Institute Laboratory, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA 2Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, USA Received May 2, 1991 Accepted June 1, 1991 Requests for reprints should be addressed to Keith W.C. Peden, Laboratory of Molecular Microbiology. Building 4, Room 310, NIAID, NIH, Bethesda, MD 20892, USA.

Key words: SV40, T antigen, amino-terminal mutants

Abstract

A series of amino-acid substitution mutants has been made with changes in the region of simian virus 40 large tumor antigen (T antigen) that is shared with the small tumor antigen (t antigen). Both single and multiple amino-acid replacements were obtained using the heteroduplex deletion loop method and sodium bisulfite as the mutagen. The mutants could be divided into five phenotypic classes on the basis of their biological properties: a) mutants whose changes did not affect their ability to propagate on permissive monkey cells, nor to transform nonpermissive rodent cells; b) mutants that were not viable, replicated their DNA to 5% or less of wild type, but were positive for transformation; c) mutants that were not viable, replicated their DNA to 5% or less of wild type, and were defective for transformation; and d) mutants that completely lost all three activities coordinately. In addition, one mutant with changes in this region, 5002, replicated its DNA to about 50% of wild type, had an impaired transformation activity, and produced virions at a level of about 4% that of wild type.

Introduction

Simian virus 40 (SV40) large tumor antigen is a multifunctional regulatory protein of 94,000 molecular weight that controls virus productive infection, transforms nonpermissive cells in culture, and causes the development of tumors in suscepti-

108

PEDEN AND PIPAS

bte animals (for reviews, see 1-3). These activities are mediated by the biochemical activities of T antigen such as an ATPase (4-6), helicase (7), specific DNA binding (8-11), and binding to the cellular proteins p53 (12,13), DNA polymerase c~(14,15), the retinoblastoma gene product (16), and possibly AP2 (17). The analysis of the effects of mutations in different regions of the T-antigen gene has revealed the presence of several distinct phenotypes: mutants that coordinately lose their ability to propagate in permissive monkey cells, to replicate their DNA, and to transform nonpermissive cells (18); mutants defective for viral DNA replication and productive infection but positive for transformation (19-23); mutants that show a trans-dominant defect for replication but are capable of transformation (24); mutants positive for viral DNA replication but defective for transformation and infectious virion production (25,26); and mutants that have a defect in the viral host-range activity and display a restricted cell type specificity for growth (27,28). So far, no function or activity has been described for the N-terminal 81 amino acids, the region of the T-antigen gene encoded by the first exon and the region shared by large T and small t antigens. Two previously reported mutants have changes in this region. Deletion mutant d11135 (18) and nucleotide substitution mutant C6-1 (19) have defects in productive infection and viral DNA synthesis. To determine the phenotypes of T-antigen mutants with amino-acid substitutions in this region, sodium bisulfite-induced mutations were introduced using the deletion loop heteroduplex method (29), and the resulting mutants were assessed for their biological properties. In this paper, we report the preliminary characterization of this series of mutants and show that alterations in this region of T antigen cause defects in several biological properties.

Materials and Methods

Mutagenesis and DNA sequencing Sodium bisulfite-induced nucleotide substitution mutations were directed to a region near the 5' end of the SV40 early region using the heteroduplex deletion loop mutagenesis procedure (22,29) and the deletion mutants dll135, which is missing SV40 nucleotides 5114-5082 (18), and d/4000, which is lacking nucleotides 5046-4974 (Pipas, unpublished), to target the deamination sites. The basepair substitutions were determined for the 5000 series mutants by DNA sequence analysis according to Maxam and Gilbert (30) and for the 5100 series mutants by the dideoxynucleotide method of Sanger et al. (31) using double-stranded DNA as the template (32). The reaction products were fractionated by electrophoresis in 8% polyacrylamide gels in the presence of 8 M urea (33). The DNA sequence changes associated with each mutant, along with their predicted effect on the primary structure of T antigen, are shown in Fig. 1.

SV40 LARGET-ANTIGEN MUTANTS

109

Plaque assays and marker rescue Growth and titration of wild-type and mutant SV40 on BSC40 and CVl cells was essentially as described (18,22,34,35). Marker rescue was done as described (I9,22,36) using the SV40 HaeIII-E fragment (nucleotides 5234-4862), the HindIII-B fragment (nucleotides 5171-4001), and the HaeIII-A fragment (nucleotides 3201-4862).

Viral DNA replication, transformation assays, and indirect immunofluorescence Replication of viral DNA was assessed by the Dpnl-resistance assay as described (37). The focus assay was used to assess the ability of the mutants to transform nonpermissive rodent cells (38). Indirect immunofluorescence was done (18,39) using a hamster polyclona] anti-T serum (40) and mouse monoclonal antibody PAb419 (41).

Results

Sequences of the amino-terminal mutants In the mutants directed by d11135 (5000 series), seven had single amino-acid substitutions, while five mutants carried multiple substitutions (Fig. 1A). Two of the mutants, 5033 and 5034, had mutations that converted Trp(24) to a TGA or a TAG nonsense codon, respectively. These two mutants were not studied further. For the dl4000-positioned mutants (5100 series), five had single amino-acid substitutions, and 12 had multiple changes (Fig. IB). All of the nucleotide changes within the targeted regions, which correspond to amino-acid residues 17-28 (5000 series) and 40-64 (5100 series) of the small and large T antigens, were C to T transitions, consistent with bisulfite mutagenesis. Four activities of the mutants were measured in order to categorize their phenotypes. These include the ability to produce a T antigen detectable by immunofluorescence; to plaque on permissive BSC40 cells at 32°, 37°, and 40°C; to replicate viral DNA; and to induce foci in nonpermissive REF52 cells.

Indirect immunofluorescence of transfected BSC40 cells Indirect immunofluorescence with a hamster polyclonal anti-T-antigen serum and the mouse monoclonal antibody PAb419 was used to assess whether the mutants produce T antigens and to determine their cellular location. Plasmid DNAs from the mutants were transfected into BSC40 cells using the DEAE dextran method,

1 I0

PEDEN AND PIPAS

5115

5081

J

i

CTA.GGT.CTT.GAA.AGG.AGT.GCC.TGG.GGG.AAT.ATT.CCT GAT.CCA.GAA.CTT.TCC.TCA.CGG.ACC.CCC.TTA.TAA.GGA (17)

Leu Gly Leu Glu Arg Ser Ala Trp Gly Ash Ile Pro

(28)

Mutant Number 5OOl

T Set

5002

T Phe

T

5003

T Val

5004

5005

A

AA Ash

5006

A

A

AA

A A A Lvs Ash Thr A

A

AAA Lvs

A Thr

~

5007

A Thr

5008

5009

A

A

A

A a~g

A Arg

A Gly

5010

5011

AA

A A fiJ-U

5012

5033

A Lys

5034

A Ara

A Ash

A Thr

A Ter

AA Glu

A Ter

A Fig. 1 A: Sequences o f point mutants isolated using d11135 as template. B: Sequences o f point mutants using d14000 as the template. The nucleotide sequence o f wild-type SV40 DNA, together with the corresponding amino-acid sequence o f T antigen, are shown across the top. The positions o f the deletion end points are indicated (nucleotides 5115 and 5081 for dl1135 and 5047, and 4973 for d14000), and mutant designations are given in the left column. All mutations are shown, but the resulting amino-acid substitutions are indicated by underlining. For the mutations directed by dt1135 (500 series), nucteotide substitutions were ascertained by DNA sequence analysis according to Maxam and Gilbert (31) after labelling at the Hindlll site at nucleotide 5163 as described (22). For those directed by d14000 (5100 series), the procedure o f Sanger et al. (32) as adapted to plasmid

SV40 LARGE T-ANTIGEN MUTANTS

111 4973

5047 1

1

AAG.GAG.T•T.CAT.CCT•GAT•AAA.GGA.GGA•GAT.GAA.GAA.AAA•ATG.AAG•AAA•ATG•AAT•ACT.CTG•TAC.AAG.AAA•ATG.GAA.GAT TTC.CTC.AAA.GTA.GGA.CTA•TTT.•CT.CCT.CTA.CTT.CTT.TTT.TAC.TTC•TTT•TAC•TTA.TGA.GAC.ATG•TTC.TTT•TAC•CTT.CTA (39) Lys Glu Phe His Pro Asp Lys Gly Gly Asp GIU Glu Lys Met Lys Lys Met ASh Thr Leu Tyr Lys Lys Met Glu Asp (64) Mutant Number

A

5101

A Leu T

5105

T

5107

T iI~

Ser

5108

5109

A

A

A ~U

k

L~ 5110

5113

5115

A Lys

TT

Ty~

A

A

5116

5118

5119

A

512o

5121

A

A

A

~

Lvs L~m

A

A

Ash

Lyre iJm

A

A ~ AA

A

~

~d~ ~

GI~

AA

A

A 16~

A

i~

L~

A L~

ZI~

A

A

A i~

A Lys

A

A L~

A

A A Ash i ~

~

5117

A L~U

A Lys A ~

GI~

5114

A ii~

A ArS

A

Glu 5112

A Lys

A ~n

A

Glu 5111

A

A Ash

A ~

A

A ~ A A Glu ~

A Lys

A Leu

A

Lys

A

A L~ A

L~ A ~

A

A

Lys A A ]J~ Lys

A Lys

A

A

Lys

Ueu

A Lys A Lvs

A lys

A

A

A

A IALa

]~

A A lie Lys

A

I~

A L~s

A ~/~

B sequencing (33) and with [c~JSS]dATP (55) was used with an oligonucleotide whose sequence was derived from SV40 nucleotides 5144-5164. Reagents were purchased as a kit from Promega Biotec (Madison, WI), and reaction products were fractionated by electrophoresis in 8% polyacrylamide gels containing 8 M urea (34).

and 24 and 48 hr later the cells were fixed and immunostained as described before (18,22). All mutants produced nuclear fluorescence, although the intensity varied among them. The reason for this variability is not known but most likely reflects the varying stability of the mutant T antigens.

Plaque assay, marker rescue, and complementation

analyses

Table 1 shows that nine mutants of the 5000 series (5001, 5003, 5006, 5007, 5008, 5009, 5010, 5011, and 5012) were able to plaque on BSC40 cells and two (5004

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PEDEN AND PIPAS

Table 1. Infectivity and transformation activities of SV40 T antigen mutants

Infectivity and complementation (pfu/dish)

Transformation of REF52 cells

Mutant

Alone

+ dllO07

Number of foci per dish

Percentage of wild type

Wild type 5001 5002 5003 5004 5005 5006 5007 5008 5009 5010 5011 5012 Wild type 5101 5105 5107 5108 5109 5110 5111 5112 5113 5114 5115 5116 5117 5118 5119 5120 5121

164, 191 123, 127 0,0 b 154, 133 0, 0 0, 0 98, 88 185, 188 121, 157 91, 94 54, 48 39, 40 47, 61 a >200, >200 >200, >200 >200, >200 0, 0 0, 0 0, 0 0, 0 0,0 0, 0 0, 0 0, 0 0,0 0,0 0,0 0,0 0,0 0, 0 0, 0

N.D. >200, >200 115, 129 >200, >200 >200, >200 133, 145 111, 160 156, 195 >200, >200 112, 160 74, 55 64, 56 56, 81 N.D. >200, >200 >200, >200 33, 45 43, 53 40, 46 51, 62 63,65 64, 40 50, 47 37, 52 61, 77 45,44 48,44 71, 52 60,58 55, 59 45, 48

72, 65, 52, 41 14, 5, 8, 11 0, 0, 0, 0, 48, 61, 62, 50 0, 0, 0, 0 0, 0, 0, 0 23, 10, 12, 14 54, 57, 49, 65 6, 8, 10, 15 22, 9, 27, 11 20, 19, 21, 13 68, 53, 46, 49 2, 4, 6, 2 86, 79, 76 55, 52, 54 44, 47, 45 56, 58, 49 42, 46, 44 0, 0, 0 17, 19, 18 2, 9, 4 21, 17, 19 32, 20, 15 22, 17, 11 0, 0, 0 0, 0, 0 0, 0, 0 0, 0, 0 0, 0, 0 0, 0, 0 0, 0, 0

100 17 0 96 0 0 26 98 17 30 32 94 6 100 67 55 67 55 0 32 6 24 28 21 0 0 0 0 0 0 0

N.D. = not done. aSmall plaque morphology. bVery small plaques appearing 7 days late.

and 5005) were defective for this activity. Mutant 5002 forms very small plaques approximately I week late. The biological and biochemical properties of this mutant have been described (26). For the 5100 series, only mutants 5101 and 5105 were viable, and 15 were defective. In all cases, the defective mutants were complemented by the late region deletion mutant d/1007 (42), and, therefore, none was similar to the trans-dominant defective class of mutants described previously (24). N o n e of the mutants was found to have a temperature-sensitive defect. The mutations were rescued by the Hind-III-B fragment (nucleotides 4002-5171) and

113

SV40 L A R G E T - A N T I G E N M U T A N T S

Wild Type ab

c

d

5111

5112

5117

5116

b c d a b c d a b c d

a b

5119

c d a b

c d

wWu

o ~

~

91~

O

SV40

O

O

~10o7

I

~

I

IIQa w~

8

Fig. 2 Replication o f SV40 mutant DNA in monkey cells. Subconfluent BSC40 monolayers were transfected with wild-type and mutant DNA (100 ng) with and without dllO07 DNA (100 ng). Hirt DNA was prepared at different times after infection, purified, digested with D p n / a n d Bcl/, fractionated by electrophoresis on a 1.2% agarose gel, transferred to nitrocellulose, and hybridized to J2P-labelled SV40 DNA (35,36), DNA was extracted at the foUowing times after tran~fection: 24 hr (lanes a), 48 hr (lanes b), 72 hr (lanes c), and 48 hr with dltO07 (lanes d). Mutant designations are given across the top, and the migration position o f fidt-length and d11007 are indicated on the right.

the HaelII-E fragment (nucleotides 4862-5191), but not by HaelII-A (nucleotides 3201-4862), thus localizing the mutations between nucleotides 4862 and 517I (data not shown). The genome between nucleotide 5191 and the large T-antigen gene splice at nucleotide 4917 was sequenced for all the mutants. Only the nucleotide substitutions shown in Fig. 1 were found.

Viral DNA replication Each mutant was tested for its ability to replicate DNA in permissive monkey cells as described previously (22,38). Viral DNA was freed from plasmid sequences by digestion with BamHI, diluted and cyclized with T4 DNA ligase, and introduced to subconfluent monolayers of BSC40 cells by DEAE dextranmediated transfection. Replicated viral DNA was detected as DpnI-resistant molecules after hybridization. All of the 5000 series mutants were positive, although 5004 and 5005 replicated their DNA to

Simian virus 40 mutants with amino-acid substitutions near the amino terminus of large T antigen.

A series of amino-acid substitution mutants has been made with changes in the region of simian virus 40 large tumor antigen (T antigen) that is shared...
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