Genetic map of the vaccinia virus Hindlll D fragment

160,

VIROLOGY

11 O-1 19 (1987)

Genetic

Map of the Vaccinia

JANNY SETO, LYN M. CELENZA, Biochemistry

Department,

State

Received

March

RICHARD University 30,

Virus HindIll C. CONDIT, of New

1987; accepted

York, May

D Fragment AND

Buffalo,

EDWARD New

York

G. NILES’

142 14

4. 1987

Seventeen ts mutants of vaccinia virus known to map to the viral HindIll D fragment (R. C. Condit and A. Motyczka, 1981, Virology 113, 224-241; R. C. Condit, A. Motyczka, and G. Spizz, 1983, Virology 128, 429-443; M. J. Ensinger and M. Rovinsky, 1983, J. Viral. 48, 419-428) have been sorted into seven complementation groups. The precise location of each mutant on the Hindlll D DNA fragment has been identified by either one-step or two-step marker rescue. By a comparison of this genetic map and the known sequence of this DNA fragment (E. G. Niles et a/., 1986, Virology 153,96-l 12; S. L. Weinrich and D. E. Hruby, 1986, Nucleic Acids Res. 14,3003-3016) each mutant has been assigned to a single gene in the HindIll D fragment. In several cases, the map position of a mutant has been localized to a region of fewer than 300 bp in length. The complementation groups are evenly distributed along the DNA. However, within a single gene, the mutants are often clustered. o 1987 Academic PVSSS. I~C.

INTRODUCTION

Moss et a/., 1981; Panicali and Paoletti, 1982; Drillien et al., 1981; Perkus et al., 1986;) and, in addition, drugresistant mutants have been isolated: 5-bromodeoxyuridine, Dubbs and Kit (1964); rifampicin, Moss et a/. (197 1); isatin-&thio-semicarbazone, Katz et a/. (1973); phosphonoacetic acid, Sridhar and Condit (1983); 2’0-methyladenosine, Raczynski and Condit (1983); CYamanitin, Villarreal et al. (1984); aphidicolin, Defilippes (1984). Based on these collections of virus mutants, recombination maps have been generated and in some cases, these genetic maps have been correlated with gross physical mapping studies (Ensinger and Rovinsky, 1983; Drillien and Spehner, 1983). With the availability of a detailed physical map of a segment of the genome, it has become feasible to attempt to associate each individual ts mutant with a single gene within that region. This approach has been successfully carried out with eight ts mutants from four complementation groups derived from the /-/indIll LJH region of the genome (Ensinger et a/., 1985). The initial goal of this project is to develop a coordinated genetic and physical map of the 16-kb vaccinia virus HindIll D fragment. To this end, we have determined the base sequence of the DNA and have identified 13 complete open reading frames (ORFs) (Niles et al., 1986; Weinrich and Hruby, 1986). Furthermore, we have undertaken the transcript mapping of each gene and have identified genes expressed early or late after infection (Lee-Chen et a/., in preparation). In this report, we present the detailed genetic map of the HindIll D fragment generated by marker rescue analysis of the 17 ts mutants previously mapped to this region of the viral genome (Condit era/., 1983; Ensinger and

Vaccinia virus, a member of the poxvirus family, possesses a double-stranded DNA genome of 180,000 bp in length, which has the capacity to encode about 200 different proteins. The vaccinia life cycle takes place in the cytoplasm of infected ceils, and as a result, vaccinia expresses its genes independently of the host nucleus. The virus encodes its own enzymes required to carry on viral transcription and RNA processing, and also, many if not all of the enzymes needed for viral DNA replication (Moss, 1985). During the past several years, substantial progress has been reported in describing the structure of the viral genome. The base sequence of about 40 kb of DNA in the well-conserved central region has been reported (Plucienniczak et al., 1985; Broyles and Moss, 1986; Rose1 et al., 1986; Niles et al., 1986; Weinrich and Hruby, 1986). Detailed transcript mapping has been carried out, yielding a working definition of the structure of early and late viral genes. A genetic approach to analyzing this virus has also been taken. Collections of ts mutants have been developed for both vaccinia (Chernos et al., 1978; Dales et a/., 1978; Condit and Motyczka, 1981; Condit et al., 1983; Drillien et a/., 1982; Drillien and Spehner, 1983; Ensinger, 1982; Ensinger and Rovinsky, 1983) and the closely related rabbit poxvirus (Sambrook et a/., 1966; Padgett and Tomkins, 1968; Lake and Cooper, 1980). Deletion and insertion mutants have been described (Fenner and Sambrook, 1966; Moyer and Rothe, 1980; ’ To whom

0042.6822/87 Copyright All rights

requests

for reprints

$3.00

Q 1987 by Academic Press. Inc. of reproduction in any form reserved.

should

be addressed.

110

VACCINIA

VIRUS

Rovinsky, 1983; Niles et al., 1986) and assign complementation group to a single gene.

HindIll

each

METHODS Cells and viruses The virus employed in this study were isolated and characterized previously (Condit and Motyczka, 1981; Condit et a/., 1983; Ensinger, 1982; Ensinger and Rovinsky, 1983). Virus stocks were prepared from BSC 40 cells infected at 31°, as described (Condit and Motyczka, 1981) and stored at -70”. Plasmids containing DNA fragments from the /-/indIll D region of vaccinia were constructed as described (Niles et a/., 1986). Ml 3 recombinants were derived either by shotgun cloning from subfragments of the HindIll D fragment or by the exonuclease III deletion method of Henikoff (1984) and were used in the DNA sequence analysis of the HindIll D fragment (Niles et al., 1986). The endpoints of the DNA inserts in the M 13 recombinants were determined either by DNA sequence analysis or by size analysis of the DNA fragment released by restriction enzyme digestion of the RF form of the phage DNA. Complementation

test

The spot complementation test described by Condit and Motyczka (1981) was used to test the ability of virus E52 and E94 complement. Marker

rescue

For virus mutants which are not leaky and have a low reversion frequency, the one-step marker rescue protocol described by Thompson and Condit (1986) was followed. Virus were diluted to a point at which the monolayer remains intact after infection and incubation at 40” for 3 days. Infected cells were transfected with 10 pg of linearized plasmid DNA or 30 pg of singlestranded phage DNA, and after 4 days at 40”, the dishes were stained with 0.1% crystal violet, and the plaques were counted. The monolayers often have a mottled character which in some cases makes exact plaque counting difficult. In addition, the plaque sizes are often heterogeneous and this contributes to the problem. As a result, in cases where these problems are apparent, the data that are presented are rounded off to the nearest 5 for numbers less than 50 and to the nearest 10 for numbers greater than 50. For all of the nonleaky viruses tested, the number of plaques formed at 40” on control dishes in the absence of DNA

D FRAGMENT

GENETIC

MAP

111

was 6 or fewer, most often 0, and presented no problem in assigning a map position. For leaky mutants, those which yield measurable virus at 40” on control dishes, a two-step protocol was followed. In these cases, cells were coinfected at m.o.i. of 0.1 and transfected with 10 pg of plasmid DNA. After 4 days, the virus was harvested and titered at 40”. The nomenclature which identifies these complementation groups has evolved since the mutants were first isolated (Condit and Motyczka, 1981; Ensinger, 1982; Condit et a/., 1983) and subsequently sorted into groups (Niles et a/., 1986). Since we now have identified the precise map location of each group, we will refer to each group by the gene in which it is located. As an example, what had previously been complementation groups 1 and 2 are now Dll and D5. In accordance with the proposal of Rose1 et al. (1986) the genes in the /-/indIll D fragment will be named Dl to D13, numbered from left to right on the physical map. We had previously reported that there are 14 open reading frames in the HindIll D fragment based on a DNA sequence 16059 bp in length (Niles et a/., 1986). Weinrich and Hruby (1986) reported a DNA sequence which contains the right 3 kb of the HindIll D fragment. They found that we overlooked a single C at position 13280 which results in a frame-shift mutation generating two open reading frames from one. Our ORFs 1 1 and 12 are actually a single gene, Dl 1. Our ORFs 13 and 14 are now genes D12 and D13. Our genes Dl 1, D12, and D13 are the same as ORFs 5, 4, and 3, respectively, of Weinrich and Hruby (1986). RESULTS The mutants viruses employed in this study were derived from several complementation groups in two collections of temperature-sensitive mutants (Condit and Motyczka, 1981; Condit et al., 1983; Ensinger, 1982). Members of each group, in each set, were tested in the spot complementation test and sorted initially into eight complementation groups (Niles eta/., 1986). During the course of this work, two mutants E52 and E94 were shown to be closely linked. In order to determine if the mutants are in the same gene, the spot test was again employed. The two mutants were shown to be noncomplementing and therefore in the same group (Fig. 1). As a result, the 17 ts mutants can be categorized into seven complementation groups (Table 1). The precise physical map position of each member of each complementation group was determined by marker rescue. For stable mutants, a single-step rescue protocol was followed (Thompson and Condit, 1986);

112

SET0

ET AL

ES4

FIG. 1. The results of the spot complementation test between the mutants serially diluted and one drop of each dilution was used to infect a monolayer, (Thompson and Condit, 1986). One drop of the highest dilution of each virus each dish. The dishes were incubated as above, and plaques were observed not complement each other but each complements the mutants C5 and C35.

for leaky mutants, a two-step approach was employed (Sridhar and Condit, 1983). Several DNA fragments were used in the rescue analysis of each mutant. Linearized plasmid DNA was often satisfactory, but for precise mapping, single-stranded circular DNA from the Ml 3 recombinants, generated during the sequencing phase of the project, were employed. A typical example of the rescue results employing either plasmid or M 13 DNA is presented in Figs. 3C and 4, for the mutant C21 from complementation group D7. The mapping results for the individual complementation groups are presented below. Complementation group D2: Group D2 mutants display a normal pattern of protein and nucleic acid synthesis during infection at 40’ (Ensinger, 1982). Two mutants, E52 and E94, can be rescued by the plasmids 791, 790, 775ab, and 775b2 (Fig. 2, Table 2). Further mapping was carried out with single-stranded DNA from M 13 recombinants in order to refine the positions

E52 and E94 are presented. Prior to the test, the virus were and after 4 days at 40” they were stained with crystal violet which permitted the monolayer to remain intact was added to after staining with crystal violet. The mutants E52 and E94 do

of these mutants (Fig. 3A, Table 3A) Mutants E52 and E94 are rescued by DNA from phage ,009 and .032 which places the lesions between 2533 and 3001, in the region of gene D2. However, the coding sequence for gene Dl extends to 2637, which is a substantial degree of overlap. In addition, a minor ORF a is located between 265 1 and 2891. In order to identify the location of the group D2 mutants, DNA sequence analysis is being carried out on mutants E52 and E94. Preliminary results have identified three base changes at 2663, 2668, and 2691 (Celenza). All three are to the right of Dl, eliminating this gene as the site for these mutations. However, they are in a region shared by D2 and ORF a. Gene D2 is transcribed both early and late after infection. There is also evidence for late expression of ORF a (Lee-Chen et a/., in preparation). Complementation group D3: Group 03 mutants are capable of carrying out normal levels of protein and nucleic acid synthesis at 40” (Condit et al., 1983). The

VACCINIA TABLE

Mutantb

HindIll

1

CHARACTERISTICS OF THE ts MUTANTS IN THE HindIll D FRAGMENT’

Complementation group

VIRUS

WHICH

MAP

Phenotype”

D2

E52 E94

Normal

D3

c5 c35

Normal

D5

Cl7 C24 E69

DNA-

D6

C46 E93

Normal

D7

c21 E45

Defective

late

Dll

c50 C36 El7

Defective

late

D13

c33 c43 El01

Normal,

rif

a ts mutants were isolated in the Condit and Ensinger laboratories and their phenotypes described (Condit et a/., 1983; Ensinger, 1982). b C, Condit; E, Ensinger. c The phenotypes are based on the rates of protein and DNA synthesis in mutant-infected cells at the nonpermissive temperature: defective late, normal early protein synthesis and DNA replication but abnormally low levels of late protein synthesis; DNA-, low levels of DNA synthesis and no late protein synthesis; normal, normal levels of early and late protein synthesis, Gene D13 was shown to be the locus for rifampicin resistance by Tartaglia et al. (1986).

mutants C5 and C35 are rescued by the plasmids 791, 775ab, and 775a3 (Fig. 2, Table 2). Single-stranded DNA from the M 13 recombinants ,019 and .028 also rescue both mutants which places the lesions between 3109 and 3412, within gene D3 (Fig. 3A, Table 3A). This gene is expressed only at late times after infection (Lee-Chen et al., in preparation). Complementation group D5: Group D5 mutants exhibit a DNA- phenotype (Condit et a/., 1983; Ensinger, 1982). Mutants in this group are rescued by plasmids 787 and 738 which places them between 4779 and 6080, within the central region of gene D5 (Fig. 2, Table 2). Plasmids 822 and 823 rescued each of these mutants poorly, suggesting that the mutations lie near to the Sal1 site at 5956. Gene D5 is transcribed early after infection (Lee-Chen et al., in preparation). Complementation group D6: Mutants in this group also exhibit normal DNA and protein synthesis at 40”

D FRAGMENT

GENETIC

MAP

113

(Condit et al., 1983; Ensinger, 1982). E93 is rescued to wild-type phenotype by the plasmids 797, 731, 823, and 823a in a single step (Fig. 2, Table 2). The mutant C46 is very leaky and requires a two-step rescue protocol in order to assign its position to 823a. This fragment extends from 6637 to 7110 on the physical map. The coding region of gene D5 extends to 6787 and that of D6 begins at 6828. In order to distinguish between these two genes, single-stranded M 13 recombinant DNA was used in a single-step rescue (Fig. 3B, Table 3B). The mutant E93 is rescued by phage ,154 DNA which contains an insert that extends from 6825 to 7045, entirely within the 5’ terminal region of D6. Gene D6 is expressed late after infection (Lee-Chen et a/., in preparation). Complementation group D7: Group D7 mutants possess a defective late phenotype (Condit et a/., 1983). The mutant C21 is rescued in a single-step protocol by plasmids 770, 801, and 801 b (Fig. 2, Table 2). A two-step protocol was employed to map the leaky mutant E45 to the fragment in the plasmid 801 b. These data place the mutations within the region between 8703 and 9242. In addition to gene D7, this fragment possesses 39 bp from the 3’ region of gene D6 and 30 bp from the 3’ end of gene D8. in order to refine the map position of the group D7 mutants, C21 was mapped by a one-step rescue protocol using Ml 3 phage recombinant single-stranded DNA (Figs. 3C, 4, Table 3C). The mutant C21 is rescued by the phage ,028 DNA which contains an insert that extends from 8920 to 9242, thus eliminating gene D6 as the location for group 3 mutants. Based on the number of possible bases of D8 in the plasmid 801 b, it is unlikely that the

I

234

5

6

7

6

9

IO

II

I2

I3

GENE

8

791 790 7610

w

d?Lef 787 622.

329a 7uwd In9

E

767 -

%I

770 601

%CG 722 776 906 -BQsts

239-L e(tplb

AL 262

FIG. 2. The results of the mutant rescues are presented. The length of the HindIll D fragment is 16,060 bp. Each gene is labeled with the number 1 to 13, and the lengths of their coding regions are drawn to scale. Early genes are filled in, late genes are stippled, and those that are expressed both early and late are lined (Lee-Chen et al., unpublished). The open boxes below the genes are the sizes of the smallest DNA fragments capable of rescuing the mutants to the wild-type phenotype. The numbers above the boxes refer to the complementation group. The numbered lines below the boxes indicate the map positions of the plasmid DNA used to rescue these mutants.

114

SET0

SUMMARY Complementation group

TABLE 2 OF PLASMID RESCUE DATA~

Plasmid

D2 791 790 791a 775ab 775b2 775a3 0 D3 791 790 791a 775ab 775b2 775a3 0 D5 787 738 822 823 0 D6 797 801 731 823 823a 0 D7 770 801 801a 801b 0 Dll 722 810 762 776 0 D13 722 776 * 806 806bx 810 0

Plaques/dish E52 200 75 0 50 20 0 0 c5 r500 3 0 500 0 50 0 Cl7 C24 200 200 6 6 2 3 0 0 0 0 E93 300 0 25 15 8 0 c21 100 30, 4 x 1 04** 1 ND 6, 1 X 103** 1, cl c50 C36 100 6 50 ND 20 6 0 0 0 0 c33 c43 200 200 200 150 100 40 20 ND 0 4 0 0

or PFU/ml E94 150 20 5 50 6 0 0 c35 150 0 0 50 0 15 0 E69 200 1 1 2 0 C46 NDb ND ND 7 x 1 05** 4 x 1 05** 6 x 1 04** E45 L 3 x 1 05** ND 1 x 105** 0 El7 20 10 ND 0 0 El01 75 50 25 ND 1 0

a In an efficient single-step rescue, the exact plaque count is often difficult to determine. As a result, for dishes that are difficult to count, the plaque count is rounded off to the nearest 5 in the plates with fewer than 50, and to the nearest 10 for plates with greater than 50 plaques. ’ Not determined. * This sample was rescued in a separate experiment with a control of 0 plaques. ** These values were derived from two-step rescues.

ET AL

two group 3 mutations lie in gene D8. Gene D7 is transcribed early after infection into two abundant mRNA species (Lee-Chen et al., in preparation). Complementation group Dl 1: Group Dl 1 mutants possess a defective late phenotype (Condit et al., 1983). In a single-step rescue, these mutants can be converted to the wild-type phenotype by DNA fragments derived from the plasmids 776, 810, and 762, which places the mutants between 12,872 and 13,628, either in genes Dl 1 or D12 (Fig. 2, Table 2). In order to distinguish between these two possibilities, singlestranded DNA from M 13 recombinants were employed in a single-step rescue (Fig. 3D, Table 3D). In addition to other phage, the mutants are rescued by DNA from phage .030 and .128. This places the mutations in the 5’-end region of ORF 11 between 13,219 and 13,484. This gene is transcribed late after infection (Weinrich and Hruby, 1986). Complementation group D13: Group D13 mutants display a normal pattern of protein and nucleic acid synthesis at 40” (Ensinger, 1982; Condit et al., 1983). Since these mutants are rescued by the plasmids 722, 776, 806, and 806bX (Fig. 2, Table 2) they must lie between 14,71 1 and 16,060, in the region of D13. This gene is expressed late after infection (Weinrich et al., 1985; Tar-taglia and Paoletti, 1985) and is the site of resistance to rifampicin (Tartaglia et al., 1986). DISCUSSION The initial goal of this project is to construct a combined genetic and physical map of the vaccinia virus HindIll D fragment. We have completed the DNA sequence analysis of this fragment and have identified 13 complete ORFs (Niles et a/., 1986; Weinrich and Hruby, 1986). Transcript mapping studies are approaching completion and each gene can be categorized according to its time of expression and the location of the 5’ and 3’termini of the steady-state mRNA (Morgan et a/., 1984; Tartaglia and Paoletti, 1985; Weinrich et al., 1985; Weinrich and Hruby, 1986; LeeChen eta/., in preparation). In this report we have completed the genetic mapping analysis of the 17 temperature-sensitive mutants known to be located in this DNA fragment (Condit et a/., 1983; Ensinger and Rovinsky, 1983; Niles et a/., 1986). Complementation testing had previously sorted the mutants into eight groups (Niles et al., 1986). The marker rescue data placed two mutants, E52 and E94, in a single 514-base fragment which contained parts of two major and one minor ORF. Since this brought the previous complementation testing results into question, we retested mutants E52 and E94 (Fig. l),

VACCINIA

VIRUS

2533

3L99

GENE

01

3z%2-.2 3464

02

D3 I

I 035 032 ,030 029 028 025

D FRAGMENT

009 ol4 ,013 oq

2827 2569

L?!%

HindIll

GENETIC

115

B I

66 I

6.6 I

I

7.0 I

I

I

72 I

kb

cng

%I

“IC

L

2sse

Ff

I I

GENE

06

05 3001 3lb3 3lM

--. 34r2 3744

C

,222 ,113 ,068 ,030 .049 ,127

D6

GENE

D7

80lb

.ooe oze

-

_____________ . . . ..

-_

006

,203 ,011 ,209 ,067 ,128 .I02 224

I. 1. L... -. L. h....

I(

L..... L 1

0

SENE

.. ..A ...A -I .I I

FIG. 3. The sizes and the map positions of the DNA Inserts in the single-stranded M 13 recombinant In each case, the solid lrne represents the region of each phage DNA which was sequenced. The extent of the insert which was not sequenced but was estimated to be part of the recombinant phage of the DNA cloned into the RF form of each phage DNA. For the phage in (A), a solrd line is drawn. sequenced. This is due to the fact that each phage was generated by the exonuclease Ill procedure known in each case. Each phage possesses a three-digit number and a prefix which is left out of the open boxes represent the genes present in each region under consideration. drawn to scale.

and we demonstrated that they are noncomplementing under these conditions. As a result, there are seven complementation groups in this region of the viral genome (Table 1). Marker rescue was employed to locate the position of each mutant on the physical map. In most cases a single-step protocol was sufficient, but for two leaky mutants, a two-step rescue was required. Often the use of double-stranded plasmid DNA was sufficient to locate the map position of a mutant (Table 2). However, in some cases, convenient plasmids were not available, and as a result, single-stranded DNA from M 13 recombinants were employed (Table 3A-D, Figs. 3A-D). This approach was particularly useful since a collection of M 13 virus containing overlapping fragments whose endpoints either were known or could be closely estimated, were available from the sequencing phase of this project. In one case in which two coding regions

6230 Ix57 .I 54 .I I I

Ml3 .a4 ,711

B

MAP

phage used in these rescues are drawn. dotted portion of each line indicates the DNA based on the analysis of the length even though the entire insert was not of Henikoff (1984) so the distal end was drawing but is described in Table 3. The

share a common sequence, gene D2 and ORF a, marker rescue is unable to assign a map position. As a result, the DNA sequence of the mutant viruses in group D2 must be determined. (See Table 4 for a summary of map position of the mutants in HindIll D fragment.) The rescue results are satisfying (Fig. 2). Mutants in each complementation group rescue to the same gene. The groups are evenly distributed along the DNA. Within a large gene, mutants in a single group are often clustered, i.e., group Dl 1, in gene Dl 1. We have assumed that the efficiency of rescue of a ts mutant would be dependent on both the size of the rescuing DNA fragment and the position of the mutation in respect to the ends of the rescuing DNA fragment. The results in both Tables 2 and 3 show that while larger DNA fragments do yield a greater number of plaques, double-stranded DNA fragments as small as

116

SET0 TABLE SINGLE-STEP

3

TABLE

RESCUES USING SINGLE-STRANDED VIRUS RECOM~INANTS~

A. Complementation

groups

,035 ,032 ,030 ,029 .028 ,025 ,009 ,014 ,013 .019 .017 ,018 ,020

M 13 virusB

D2 and D3

E52

E94

c5

c35

4 110 120 ND ND ND 150 4 4 ND ND ND ND

2 25 96 ND ND ND 300 6 5 ND ND ND ND

ND

ND

6 8 3 46 55 ND ND ND 54 2 ND ND

8 2 2 29 44 ND ND ND 29 11 3 ND

4

0

2

6

>500 22 4 ND

>500 20 0 z-500

B. Complementation

>500

>500 0

>500 ND group

6 8 0

,057 .154 ,111 Plasmids

17 7 0

823 823a 0 C. Complementation Ml3 virusd ,008 ,028 .006

group

D7

Plaques/dish c21 200 150 0

Plasmids 801 810b 0

Dl 1

3 120 ND

,014 ,711 ,222 ,113 ,068 ,030 .049 ,127 .203 ,011 ,209 .067 .128 ,102 ,224 0 Note. ND means that this B Single-step rescues were per dish. * Each virus has the prefix the Table. c Each virus is named with d Each virus has the prefix e Each virus has the prefix the Table.

c50 75 50 30 15 10, 5, 3, 1, 2 1 6. 1. 4, 6, 20,

El7

7 7 4 1

3 0 15 40 60

2, 2

15 20 25 10 10 10 1 1 ND ND 1 2 4 8 25 2

rescue was not attempted. carried out using 30 pg of M 13 DNA R1220EX

prior to the number

listed

in

the prefix R13400. R13600 before the number in the Table. BM3205 prior to the number listed in

D6

Plaques/dish E93

Ml3 virus=

group

Plaques/dish

Plasmids 0 775ab 775b2 775a3 791

3-Continued

D. Complementation

M 13

Plaques/dish Ml3 virusb

ET AL.

250 77 0

500 bp and single-stranded M 13 phage recombinants with inserts as small as 300 bases rescue quite well, in a single step. Clearly, although either of these factors may have an effect on the rescue efficiency, satisfactory results can be obtained with very small DNA fragments. These results assign a number of interesting phenotypes to specific genes and gene products. Mutants in two complementation groups exhibit a defective late phenotype (Table 1) in which early genes are expressed at normal levels but, at 40°, late gene expression is lower than that found in wild-type infected cells (Condit and Motyczka, 1981; Condit et al., 1983). Group Dl 1 mutants map to the 5’ end of gene Dl 1 (Fig. 2). This gene was recently shown to encode the nucleic aciddependent ATPase activity (Rodriguez et a/., 1986). The function of this enzyme is yet to be determined but it has a phenotype which suggests that it either directly or indirectly plays a role in late gene expression. It is interesting to note that the loss of its activity has no apparent effect either on the expression of early genes or on viral DNA replication (Condit and Motyczka, 1981; Condit et al., 1983). Several lines of evidence demon-

VACCINIA

plasmid

VIRUS

HindIll

0

.008 thaw FIG. 4. A typical example of the results of a single-step is presented. The whole numbers refer to plasmid DNA. in Table 36.

SUMMARY

Complementation group D2 D3 D5 D6 D7 Dll D13

4

OF THE MAP POSITIONS OF THE MUTANTS IN THE HindIll D FRAGMENT

Gene” 2 3 5 6 7 11 13

GENETIC

MAP

801

801b

.028

,006

rescue employing either double-stranded plasmid DNA or single-stranded The decimal numbers identify the M 13 phage DNA used. The results

strate that this gene is expressed at late times after infection (Paoletti et a/., 1974; Rodriguez et a/., 1986; Weinrich and Hruby, 1986). Mutants in group D7 map to gene D7 (Fig. 2). It is likely that 07 encodes one of the small subunits of the viral RNA polymerase. Jones and Moss (unpublished

TABLE

D FRAGMENT

Endpoints

Midpoint

2,661-2,691 3,109-3,412 4,779-6,080 6.828-7.045 8,920-9,242 13,219-13,484 14,71 l-16,035

2,676 3,261 5,430 6,937 9,081 13,355 15,373

“The term ORF will be replaced by gene since these regions of the HindIll D fragment are now known to encode functions essential for virus growth. In keeping with the nomenclature of Rose1 et al. (1986) each gene number will be preceded with the letter D to designate that the gene resides in the HindIll D fragment. In the future, the mutants in each complementation group will be referred to by their gene location; i.e., those in group Dl 1 will be identified as being in gene Dl 1.

117

M 13 DNA are tabulated

observation, presented at the poxvirus workshop, 1984) have mapped a 21-kDa RNA polymerase subunit to this region of the HindIll D fragment. Condit and Thompson (in preparation) have demonstrated that the defective late phenotype can be associated with an alteration in the viral 22-kDa RNA polymerase subunit. Since group D7 mutants have a defective late phenotype and D7 encodes a protein similar in size to that mapped by Jones and Moss, it is likely that the group D7 mutants lie in an RNA polymerase subunit encoded by gene D7. This gene is expressed at high levels at early times after infection (Lee-Chen et al., in preparation). Mutants in group D13 map to gene D13 (Fig. 2) and synthesize normal levels of protein and nucleic acid at 40” (Condit et a/., 1983; Ensinger, 1982). This gene has recently been shown to encode the late protein L65 (Weinrich et al., 1985; Tartaglia and Paoletti, 1985). Tar-taglia et a/. (1986) have demonstrated that it is also the site of rifampicin resistance. Virion maturation is inhibited by rifampicin addition (Moss et a/., 1969), implying that the product of gene D13 plays a role in this aspect of the virus life cycle. Of the 13 genes in the HindIll D fragment ts mutations are available in only 7. We do not know if the other genes are essential for virus growth, but we feel that it is unlikely that the genetic map is saturated. As an example, gene Dl encodes the large subunit of the

118

SET0

viral guanyltransferase (Morgan et al., 1984). It would seem to be a likely candidate for an essential viral gene. Site-directed mutagenesis of this gene and the other HindIll D fragment genes is being employed in order to determine if each of these genes is essential.

ACKNOWLEDGMENTS We thank Dr. Marcia Ensinger for providing ts mutants from her collection which map in the Hindlll D fragment. We also acknowledge the contributions that she has made to our understanding ofvaccinia virus genetics.

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