Phenotypic characterization and physical mapping of a temperature-sensitive mutant of autographa californica nuclear polyhedrosis virus defective in DNA synthesis

VIROLOGY

138,694

(1984)

Phenotypic Characterization and Physical Mapping of a TemperatureSensitive Mutant of Autographa californica Nuclear Polyhedrosis Virus Defective in DNA Synthesis JAMES D. GORDON Department of Microbiology and Immunology,

AND

ERIC B. CARSTENS’

Queen’s University, Kingston,

Ontario

K7L SN6, Canada

Received April 28, 1984 accepted June 18, 1984 A ts mutant of Autograph californicu nuclear polyhedrosis virus (AcMNPV), ts8, was shown to he defective in viral DNA synthesis at the nonpermissive temperature. TsS-infected cells synthesized only early viral polypeptides at the nonpermissive temperature, and in contrast to wild-type (WT)-infected cells, showed no inhibition of host cell protein synthesis. The effect of the mutation on viral DNA synthesis was not immediately reversed after shifting infected cells down from the nonpermissive temperature to the permissive temperature; rather, a delay of several hours occurred before viral DNA synthesis was detected. The rate of accumulation of viral DNA in ts8infected cells failed to increase after a shift from the permissive temperature to the nonpermissive temperature. This indicated that the ts8 mutation was involved in the synthesis of proteins required for viral DNA synthesis. The mutation was mapped by marker rescue to the region lying between 60.1 and 62.0% of the AcMNPV physical map. 0 1984 Academic Press, Inc.

(Carstens et aL, 1979; Dobos and Cochran, 1980; Wood, 1980; Maruniak and Summers, Autographa califbrnica nuclear polyhe- 1981). The early phase includes events drosis virus (AcMNPV) has become the prior to the initiation of viral DNA synmodel system in the study of the nuclear thesis. The middle phase involves the synpolyhedrosis virus group of the family thesis of structural polypeptides and the Baculoviridae. The genome of AcMNPV is release of extracellular virus. The late a covalently closed circular molecule of phase involves the de novo synthesis of 128 kbp for which physical maps have intranuclear membranes and the occlusion been prepared (Miller and Dawes, 1979; of virus into polyhedra. Smith and Summers, 1979; Vlak, 1981; A number of hybridization selection Cochran et al, 1982; Vlak and Smith, and in vitro translation studies have iden1982). tified the map location of at least 35 viral The infectious cycle of AcMNPV in tis- polypeptides (Vlak et al, 1981; Esche et sue culture is quite complex with the aL, 1982; Smith et aL, 1982; Rohel et al, release of extracellular nonoccluded virus 1983). In vitro translation of virus-specific (NOV) followed by the occlusion of virus RNA harvested at 6 hr after infection has particles into polyhedral inclusion bodies identified six early polypeptides which later in infection (for review see Granados, could be localized to two regions on the 1980). Numerous studies on the time AcMNPV map (Esche et aL, 1982; Smith course of protein synthesis in infected et aL, 1982). It has also been shown that cells have revealed that at least 35 virus- cytoplasmic RNA labeled at early times specific polypeptides can be identified dur- after infection hybridized to the majority ing three phases of the infectious cycle of the AcMNPV genome (Erlandson and Carstens, 1983). In addition, at least 35 ’ Author to whom requests for reprints should be early transcripts can be detected by hyaddressed. bridization of 32P-labeled viral DNA fragINTRODUCTION

69

0042-6822/34 $3.00 Copyright All rights

0 1984 by Academic Press, Inc. of reproduction in any form reserved.

70

GORDON

AND

ments to Northern blots of cytoplasmic RNA harvested at early times after infection (Erlandson et al, manuscript in preparation). Thus, although a large portion of the AcMNPV genome is transcribed very early after infection, only a small portion of the transcripts have yet been translated in vitro. Several groups have been involved in the isolation and characterization of temperature-sensitive mutants of AcMNPV (Brown et aZ., 1979; Lee and Miller, 1979; Brown and Faulkner, 1980; Duncan and Faulkner, 1982). Two reports have described some characteristics of ts mutants affecting viral DNA synthesis (Brown et CLL,1979; Miller et aL, 1983). The present study outlines the further characterization of the DNA negative mutant, ts8, including the effect of the mutation on viral DNA replication, protein synthesis, and on the temporal control of the viral replication cycle. The mapping of the mutation by marker rescue is also described. The results show that ts8 is defective in an early event and is unable to advance beyond the early phase of infection at the nonpermissive temperature. MATERIALS

AND

METHODS

Virus and cells. Monolayers of Spodoip tern frugiperdu (Sf) cells were maintained by passage in TC-100 media supplemented with 10% fetal calf serum and 50 kg/ml gentamycin (a gift of Schering Canada Inc.). AcMNPV strains HR-3 (WT) and MPts(NOV)D8g(SF) (Brown et uL, 1979), designated ts8A (Erlandson et uL, submitted for publication) were obtained from P. Faulkner, Queen’s University. Virus stocks were plaque purified two times prior to passage. WT was passaged at 28” while ts8 was passaged at 25”. Nonoceluded virus was purified from infected cell culture fluid by centrifugation as previously described (Carstens et uL, 1979). In all studies reported here, third passage virus was used as inoculum. Infectious stocks were titrated by plaque assay (Brown and Faulkner, 1978) or extinction dilution assay (Brown and Faulkner, 1975) in Sf cells at 25’.

CARSTENS

Pur$kution of DNA. DNA was purified from extracellular virus as previously described (Tjia et uL, 1979). Plasmid DNA containing inserts of AcMNPV restriction endonuclease fragments (Cochran et aL, 1982) was purified according to the base/ acid extraction procedure described by Thompson et al. (1983). Electrophoresis and electroelutim of DNA. DNA was digested with restriction endonucleases in 33 mM Tris-OAc, pH 7.9, 66 mM KOAc, 10 mM MgOAc, 0.5 mM dithiothreitol, 0.1 mg/ml bovine serum albumin (TA buffer) (O’Farrell et al, 1980) at 37” for at least 2 hr. Restricted DNA was electrophoresed through agarose gels of various concentrations as previously described (Carstens, 1982). Individual bands were excised from gels and the DNA was purified by electroelution using concentrator cups (ISCO, Lincoln, Nebr.). After electroelution for 4 hr in 1 mM Tris, 5 mMglycine, 0.1% SDS at 100 V (Allington, 1978) the DNA was extracted four times with buffer-saturated butanol (5 M NaCl, TE, pH S.S),once with chloroform:amyl alcohol (24:1), and then precipitated with 2 vol of ethanol. The precipitated DNA was resuspended in distilled water. Trunsfection with puti~ed DNA. Monolayers of Sf cells were transfected with AcMNPV DNA using the calcium phosphate procedure of Graham and Van der Eb (1973) as previously described (Carstens et uL, 1980). The cells were treated for 3 min with 25% dimethyl sulfoxide (DMSO) in HEPES-buffered saline (Stow and Wilkie, 1976) at 6 hr after transfection. The monolayers were washed two times with standard media then incubated at 25”. The supernatant was collected from infected monolayers after 6 or 7 days incubation and the titer of extracellular virus was determined using the extinction dilution method (Brown and Faulkner, 1975) at both 25 and 33”. Protein gel electrophoresis. Infected Sf cells were pulse-labeled with [%]methionine (New England Nuclear, Lachine, P.Q.) at various times after infection as previously described (Carstens et al, 1979). Pulse-labeled cell extracts were analyzed

DNA

NEGATIVE

BACIJLOVIRUS

by SDS-polyacrylamide gel electrophoresis as described by Carstens and Weber (1977). Following electrophoresis, gels were fixed overnight in 40% methanol, 7% acetic acid, then dried and processed for autoradiography using Kodak XAR film. Cell spot hybridization. Intracellular viral DNA was detected by a cell spot hybridization technique. Monolayers of infected Sf cells were harvested by scraping, the cells were transferred to l&ml tubes, and pelleted for 1 min at 12,000 g. The cell pellet was resuspended in 200 ~1 0.01 M Tris, pH 7.5,l mM EDTA. Samples were stored at -20’ until used for analysis. Aliquots of harvested cells were spotted onto “Gene Screen” filters (NEN) and intracellular DNA was bound to the filters using the procedure of Brandsma and Miller (1980). Known amounts of purified viral DNA were also spotted onto the filters in order to quantitate the amount of viral DNA present in each cell spot. The filters were probed with 32Plabeled viral DNA prepared by nick translation (Rigby et aL, 1977) as previously described (Tjia et aL, 1979). Prehybridization and hybridization conditions used were those previously described (Carstens, 1982). The filters were washed as described by the manufacturer. Following autoradiography, individual spots were cut out and the amount of =P-labeled viral DNA bound was determined by liquid scintillation counting. RESULTS

Protein synthesis in iqfected cells. Intracellular protein synthesis in monolayers of Sf cells, infected with WT or ts8 at a multiplicity of infection (m.o.i.) of 5 PFU/ cell was studied by pulse labeling with r5S]methionine at various times postinfection (p.i.). The protein synthesis profile obtained for WT- or ts&infected cells at 25” was very similar to that described by Carstens et al. (1979) with the appearance of at least 25 virus-induced polypeptides by 36 hr p.i. (Fig. 1). Synthesis of viralinduced polypeptides was first detected at 4 hr p.i. with the synthesis of polypeptides

MUTANT

71

of 30K and 31K molecular weight. Polypeptides of similar migration were present in lysates of mock-infected cells but these polypeptides were greatly enhanced on virus infection. It is not known if the polypeptides observed in infected cells represented overproduction of cellular polypeptides or synthesis of distinct viral polypeptides. At 8 hr p.i., a polypeptide comigrating with the 43K marker was seen. The synthesis of the 31K and 43K polypeptides continued throughout the infectious cycle while that of the 30K polypeptide began to decline by 24 hr p.i. and had ceased by 32 hr p.i. The synthesis of host polypeptides also began to decline at 24 hr p.i. and had, for the most part, ceased by 36 hr p.i. The time course of protein synthesis in WT-infected cells incubated at 33” was similar to that at 25” although most polypeptides appeared 4 to 8 hr earlier (Fig. 1). The time course of protein synthesis in ts&infected cells at 33” was very different. Only the three earliest polypeptides (30K, 31K, 43K) were synthesized during the time studied (Fig. 1). In contrast to WT-infected cells where the synthesis of the 30K polypeptide ceased by 24 hr p.i., all three early polypeptides were synthesized throughout the time studied in ts8infected cells at 33’. There was no inhibition of host protein synthesis nor any indication of the synthesis of late viral polypeptides in the ts&infected cells. These experiments were repeated using [3H]leucine as a radioactive precursor; however, no additional virus-induced polypeptides were observed in ts&infected cells at 33” (results not shown). DNA synthesis in irlfected cells. The time course of DNA synthesis ‘in virusinfected cells was monitored by cell spot hybridization. Monolayers of Sf cells were infected with WT or ts8 at an m.o.i. of $5 PFU/cell. After a 1.5-hr adsorption period at room temperature, the inoculum was removed, the monolayers were washed once with phosphate-buffered saline (PBS), and overlayed with standard medium. The infected cells were incubated at 25 or 33’ beginning at 2 hr p.i. Representative ‘monolayers were harvested at

72

GORDON

AND

La-L

WT

4M 4

6 121620242*32364*&

we

CARSTENS

M 4 4

33°C M 8 12 16 20 24 26 32 364636

43 31 30 -25.5

Ts8

:

4

2 5% 8 12 16 20 2428323648;

M: 4 4

33-c 8 12 16 202428

323648

3Ms

-92.5

-25.5

FIG. 1. Protein synthesis in infected cells. Monolayers of Sf cells were infected with WT or ts8 at an m.o.i. of 5 PFU/cell. After a 2-hr adsorption at room temperature the monolayers were washed once with PBS then incubated at 25 or 33”. At the indicated times postinfection, the cells were pulse-labeled with [%]methionine for 1 hr at 33’ or 1.5 hr at 25“. Whole cell extracts from pulse-labeled-infected or mock-infected’ (M) cells were analyzed by SDS-polyacrylamide gel electrophoresis. The initial appearance of polypeptide bands during infection is indicated by arrows. Molecular weight markers included phosphorylase B (92.5K), bovine serum albumin (68K), ovalbumin (43K), cY-chymotrypsinogen (25.710, and ,!I-lactoglobulin (18.4K). In these studies, the zero time point is taken as the time that the viral inoculum was added to the cells prior to the adsorption period.

regular times after infection as described under Materials and Methods and the level of intracellular viral DNA was determined by cell spot hybridization. The

amount of viral DNA (in nanograms) was determined by comparison of =P counts bound to individual spots with a standard curve prepared by titration of counts ob-

DNA NEGATIVE

BACULOVIRUS

MUTANT

73

tained with known amounts of purified continuously at 33“ did not appear to WT DNA. The standard curve was linear replicate viral DNA, we wanted to deterover the entire range of counts observed mine if there were conditions under which (data not shown). The time courses of ts8 DNA replication could occur at 33”. viral DNA synthesis in WT- and ts8- We therefore carried out a series of teminfected cells at 25” were similar (Fig. 2). perature shift-up experiments and meaViral DNA began to accumulate at lo-12 sured the amount of viral DNA in these hr p.i. and continued to accumulate cells at various times after the shift. throughout the 28-hr time period studied. Representative monolayers of Sf cells were Viral DNA did not accumulate to as high infected with WT or ts8 as previously a level in ts8-infected cells incubated at described and were transferred from 25 25” as in WT-infected cells at the same to 33” at 4, 8, 12, and 16 hr p.i. At 4-hr temperature. The time course of DNA intervals following the temperature shift, synthesis in WT-infected cells incubated the monolayers were harvested and asat 33” was somewhat accelerated over sayed for viral DNA by cell spot hybridthat observed at 25’; viral DNA began to ization. Control cells were maintained at accumulate at 8 hr p.i. and again continued 25” throughout the time studied and harto accumulate throughout the time stud- vested at the same times for comparison. ied. In contrast, there was no increase in Viral DNA accumulated to higher levels the level of viral DNA in ts8-infected cells in WT-infected cells shifted to 33” than incubated at 33’ during the time studied. in control WT-infected cells maintained DNA synthesis following temperature at 25” (Fig. 3A). The final rate of accushift up. Since ts&infected cells, incubated mulation of DNA synthesized in shifted cells, that observed 8-12 hr after a temperature shift at 4 or 8 hr p.i. or 4-12 hr after a shift at 12 or 16 hr p.i. was very similar to that observed in control cells from 20 to 28 hr p.i. at 25”. The steadystate level of viral DNA which we have measured in these experiments is a function of the rate of synthesis, the rate of turnover (degradation) and the rate of transport from the infected cells via budding of extracellular virus. Although we do not have direct evidence yet, we suspect that the increased accumulation of viral DNA likely resulted from an initial increase in the rate of DNA synthesis in the shifted cells rather than from an overall increase through the time after the shift since the rates of accumulation of DNA at the two temperatures were FIG. 2. DNA replication in infected cells. Monolay- very similar. ers of 1 X lo6 Sf cells in 35-mm dishes were infected In ts&infected cells, a shift from 25 to with WT or ts8 as described in Fig. 1. At the 33” did not result in an increase in the indicated times after infection, monolayers were rate of accumulation of viral DNA synharvested by scraping and transferring the cells to thesis following the shift. In ts&infected a 1.5-ml tube. After centrifugation for 1 min at cells shifted to 33” at 4 hr p.i., a time at 12,000g the cell pellets were resuspended in TE, pH which no viral DNA synthesis had yet 7.5. These harvested cells were assayed for intracellular viral DNA by cell spot hybridization as de- occurred (Fig. 2), there was no accumuscribed under Materials and Methods. The results lation of viral DNA during the period are expressed as ng of viral DNA in 5~1 aliquots following the shift (data not shown). In containing 2500 cells: WT-infected cells at 25” (O), ts&infected cells shifted to 33” at 8 hr 33’ (0), ts8-infected cells at 25’ (D), 33” (0). p.i., about the time of the initial appear-

74

GORDON AND CARSTENS

FIG. 3. DNA synthesis in WT- and ts8-infected cells following temperature shifts from 25 to 33”. Monolayers of Sf cells were infected with WT (A) or ts8 (B) as described for Fig. 1 and incubated at 25”. At 4, 8, 12, and 16 hr pi., monolayers were shifted to 33” by replacing the incubation media with fresh media prewarmed to 33’. Shifted monolayers were incubated at 33’. At 4-hr intervals following the shift, representative monolayers were harvested and the level of intracellular viral DNA was assayed as described for Fig. 2. Control WT- or ts8-infected Sf cells were maintained at 25” and harvested at the same times. These curves therefore represent the time course of viral DNA at this temperature.

ante of viral DNA at 25”, there was a slight increase in the amount of viral DNA during the period following the shift; however, it was not visible until 12 hr after the shift (Fig. 3B). In cells shifted at 12 or 16 hr p.i., when significant levels of viral DNA were present in the infected cells at 25”, there was a significant increase in the level of viral DNA following the shift but the rate of accumulation after the shift was low, similar to the rate seen at 25’ prior to the shift rather than to the rate observed in control WT-infected cells at the times studied (Fig. 3B). DNA q&he&s fo&wing &mp@rature sh@l ddwn. It was of interest to determine

if the effect of the mutation on DNA synthesis was reversible after a shift from the nonpermissive temperature to the permissive temperature. For this study, the shift down was carried out at 12 hr p.i., since this time was significantly after the initial appearance of viral DNA to allow for the accumulation of the defective ts8 gene product. Monolayers of Sf cells were infected with ts8 and were incubated at 33”. At 12 hr p.i., the incubation media was removed, similar media maintained at 25” was added, and the cells were then incubated at 25”. At the time of the shift and at regular times thereafter, the cells from representative monolayers were harvested. In addition, at the time of the shift (12 hr p.i.), monolayers of Sf cells were infected with WT and incubated at 25’; these monolayers were also harvested at regular intervals to allow comparison of the time course of DNA synthesis after the shift with that in the early stages of WT infection at 25”. The level of viral DNA in harvested cells was determined by cell spot hybridization. As expected, there was no increase in the level of viral DNA in t&-infected cells maintained at 33” (see Fig. 2). In contrast, viral DNA did accumulate in t&infected cells shifted from 33 to 25” (Fig. 4). Viral DNA was not detected in the t&infected cells immediately after the temperature shift, but there was a dramatic increase in accumulation of viral DNA about 4 hr after the shift down. This suggested that the mutated gene product synthesized at 33” may not be functional at 25’ either, and that further protein synthesis was required before DNA synthesis could begin. Alternatively, the conformation of the mutated protein could revert to an active form at 25”, but further protein synthesis was still required to initiate viral DNA synthesis. Physical mapping of the mutation. The mutation site in ts8 was located on the physical map of AcMNPV by marker rescue. Monolayers of Sf cells were transfected with a mixture of whole ts8 DNA and various restriction fragments of WT DNA. The amount of ts8 DNA used represented about 1000 PFU per plate based

DNA

NEGATIVE

BACULOVIRUS

MUTANT

75

period. At higher dilutions, cell growth with cellular hypertrophy and vacuolation was observed as previously reported (Brown et UC, 1979). When supernatants containing a mixture of ts+ and ts- recombinants were titrated at 33”, polyhedra formation was superimposed on this nonpermissive cytopathic effect. No polyhedra were observed at low dilution likely due to infection of all cells with ts8. At higher dilutions, polyhedra were clearly observed in some cells while the large vacuoles characteristic of ts8 were seen in others. For the purpose of these titrations, it was FIG. 4. DNA synthesis in ts.&infected cells following assumed that if tsf recombinants were a shift from 33 to 25” at 12 hr p.i. Monolayers were present in high enough levels in high infected as described in Fig. 1 and incubated at 33”. dilutions to allow polyhedra formation, At 12 hr p.i., monolayers were shifted to 25” by they would also be present in lower diluremoving and replacing the media with fresh media tions even though polyhedra were not at 25”. The shifted monolayers were incubated at visible. 25’ and representative monolayers were harvested Transfection with ts8 DNA and WT at various times after the shift and assayed for total DNA digested with EcoRI demonstrated viral DNA as described in Fig. 2. In this study, that optimum levels of ts+ recombinants t = 0 represented the time of the shift from 33 to could be obtained when a lo-fold molar 25”. The time course of DNA synthesis following infection at 25” of Sf cells with WT is also shown. excess of the appropriate restriction fragTs8-infected cells shifted from 33 to 25’ at 12 hr pi. ment was used (data not shown). All (O), WT-infected cells following infection at 25’ (m). further studies were carried out using this molar ratio. Initial mapping studies were carried out using SmuI fragments on titration studies using purified Ac- of WT DNA purified from agarose gels by MNPV DNA (Carstens et aL, 1980). The electroelution. Transfection of Sf cells supernatant was collected from trans- with ts8 DNA and WT SmaI fragment C (49.4-64.8s) resulted in the production of fected cells following 6-7 days incubation at 25” and the titer of extracellular virus extracellular virus with a high proportion was determined at 25 and 33” by the of the ts+ phenotype (Table 1). Transfecextinction dilution method (Brown and tion with ts8 DNA alone or with any of Faulkner, 1975). Recombination between the other SmaI fragments resulted in the production of only ts- progeny. This inthe t&3 genome and the WT fragment containing the homologue of the mutated dicated that the ts8 mutation was located region would yield tsf virus capable of within a region mapping between 49.4 and replication at 33 and 25” whereas recom- 64.8% (Fig. 5). The low level of ts+ progeny bination with fragments from other re- observed after transfection with ts8 DNA gions of the genome would yield ts- virus and WT SmaI fragment D was likely due that was capable of replication only at to contamination of the fragment with 25”. Titration plates were screened for SmaI C as it was not observed with other the presence of polyhedral inclusion bodies preparations of this fragment. in infected cells as an indication of ts+ Further mapping studies were carried recombinants. Titration at 33” of the out using EcoRI fragments of AcMNPV supernatant from cells transfected with cloned into the plasmid vector pBR322. Four EcoRI fragments (EcoRI C, G, W, ts8 DNA alone showed a nonpolyhedron dependent cytopathic effect (data not and D) map within SmuI fragment C shown). At low dilution, no growth of the (Cochran et al, 1982). Clones containing fragments C, W, and D were obtained cells was observed over the incubation

%7=&d HOURS

76

GORDON

AND

CARSTENS

TABLE

1

MARKERRESCUEOF ts8 WITHELECTROELUTEDSWZUI FRAGMENTS Purified viral DNA WT tst3 t&3 tss tst? ts8 ts8 a Digested

WT DNA fragment

Titer 33” (PFU/ml)

Titer 25” (PFU/ml)

-

7.3 x lo6 <60 <60 <60 5.9 x 10” 9.7 x lo* 2.1 x lo6 <60

2.3 X lo7 5.9 x lo6 2.7 x lo6 3.6 X lo6 3.3 x lo6 7.7 x lo6 5.9 x lo6 <60

SW& A Sm.uI B S?noI c SmaI D WT” WT”

Sma I

3.2 <3.0 <3.0 13.0 1.8 1.3 3.6

X x x x X x

10-i 1o-5 1om5 1o-5 10-z 1om4 X lo-* -

with SmaI.

progeny was obtained following transfection with ts8 DNA and the plasmid bearing EcoRI fragment D (60.1-68.3s) (Table 2). Thus the mutation in ts8 lies in the region shared by EcoRI fragment D and SmaI fragment C between 60.1 and 64.8 map units, a region of 6.0 kbp (Fig. 5). The region of overlap between SmaI fragment C and EcoRI fragment D is cleaved into three fragments by Hind111 (Fig. 5). These fragments were purified from gels of the EcoRI fragment D clone after digestion with EcoRI and Hind111 or Hind111 and SmuI. These fragments were denoted according to the flanking

from a library of WT clones (Cochran et a& 1982) while the EcoRI G clone was obtained from a library of clones derived from the AcMNPV polyhedron morphology mutant M5 (Carstens, 1982; unpublished work). The plasmids used are identified in Table 2; each clone is identified by the source of the DNA and the designation of the inserted restriction fragments. In two cases, the plasmid used contained more than one inserted AcMNPV fragment but only one mapped within SwuzI C. Transfections were carried out with plasmid digested previously with EcoRI to excise the cloned fragment. Ts+ Eco RI

Ratio 33/25”

IRO

I 1

A

.

J

1

KTMN ,‘I,

FVU

A

’ _.-/Kbp

,-r,

c* ,

C D*- *-I

% Genome

1 495

Smal C b Hmd III C pAc Eco D 19 pAcEH3

D

QL

c

,

,

nz iif

,

,

,

,

,

,

,

Et 88

,

-.

-. ,

,

B A

-.

-== E-F,

i I

597 620 62.7 640 601 62.6

8

SXP

-. ,

, -._, i

(

20

ri?C 1 II

WW

-1

527 533 529

,

15

IO

H I

E

-. -. s

.’

5

0

GW

25

=- -ay =:pg .

66.9 662 663

I 4 -

FIG. 5. Map location of the ts8 mutation site summarizing the results of marker rescue studies outlined in Tables 1, 2, and 3. The cleavage sites for the restriction endonucleases SmaI, EcoRI, and Hind111 of AcMNPV DNA (HR3, Cochran et &, 1982) are indicated. Ts+ recombinants were obtained following transfection with ts8 DNA and SmaI fragment C, EcoRI fragment D, Hind111 fragment C, and the 2.6 kbp EcoRI-Hind111 fragment denoted EH3 localizing the mutation site to the region between 60.1 and 62 map units.

DNA

NEGATIVE

BACULOVIRUS TABLE

77

MUTANT

2

MARKER RESCUE OF ts8 WITH CLONED EcoRI FRAGMENTS Purified viral DNA WT ts8 tati ts8 ts8 ts8 ts8 ’ Digested

WT DNA fragment

Titer 33’ (PFU/ml)

Titer 25’ (PFU/ml)

Ratio 33/25”

-

3.4 x lo7 <60 <60 <60 <60 7.3 x lo6 4.4 x lo6

7.3 9.5 9.5 5.7 5.7 9.5 5.7

4.7
pAcEcoCX18” pM5EcoG” pAcEcoNPW2.4” pAcEcoD19” WT”

x x x x x x x

10’ lo7 lo1 lo7 lo7 10’ 10’

x x x x x x x

10-l 10-O 1o-6 10-6 1o-6 1om3 10-3

with EcoRI.

restriction sites and their migration in agarose gels. Fragment EH3 mapped between the EcoRI site at 60.1% and the Hind111 site at 62.0%; EH5 mapped between Hind111 sites at 62.0 and 62.6% and corresponded to Hind111 fragment W (Cochran et aL, 1982); HS2 mapped between the Hind111 site at 62.6% and the SmaI site at 64.8% (Fig. 5). Only cotransfection of Sf cells with ts8 DNA and the fragment EH3 yielded ts+ progeny (Table 3). This indicated that the mutation must

TABLE

lie between 60.1 and 62.0% at the left end of EcoRI fragment D, a region of 2.6 kbp. This result was confirmed by marker rescue with Hind111 fragment C which overlaps EcoRI D only in this region (Table 3). Rescue of ts8 with WT DNA digested with EcoRI was more efficient than with the same DNA digested with Hind111 (Table 3). Likewise, rescue with the EcoRI D clone digested with EcoRI was more efficient than it was using the clone digested

3

FINE MAPPING OF ts8 WITHIN EcoRI FRAGMENT D Purified viral DNA WT ts8 ts8 ts8 ts8 ts8 ts8 ts8 ts8 t&3 ts8 ts8 ts8

WT DNA fragment

Titer 33’ (PFU/ml)

Titer 25” (PFU/ml)

Ratio 33/25”

-

5.9 x lo6 <60 1.9 x lo5 3.9 x 10’ 6.5 X 10’ <60 40 2.1 x lo5 <60 7.3 x 10’ 1.1 x lo6 6.5 X 10” <60

3.9 5.1 3.1 2.7 1.9 2.7 1.4 5.0 3.3 3.1 1.2 2.3 3.0

1.5 <5.0 6.1 1.4 3.4 <5.0 15.0 4.2 15.0 2.4 9.2 2.8 <5.0

pAcEcoD19” pAcEcoD19” EH3 EH5 HS2 H&d111 C (WT) Hind111 C (ts8) pEH 3” WT” WTb ts8”

a Digested with EcoRI. b Digested with HindIII. ‘Digested with EcoRI and HindIII.

x x x X x x x x x x x x x

106 lo6 106 lo6 lo6 lo6 106 106 lo6 lo6 106 lo6 lo6

x x X x x x x X x X x X x

lo0 1o-5 1O-2 10-Z 10-z 10-5 10-s lo-’ 10-b lo-’ 10-Z 1O-z 10-b

78

GORDON

AND

with EcoRI and Hind111 (Table 3). This may indicate that the mutation site lies close to the Hind111 site at 62.0%. DISCUSSION

The time course of protein synthesis in WT- and t&-infected cells at 25” was similar to that described at 28” (Carstens et al, 1979). At least 25 viral-induced polypeptides could easily be seen synthesized during overlapping early, middle, and late phases. The time course observed in WT-infected cells at 33’ was similar to that observed at 25’ but the initial appearance of most viral-induced polypeptides was shifted to earlier times. The time course of polypeptide synthesis was greatly altered in ts&infected cells at 33”. The synthesis of host cell polypeptides continued throughout the time studied while the majority of the viral-induced polypeptides were not detected. Only three early polypeptides (30K, 31K, 43K) were detected at the nonpermissive temperature. The doublet of early polypeptides migrating just above polyhedrin has usually been resolved as a single band in other reports (Dobos and Cochran, 1980; Kelly and Lescott, 1981; Maruniak and Summers, 1981); as a result, the differential expression of these two polypeptides has been overlooked previously. Synthesis of the 30K polypeptide was shut off by 32 hr p.i. under permissive conditions while that of the 31K polypeptide continued throughout the time studied. In ts&infected cells at 33”, the synthesis of both these polypeptides continued throughout the time studied. These results indicate that at 33”, ts8 was blocked in a function necessary for the transition out of the early phase of infection. Late events such as the formation of polyhedra and the release of infectious virus were not observed at 33” (data not shown). Using these criteria, the ts8 mutation was not leaky. The synthesis of viral DNA in ts8infected cells was previously studied by hybridization of intracellular DNA purified from infected cells pulse-labeled with [3H]thymidine to viral DNA immobilized on nitrocellulose filters (Brown et al, 1979).

CARSTENS

We studied viral DNA synthesis in infected cells by hybridization of in vitro 32P-labeled viral DNA to total cellular DNA from infected cells spotted onto “Gene Screen” filters (Brandsma and Miller, 1980). Our results confirm the previous result and extend their observations to compare the time course of DNA synthesis in WT- and ts8-infected cells at 25 and 33”. Viral DNA appeared earlier, accumulated faster, and to higher levels in WT-infected cells maintained at 33” than in WT-infected cells maintained at 25”. Brown (1979) showed that Spodoptera frugiperda cells grew faster at the elevated temperature than at 25” or at the normal incubation temperature of the cells which is 27”. In contrast, it was shown that the yield of extracellular virus was greater at 25 or 27” than at 33” due to a premature drop in the level of NOV release at 33” at late times after infection. Initially, the time course of NOV release at 33” was indistinguishable from that at 2’7” (Brown, 1979). These results combined with our work on DNA synthesis suggest that incubation of cells at 33” initially enhances the rate of events in infection but eventually results in a decrease in the level of extracellular virus. Whether this enhanced rate of accumulation of viral DNA is due to the effect of an elevated temperature on the kinetics of the enzyme systems involved in DNA synthesis has yet to be directly demonstrated. Viral DNA did not accumulate to as high a level in ts8-infected cells incubated at 25” as in WT-infected cells at the same temperature. Since identical m.o.i.‘s were used in these experiments, this suggests that either the mutation exerts some effect on DNA synthesis at the permissive temperature, or there is a high level of noninfectious virions present in ts8 virus stocks which inhibit the normal infectious cycle at 25”. Production of defective genomes has been observed on passage of ts8 at 25” (Erlandson and Carstens, submitted) but it is unclear as yet whether this effect is directly related to the phenotype of ts8. On the basis of studies with metabolic inhibitors, Kelly and Lescott (1981) have

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divided the infectious cycle of Trichoplmia The delayed onset of DNA synthesis ni nuclear polyhedrosis virus into four after a shift from the nonpermissive temphases denoted a, b, c, and d Inhibition perature to the permissive temperature of transcription or translation during any (Fig. 4) indicates that the synthesis of stage prevented the subsequent stage from some viral polypeptides must take place occurring. Reversal of the block allowed before DNA synthesis can begin. The fact subsequent stages to occur. They suggest that viral DNA appeared sooner and acthat a cascade mechanism similar to that cumulated more rapidly in the cells shifted described for herpes simplex virus (Honess down from 33’ than during a normal and Roizman, 1974) also occurs in bacu- virus infection at 25” suggests that a pool lovirus-infected cells. In this model, a of active a proteins made at 33” may be polypeptides would be synthesized soon available once the defective gene product after infection and would be necessary for is synthesized at 25”. Whether synthesis the synthesis of b polypeptides which, for of both a and b polypeptides or just that the most part, would be involved in the of b polypeptides is required for DNA synthesis of viral DNA. Expression of b synthesis is not clear at this time. polypeptides would also be required for Cotransfection of S&twa fiperdu the synthesis of c polypeptides, which for cells with ts8 DNA and various purified the most part, would be virion structural WT restriction fragments yielded ts+ components. The fourth phase d polypep- progeny only when DNA from the region tides including the polyhedrin protein, between 60.1 and 62.0 map units was would be involved in the occlusion of the present. This region has been shown to virus. be highly expressed as cytoplasmic RNA On the basis of this model, the present at early times postinfection (Erlandson work suggests that the gene product de- and Carstens, 1983). Northern blots of fective in t&-infected cells belongs to the early RNA probed with this 2.6-kbp DNA a class. Only the a phase polypeptides fragment identified a 4.6-kb transcript were observed and viral DNA synthesis originating from this region of the genome was not detected in t&-infected cells at (Erlandson and Carstens, manuscript in 33’. Further evidence for this hypothesis preparation) although hybridization sewas obtained from the temperature shiftlection and in vitro translation experiup experiments. ments have failed to identify an early Shifting the temperature of incubation polypeptide encoded by this region (Esche from the permissive temperature to the et al, 1982; Smith et a& 1982). Further nonpermissive temperature prevented any studies to identify the gene product that increase in the rate of DNA synthesis in is defective in ts8 are currently underway. t&infected cells after the shift (Fig. 3). A decrease in the efficiency of rescue This could result from the inactivation of using WT DNA or EcoRI fragment D the mutated gene product at the nonper- DNA digested with Hind111 compared missive temperature preventing the for- with that observed with the same DNA mation of any more active b polypeptides digested with EcoRI (Table 3) suggests including those needed for DNA synthesis. that the mutation may lie close to the This inactivation would not affect b poly- Hind111 site at 62.0%. peptides synthesized before the shift, alMiller et al. (1983) have reported the lowing a low level of viral DNA synthesis characterization of a mutant of AcMNPV fixed at the rate occurring at the time of with a similar phenotype. Their mutant, the shift. If the shift was carried out tsB821, was defective in DNA and late prior to the initiation of DNA synthesis, protein synthesis but was mapped by no viral DNA was detected at any time marker rescue to a region lying between after the shift. This indicates that the 90.7 and 1.9% of the genome (Miller, 1981), mutated gene product is one required a region quite distinct from that discussed throughout infection for the synthesis of in the present paper. The fact that two b polypeptides. mutations mapping in two distinct regions

80

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of the genome are required for the onset of the DNA synthesis indicates that at least two regulatory functions are involved or that the early regulatory protein consists of at least two polypeptides.

CARSTENS, E. B., and WEBER, J. (1977). Genetic

analysis of adenovirus type 2. Pleiotropic effects in an assembly mutant. J. Gen ViroL 37,453-474. COCHRAN,M. A., CARSTENS,E. B., EATON, B. T., and FAULKNER,P. (1982). Molecular cloning and physical mapping of restriction endonuclease fragments californica nuclear polyhedrosis of Autogrupha ACKNOWLEDGMENTS virus DNA. J. Viral 41.940-946. DOBOS,P., and COCHRAN,M. A. (1980). Protein synWe thank M. Hough and J. Bekkedam for their thesis in cells infected by Autographa califbrnica excellent technical assistance and M. A. Erlandson nuclear polyhedrosis virus (AC-NPV): The effect for useful discussions during the course of this work. of cytosine arabinoside. Virology 103,446-464. This work was supported by a grant from the DUNCAN,R., and FAULKNER, P. (1982). BromodeoxMedical Research Council of Canada. yuridine-induced mutants of Autographa c&fornica nuclear polyhedrosis virus defective in occlusion body formation. J. Ga. ViroL 62,369-373. REFERENCES ERLANDSON,M. A., and CARSTENS.E. B. (1983). ALLINGTON, W. B., CORDRY,A. L., MCCULLOUGH, Mapping early transcription products of AutograG. A., MITCHELL,D. E., and NELSON,J. W. (1978). pho californico nuclear polyhedrosis virus. Viirdogy 126,398-402. Electrophoretic concentration of macromolecules. ESCHE,H., LUBBERT,H., SIEGMANN,B., and DOERFLER, Anal. Biochem 85.188-196. BRANDSMA, J., and MILLER, G. (1980). Nucleic acid W. (1982).The translational map of the Autographa spot hybridization: Rapid quantitative screening caZ@rnica nuclear polyhedrosis virus (AcNPV) genome. EMBO J. 1.1629-1633. of lymphoid cell lines for Epstein-Barr viral DNA. GRAHAM, F. L., and VAN DER EB, A. J. (1973). A new Proc. NatL Acad Sci. USA 77,6851-6855. BROWN,M. (1979). Genetic analysis of the nuclear technique for the assay of infectivity of human polyhedrosis virus of Autographa CaliforniccL Ph.D. adenovirus 5 DNA. Virology 52,456-467. thesis, Queen’s University, Kingston, Ontario. GRANADOS,R. R. (1980). Infectivity and mode of BROWN,M., CRAWFORD,A. M., and FAULKNER, P. action of baculoviruses. BiotechnoL Bioew. 22, (1979). Genetic analysis of a baculovirus Autogm1377-1405. pha col@rniuz nuclear polyhedrosis virus. I. Iso- HONESS,R. W., and ROIZMAN,B. (1974). Regulation lation of temperature-sensitive mutants and asof herpes virus macromolecular synthesis. I. Cascade regulation of the synthesis of three groups sortment into complementation groups. J. ViroL 3l,lSO-198. of viral proteins. J. Vird 14.8-19. BROWN,M., and FAULKNER,P. (1975). Factors affect- KELLY, D. C., and LESCOTT,T. (1981). Baculovirus replication: Protein synthesis in Spcdoptera fruing the yield of virus in a cloned cell line of giperda cells infected with Trichoplusia ni nuclear !l’richoplusia ni infected with a nuclear polyhedrosis polyhedrosis virus. Microbiologica 4, 35-57. virus. J. Invertebr. Pathol 26,251+X7. BROWN, M., and FAULKNER, P. (1978). Plaque assay LEE, H. H., and MILLER, L. K. (1979). Isolation, complementation, and initial characterization of of nuclear polyhedrosis viruses in cell culture. temperature-sensitive mutants of the baculovirus AppL Environ. MicrcbioL 36,31-35. BROWN, M., and FAULKNER, P. (1980). A partial Autographn u&fur&a nuclear polyhedrosis virus. J. ViroL 31, 240-252. genetic map of the baculovirus Autographa calif&-n&xc nuclear polyhedrosis virus, based on re- MARUNIAK, J. E., and SUMMERS,M. D. (1981). Aub graphu californico nuclear polyhedrosis virus combination studies with ts mutants. J. Gen Viral phosphoproteins and synthesis of intracellular 48,247-251. proteins after virus infection. Virdogy 109. 25-34. CARSTENS, E. B. (1982). Mapping the mutation site MILLER, L. K. (1981). Construction of a genetic map of an Autographa calzfornica nuclear polyhedrosis of the baculovirus Autographa ca&fornicu nuclear virus polyhedron morphology mutant. J. ViroL 43. polyhedroais virus by marker rescue of tempera809-818. ture-sensitive mutants. J. ViroL 39, 973-976. CARSTENS, E. B., TJIA, S. T., and DOERFLER, W. (1979). Infection of Spodoptwa frugiperda cells MILLER, L. K., and DAWES, K. P. (1979). Physical map of the DNA genome of Autogmpha califmica with Autographa colifornica nuclear polyhedrosis nuclear polyhedrosis virus. J. Vied 29, 1044-1055. virus. I. Synthesis of intracellular proteins after MILLER, L. K., TRIMARCHI, R. E., BROWNE,D., and virus infection. Virdogy 99.386-398. PENNOCK,G. D. (1983). A temperature-sensitive CARSTENS,E. B., TJIA, S. T., and DOERFLER, W. mutant of the baculovirus Autogmpha ca&fornica (1980).Infectious DNA from Autographa californim nuclear polyhedrosis virus defective in an early nuclear polyhedrosis virus. Virology 101, 311-314.

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function required for further gene expression. Virdogy 126,376380. O’FARRELL, P. H., KUTTER, E., and NAKANISHI, M. (1980). A restriction map of the bacteriophage T4 genome. Md Ga Cm.&. 179,421-435. RIGBY, P. W. J., DIECKMAN, M., RHODES, C., and BERG, P. (197’7).Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol Bid 113,23’7-251. ROHEL, D. Z., COCHRAN, M. A., and FAULKNER, P. (1983). Characterization of two abundant mRNAs of Autographa c@brnica nuclear polyhedrosis virus present late in infection. virology 124, 35’7-

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THOMPSON,J. A., BLAKESLEY, R. W., DORAN, K., HOUGH,C. J., and WELLS,R. D. (1983). Purification of nucleic acids by RPC-5 analog chromatography: Peristaltic and gravity-flow applications. In “Methods in Enzymology” (R. Wu, L. Grossman, and K. Moldave, eds.), Vol. 100, pp. 368-399. Academic Press, New York. TJIA, S. T., CARSTENS,E. B., and DOERFLER,W. (1979). Infection of Spodqptwa frugiperda cells with Autographa ca&fornica nuclear polyhedrosis virus. II. The viral DNA and the kinetics of its replication. Vi’irdogy 99, 399-409. VLAK, J. M. (1981). Mapping of Barn HI and Sma I 365. DNA restriction sites on the genome of the nuclear SMITH, G. E., and SUMMERS,M. D. (1979). Restriction polyhedrosis virus of the alfalfa looper, Autograph maps of five Autographa californica MNPV varicalifmica J. Invert&r. Pathol. 36.409-414. ants, Trichoplti ni MNPV, and Go&rid meUoneUa VLAK, J. M., and SMITH, G. E. (1982). Orientation of MNPV DNAs with endonucleases Sma I, Kpn I, the genome of Autographa califmica nuclear Barn HI, Sac I, Xho I, and Eco RI. J. Viral. 30. polyhedrosis virus: A proposal. J. Vi& 41, 1118828-838. 1121. SMITH, G. E., VLAK, J. M., and SUMMERS,M. D. VLAK, J. M., SMITH, G. E., and SUMMERS,M. D. (1982). In vitro translation of Autographn califbr(1981). Hybridization selection and in vitro transnica nuclear polyhedrosis virus early and late lation of Autographa californica nuclear polyhemRNAs. J. ViroL 44.199-208. drosis virus mRNA. J. Viral. 40, 762-771. STOW,N. D., and WILKIE, N. M. (1976). An improved WOOD,H. A. (1980). Autograph californica nuclear technique for obtaining enhanced infectivity with polyhedrosis virus-induced proteins in tissue culherpes simplex virus type I DNA. J. Gen Viral ture. Virology 102, 21-27. 33,447-458.