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Jan 12, 1979 - adenovirus transformed rat cells lose their permissiveness while cells of ... reduction in number of progeny to a completely abortive infection.
J. gen. ViroL 0979), 44, 297-3o9

297

Printed in Great Britain

Modulation of Herpes Simplex Virus Replication in Adenovirus Transformed Cells By RICHARD STENBERG*, D A V I D S P E C T O R t AND L E W I S PIZER* Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19 ~74, U.S.A.

(Accepted 12 January 1979) SUMMARY

The ability of herpes simplex virus type I to replicate in cells transformed by adenovirus type 5 is strongly dependent on the origin of the cells. Studies show that adenovirus transformed rat cells lose their permissiveness while cells of hamster or human origin retain their ability to replicate HSV although at a reduced level when compared to the untransformed parent cells. One line ofadenovirus transformed rat cells, io7, demonstrates thermosensitive events, allowing HSV to replicate at 34 °C but not at 37 °C. Analysis of the biochemical events taking place at 37 °C showed that virus~specific DNA synthesis was greatly reduced but that all of the late virus structural proteins could be observed after SDS-polyacrylamide gel electrophoresis. It was also demonstrated that shut-off of host macromolecular synthesis appeared to be less efficient after HSV infection of lO7 cells than after infection of more permissive cells such as the non-transformed REF line. Collectively the data show that interactions between HSV and the host cell are perturbed when the cell is transformed by type 5 adenovirus. The degree of perturbation ranges from a slight reduction in number of progeny to a completely abortive infection. INTRODUCTION

The replication of herpes simplex virus (HSV) has been extensively studied in a variety of cultured mammalian cell types. This virus is considered to have a wide host range (Darlington & Granoff, 1973) and when HSV is introduced into a permissive cell system, the virus replication that rapidly takes place results in cell death and the release of infectious progeny (Roizman & Furlong, ~974). During the past few years, reports have come from several laboratories describing the restricted ability of HSV to replicate in cells transformed by other DNA viruses. Nordaa et al. (I966) have reported that SV4o transformed hamster and human lines, when compared to non-transformed control cells, have an increased resistance to infection by HSV. However, Rapp & Trulock (I97o) have shown that a line of SV4o transformed African green monkey cells are completely permissive to HSV replication. In addition, a relationship was shown between the degree of oncogenicity of transformed hamster cell lines and their ability to support HSV replication. Cells that contain high levels of SV4o T-antigen and which were highly tumorigenic were more resistant to HSV than were cells which had lower levels of T-antigen and were less tumorigenic (Geder et al. 1971). This result would seem to indicate * Present address: Department of Microbiology and Immunology, University of Colorado Medical

Center, Denver, Colorado 8o262, U.S.A. t Present address: Department of Pathology, Washington, University School of Medicine, St Louis, Missouri 63110, U.S.A. 0o22-I317/79/0000-3450 $02.00 ~) 1979 SGM

298

R. STENBERG, D. SPECTOR AND L. PIZER

that a varying degree of permissiveness exists within the SV4o transformed cell system. In the case of another DNA virus, Sprecher-Goldberger et al. (2973) have shown that transformation of hamster cells by adenovirus type I 2 causes restriction of HSV replication and have provided evidence that the non-permissiveness is dependent on temperature. In this case, HSV replicated to a greater extent at 35 °C than at 37 °C, the normally permissive temperature. These authors suggest that either the transforming virus within the cell directly modifies the ability of the HSV genome to replicate or alters a specific cellular function on which the virus is dependent. In another case, Tucker & Docherty (2975) showed that HDC-17 cells, when transformed by simian adenovirus, lost their ability to support the replication of HSV. This paper examines the ability of herpes simplex virus type ~ (strain HF) to replicate in adenovirus type 5 transformed cells with the purpose of establishing whether the presence of an integrated adenovirus genome or the cell type plays the major role in restricting the ability of HSV to replicate. We have paid particular attention to an adenovirus type 5transformed rat cell, line 1o7, in which thermosensitive events occur that affect HSV replication. We measured the ability of HSV to produce progeny, synthesize virus DNA and virus proteins in the 2o7 cells and compared these cells with less restricted lines. METHODS

Cells and virus. The following cell lines (source in parentheses) were used in this investigation: io7, a continuous line derived by the transformation of Fisher rat embryo fibroblasts with H5 ts 1o7, an adenovirus type 5 temperature sensitive mutant (Dr H. S. Ginsberg); FER, a line similarly derived with wild type adenovirus type 5 (Dr H. S. Ginsberg); 293-3I, a continuous line of adenovirus type 5-transformed human embryonic kidney cells (Dr R. Weinmann); HT 14a, a continuous line of adenovirus type 5 transformed hamster embryo cells (Dr P. Sharp); T637 , a continuous line of adenovirus type 12 BHK cells (Dr W. Strohl); KB, a continuous line derived from a human epidermoid carcinoma (Dr G. Cohen); rat embryo fibroblasts (REF; Microbiological Associates); BHK 21/Cl 3 (Virology Institute, Glasgow); and W~-38, human embryonic lung fibroblasts (Dr B. Howard). All cells were grown in Eagle's MEM (Eagle, 2959) supplemented with 5 % foetal bovine serum, too units/ml penicillin, 200 t~g/ml streptomycin and 2 mM-glutamine. The 1o7 and HT 14a ceils were grown in the same medium containing o'9 mM-calcium and o.l mM nonessential amino acids (Freeman et al. 2967). Herpes simplex virus type I strain HF was passaged at low multiplicity (less than o.I p.f.u./cell) in KB cell monolayers in media supplemented with o.l mM-arginine and virus was titrated on BHK/CJ3 monolayers as previously described (Flanagan, I967). All infections were performed at varying multiplicities in the appropriate growth medium containing o.l mM-arginine. Virus was adsorbed for 2 h, removed, the cells washed twice with media and overlaid with media containing o.I mM-arginine and 2o m~-Hepes buffer, pH 7"4. Tn order to assay for infectious progeny, cells and media were harvested at appropriate times and stored for less than 7 days at -2o°C. Samples were thawed, the virus released by sonic treatment and titred on BHK 21/C23 cells. Quantification o f H S V - D N A . A modification of the procedure described by Cohen et aL 097I) was used with 3H-thymidine (methyl-3H-thymidine, Io Ci/mmol, New England Nuclear) or 3H-hypoxanthine (9"o Ci/mmol, New England Nuclear) as the label. At various times after infection, cells were labelled for 2 h periods with 25 tzCi/ml of 3H-hypoxanthine or IO tzCi/ml 3H-thymidine. Cells were washed and harvested in TEN buffer (50 mM-tris, pH 8-2; o'I5 M-NaC1, io mM-EDTA), SDS was added to a final concentration of 1% and the mixture was incubated at 37 °C for 3o min. Heat-treated pronase was added to a

Modulation o f herpes simplex virus replication

299

final concentration of ~ mg/ml and the incubation was continued for 2 h at 37 °C and then overnight at room temperature. Samples were extracted at room temperature with equal vol. of phenol saturated with T E N buffer followed by extraction with an equal vol. of chloroform. Extracts labelled with 3H-thymidine were adjusted to a density of 1.71I g/ml with solid CsC1 in preparation for equilibrium centrifugation. Extracts labelled with 3H-hypoxanthine were precipitated by the addition of NaC1 to 0"3 M and two vol. of ice-cold absolute ethanol. The precipitate was resuspended in o-1 x SSC (I 5 mM-NaCI, ~'5 mu-sodium citrate) and incubated for 3o min at 37 °C with heat-treated RNase A at a final concentration of I mg/ml followed by an additional incubation with Io units/ml RNase T1 for 3o min. The extensive RNase treatment was necessary because 9° to 95 % of the 3H-hypoxanthine was incorporated into R N A which obscures quantification of D N A in CsCI density gradients. RNase treated extracts were adjusted to a density of t'7I ~ g/ml with CsCI and samples were centrifuged in a Beckman type 40 rotor for 72 h at 3oooo rev/min. Gradients were fractionated on to Whatman filter paper discs, dried, washed twice with IO% trichloroacetic acid (TCA) and twice with 95 % ethanol. The per cent of virus D N A synthesis was calculated by comparing radioactivity in the dense virus peak (I'73 g/ml) to total radioactivity in virus and host (1"7o5 g/ml) peaks. Densities were determined by refractive index measurements across parallel gradients. Assay of HSV-specific proteins. At appropriate times p.i., cells were washed three times with methionine-free, serum-free MEM and labelled for 2 h with IO p.Ci/ml of zsS-methionine (2oo Ci/mmol; Amersham-Searle) in the same medium. To stop incorporation, the cells were washed with ice-cold Dulbecco's PBS and harvested in o'6 ml lysis solution (1% SDS, 3 % fl-mercaptoethanol, 50 mM-tris, pH 6.8). Equal amounts of protein were diluted ~ : a in sample buffer (5% SDS, 8o mM-tris, pH 6.8, 1o% glycerol, 3 % fl-mercaptoethanol, o-1% Bromophenol blue), boiled for z min and subjected to electrophoresis on I5 % (w/v) SDSpolyacrylamide gels (Maizel, I97I). Gels were stained in 0.2 % Coomassie blue. Destained, dried gels were subjected to autoradiography on Kodak X-ray film.

RESULTS

The effect of temperature on HSV replication in adenovirus transformed cell lines We first assessed the effect of incubation temperature on HSV production in 107 cells by carrying out growth curves at 34 and 37 °C. Cells were infected at adsorbed multiplicities of 2o to 25 p.f.u./cell. Indirect immunofluorescent studies using antisera prepared against HSV-infected KB cells have shown that under these conditions all the cells were infected (data not shown). The growth curves shown in Fig. I (a, b) and Table I suggest that the ability of this cell line to replicate HSV is strongly dependent on temperature with the more permissive state being at 34 °C. In order to determine whether this ts phenomenon was a characteristic of the transforming properties of the adenovirus genome or if it was due to the parent REF cells, growth curves of HSV in R E F cells were performed at 32 ° and 37 °C. As shown in Fig. 1 (c, d) and Table l, there was a slight difference in the amount of virus produced per cell at the two temperatures but this was due to a faster rate of replication at 37 °C followed by thermal inactivation of the released progeny virus at later times. Nevertheless, at 37 °C the titres obtained from REF cells were considerably higher than those obtained from the Io7 cells. These results indicate that the temperature dependent restriction in ~o7 cells is a result of the transformation of REF cells by adenovirus. Expanding on these observations, we examined the ability of HSV- l to replicate in other lines of adenovirus transformed cells. These results are shown in Table I. FER, a line

300

R. S T E N B E R G , Table

AND

L. P I Z E R

Replication of HSV in rat, hamster and human cells

I.

Temperature (°C)

Cell line

D. S P E C T O R

Input , - - ~ ' - ~ Total p.f.u, p.f.u./cell

R a t cells IO7 IO7 FER FER REF REF

34 37 32 37 32 37

8-4 x 8. 4 x 2'4 x 2'4x 8.6x 9.2 x

H a m s t e r cells T637 HTI4a HTl4a BHK/CI 3

37 32 37 37

4 " 8 x 1o 7 4'8 x Io 7

H u m a n cells 293-3 I WI-38

37 37

I

107 107 io s I0 s 106 io 6

20 20 5 5 20 20

3"0 x IO s

IO II

I

(a)

107 at 37 °C

2-6 x 2"1 x 3.2 x 2"6x 2.8x 3.4 x

lO7 io~ i07 107 1o e 10 e

1"5 x 107

B urs t 0 2 to 24 h) r

Tot a l p.f.u, I ' 2 X 108

3"6 x Io 7

p.f.u./cell 22

4.2 x i o 7 8.i x i o v 7"8 x i o 7

3"6 4"o 0.8 290 [70

3"2 × 109 I' 4 x IO s 6"6 x IO7

IO0 31 I4

2"0 x I 0 s

II

6"I X 106 6-8 x 1o 6

I ' I X 107

8

I ' I X 106

2"8 X I 0 s

207

2 . 6 x 1o6

II 25

L e s s t h a n IO

I'O × 107 9"OX IO 7

42 I13

2"0 x IO 7

I

Eclipse (3 to 4 h) T o t a l p.f.u.

I

I

8"OX 105

I

I

I

I

(b) 107 at 34 °C

5 × 10 s

.---4

t0s

5 x10 7

.q

5

x

I

I I

(c)

REF at 37 °C

I

I

I

I

t

I

I

(d) REF at 32 °C

I07

10 7

I

1

I

I

I

le

l

I

I

I

3

6

9

12

24

3

6

9

12

24

Time p.i. (11) Fig. I. G r o w t h curves of HSV-I in lO7 cells a n d R E F cells. Infections were p e r f o r m e d at hi gh (a, c) a n d lo w (b, d) temperatures. Cells infected at an m.o.i, of zo p.f.u./cell were ha rve s t e d at the indicated times a n d infectious virus titred on B H K 2 I / C t 3 cells. The n u m b e r of p . f . u . / p l a t e was d e t e r m i n e d by m e a s u r i n g cell-associated and free virus.

3Ol

Modulation o f herpes simplex virus replication

I I I I ,j,..-\'¢ J

--

i

2

4

6

t 8

10

20

Time (h)

Fig. 2. G r o w t h curve o f HSV- r in 293-31 cells at 37 °C. Cells infected at an m.o.i, o f t t p.f.u./cell were harvested at the indicated times a n d infectious virus titred on B H K 21/C13 cells. T h e n u m b e r o f p . f . u . / m l was determined by m e a s u r i n g cell-associated a n d free virus.

similar to lO7 (REF cells transformed by adenovirus type 5), showed a strong restriction on HSV-l replication. Again, indirect immunofluorescent studies using antisera prepared against HSV infected KB cells have shown that under these conditions, 9o to 95 % of the cells were infected. These cells showed marked thermosensitivity and even at 32 °C might be classified as non-producers. As the temperature was increased, the FER cell line produced barely detectable amounts of virus. The effect of temperature on HSV replication in transformed rat cells was reminiscent of the work of Sprecher-Goldberger et al. 0973) with adenovirus type 12 transt'ormed BHK cells. In contrast to the work of the above authors, our strain of HSV-I replicated in a line of adenovirus type I2 transformed BHK cells (T637) producing nearly as much as the control non-transformed B H K / C l 3 cells (Table l). We also studied a line of hamster embryo cells transformed with adenovirus type 5 (HT T4a) and found that this line replicates H SV at either 32 ° or 37 °C (Table I). This result suggests that transformed hamster cells are more permissive than the analogous rat cell lines. The adenovirus type 5 transformed human line (293-31) was also studied and shown to be permissive for HSV infection (Fig. 2 and Table ~). This line which had been transformed with fragments of adenovirus D N A (Graham et al. 0977)) replicated HSV-I to a slightly lesser extent than human control cells (W 1-38). The virus growth curve in these cells shows that HSV is rapidly eclipsed and has a latent period characteristic of permissive cells. These results with the 29z-31 ceils and the HT 14a cells show that adenovirus type 5 transformation does not necessarily lead to cell lines restricted or ts for HSV replication. DNA replication under restrictive conditions It has been suggested that the inability of HSV to replicate in certain transformed cell lines may be due to the inability of the cell to synthesize virus D N A (Docherty et al. 1972; Sprecher-Goldberger et al. 1973; Tucker & Docherty, 1975). However, in previous experiments designed to investigate this question DNA was labelled with thymidine and this II

VIR 44

R. S T E N B E R G , D. S P E C T O R

3o2

I

2-4 h 6 -

I

I

/~

(a)

A N D L. P I Z E R I

I

2-4 11

34 °C

I

(d)

-

4

2

/

' R

5-7 11

(b)

,

4

X

4

10

20

30

10

20

3O

Fraction Fig. 3. D N A synthesis in IO7 cells infected with HSV. 1o7 cells infected at an m.o.i, of 2o p.f.u./cell at 34 ° (a, b, c) and 37 °C (at, e, f ) were labelled with z5 #Ci/ml 3H-hypoxanthine at the times indicated, the samples were extracted as described in the text and subjected to CsCI density centrifugation. Decreasing density is shown from left to right.

Table 2. Quantification of HSV and cellular DNA Conditions of infection

Radioactivity in D N A

Temperature Total Virus D N A Fraction of Cellular D N A Fraction of (°C) Multiplicity Time (h) c t / m i n × to -4 c t / m i n x lO-4 total ( ~ ) c t / m i n × IO-4 total ( ~ ) 34

Low

37

Low

34 37

High High

2-4 5-7 8-Io 2- 4 5-7 8-Io 5-7 5-7

7"4 7"3 7"8 7"8 4"7 3"6 7"4 8. i

I. 4 2'7 2"9 I "6 I'O I-I 3"I 3"5

19 37 37 20 22 31 42 43

6.0 4"6 4"9 6.2 3"7 2'5 4"3 4'6

81 63 63 80 78 69 58 57

Modulation of herpes simplex virus replication (a)

I

1

I

303

--[

1.73

50-

---

40-

--

1,77_ E

1-75 "~ 1.73 1.71 ~= c~

30-

1.69

20-

-

| (b) 25 2O 15 10 5

10

20 30 Fraction numbe~

413

Fig. 4. D N A synthesis in (a) R E F (5 to 7 h p.i.) and (b) 293-31 (3 to 5 h p.i.) cells infected with HSV at 37 °C. Ceils were infected at an m.o.i, o f 2o p.f.u./cell and midway through the virus replicative cycle the cells were labelled with io # C i / m l SH-thymidine. Samples were prepared as described in the text and subjected to CsCI density centrifugation.

procedure may give invalid measurements of the amount of D N A made if there was a difference between the cell types in the expression of HSV-specific thymidine kinase. We avoided this problem by using radioactive hypoxanthine to label D N A by way of adenine and guanine nucleotides. The entry of these nucleotides is not dependent on thymidine kinase or any other known virus pathway. Virus D N A synthesized in lo7 cells after infection at 34 ° and 37°C was quantified by analysis on CsC1 gradients. The results in Fig. 3 and Table 2 show that at 34 °C (Fig. 3a, b, c), virus D N A synthesis reaches a maximum at 5 to 7 h p.i. and continues at the same level for Io h. Total D N A synthesis remained constant from 2 to l o h p.i. and host cell D N A synthesis failed to shut-off to any great extent, remaining at 5o % of that found in mockinfected cultures at Jo h p.i. (data not shown). At 37°C (Fig. 3 d, e,f), the production of radioactive virus DNA reached a normal low level by 2 to 4 h p.i. but higher levels of virus DNA synthesis were not obtained at later times. The apparent increase in virus D N A synthesis at 37°C seen between 8 to IO h (Table z) was due to a decrease in total D N A synthesis rather than an increase in the amount of virus DNA produced. For comparison, the pattern of DNA synthesis in two permissive cell lines (REF and 293-3 I) was determined after HSV infection (Fig. 4)- Only virus D N A was synthesized in the REF cells (Fig. 4a), but in the Ad 5 transformed cells both virus and cellular DNA were synthesized (Fig. 4b). IIm2

3o4

R. S T E N B E R G , D. S P E C T O R (a)

(b)

(c)

(d)

A N D L. P I Z E R (e)

V

-55K -130K

V

7,

-59K

-45K

-35K

Fig. 5. SDS-polyacrylamide gel electrophoresis of t 37 and KB cells infected with HSV. Cells were infected (5o p.f.u./cell) as described in the text and at the appropriate times were washed with methionine-free, serum-free MEM and labelled in the same media with Io #Ci/ml 35S-methionine. Polypeptides were displayed on l o ~ SDS-polyacrylamide gels as described in the text. Virus proteins are designated V.

Synthesis of HS V specific proteins The capacity of ~o7 cells to synthesize virus proteins was analysed by means of polyacrylamide gel electrophoresis of radioactive cell extracts. Polyacrylamide gel electrophoresis of 35S-methionine labelled polypeptides obtained after infection of 1o7 cells at 37°C is shown in Fig. 5- Panel (a) represents uninfected control cells. Panels (b), (c) and (d) represent

Modulation of herpes simplex virus replication (a)

(b)

(c)

(d)

(e)

305

(D

-155K

V V

-130K

V

V

-59K

-45K :: L : : :

z

g~

--35K

:.:? :

½

....

~~{~:

~:~i~ ¸

Fig. 6. SDS-polyacrylamide gel electrophoresis of 293-31 and KB cells infected with HSV. Cells were infected at an m.o.i, of z5 p.f.u./cell and labelled for 2 h with to #Ci/ml zsS-methionine. Panels (a) and ( f ) represent KB cells labelled 8 h p.i. Panels (c), (d), (e) represent z93-31 cells infected and labelled at 4 (c), 7 (d), and IO (e) h p.i. Panel (b) represents uninfected 293-3I cells. Extracts were subjected to electrophoresis as described in the text.

proteins synthesized from 2 to 4, 4 to 6 and 6 to 8 h p.i. respectively. Panel (e) represents HSV infected KB cells synthesized late in infection (8 to lo h). Comparison of (b), (c) and (d) with (e) demonstrated that all the HSV-specific polypeptides found in the KB extract are present in the 1o7 cells infected at the non-permissive temperature (37 °C). From this result, one can conclude that the absence of virus structural proteins (those made late) is not the reason for the low yield of infectious progeny virus found at 37°C. For comparison the

306

R. STENBERG, D. SPECTOR AND L. P I Z E R (a)

(b)

(c)

(d)

(e)

-155K

m v

-130K

v

-59K

%

,

-45K

:!J!'JJ!i/ < ?, ?



, ~

-35K

Fig. 7. SDS-polyacrylamide gel electrophoresis of REF and KB cells infected with HSV. Cells were infected at an m.o.i, of z5 p.f.u./cell and labelled for 2 h with to/~Ci/ml a~S-methionine. Panel (a) represents uninfected REF cells. Panels (b), (c), (d) represent infected REF cells labelled at a, 4 and 6 h p.i. respectively. Panel (e) represents KB cells labelled 8 h p.i. Samples were subjected to electrophoresis as described in the text. radioactive polypeptides made in R E F cells and 29i-31 cells at different times after infection were analysed (Fig. 6 a n d 7). With b o t h cells there was a rapid disappearance of cellular polypeptides from the gel pattern a n d a marked overall drop in m e t h i o n i n e i n c o r p o r a t i o n into proteins (data n o t shown). A n interesting p o i n t is that host protein synthesis in 1o7 cells was n o t shut-off after infection to the extent found in the permissive R E F cells.

Modulation o f herpes simplex virus replication

307

Effect of increased multiplicity of infection on H S V replication Several laboratories have described situations where the restricted growth of HSV could be overcome by increasing the m.o.i. (Aurelian & Roizman, 1965; Doller et al. 1973)- We measured DNA synthesis at 5 to 7 h p.i. and the production of infectious progeny virus after infection of IO7 cells at a higher multiplicity (ioo to 13o p.f.u./cell). The gradients showed that at 34 and 37 °C, the amount of virus DNA synthesized was virtually the same and was increased over that produced at the lower m.o.i. (Table 2). This result suggests that increasing the m.o.i, would eliminate the ts phenomenon. To examine this possibility, a growth curve was carried out, on line IO7, at 37°C with an input m.o.i, of 13o p.f.u./cell. The result of this experiment shows that 1.4 p.f.u./cell is produced. The values for the virus eclipse and burst are comparable to those found at the lower m.o.i. These data indicate that despite the fact that the higher input multiplicity could overcome the defect in DNA synthesis, it was not able to increase the yield of infectious virus. Whatever the defect in the io 7 cells, it is not likely that it can be overcome by increasing the amount of early virus products by providing a larger number of virus genomes. DISCUSSION

The replication of herpes simplex virus requires a coordinated production of virus proteins and the subsequent assembly of the replicated DNA with the structural proteins of the virion (Honess & Roizman, J973, x974). The herpes virus genome is large and many virus products are synthesized. Therefore, one needs to be concerned with a series of reactions that require complex interactions between the cells' metabolic machinery and the virus. It is clear that disruption of any of these interactions by modification of the cells or by virus mutations will upset the coordinated synthesis of virus macromolecules and reduce the yield of virus progeny. There are reported instances where herpes simplex mutants have an altered host range suggesting that virus genes control the interaction with the cell (Koment & Rapp, 1975). Furthermore, there is an interesting recent report by Yanagi et al. (I978), that describes the inability of herpes simplex virus to replicate in cell mutants altered in their ability to complete the cell cycle. Studies with these two types of mutants indicate that changes within the virus or within the cells' regulatory mechanisms can disrupt the basis for a productive infection. This paper focuses on the influence exerted by the cell on herpes simplex virus type t replication. Cells of different origins transformed by type 5 adenovirus show different capacities to support HSV replication suggesting that even though HSV contains an appreciable amount of genetic information for synthetic reactions, virus processes are strongly dependent on the cell type infected. Our results clearly show that adenovirus-transformed human and hamster cells maintain their permissiveness for herpes simplex virus. Rat cells transformed by the same virus are either non-permissive when infected with HSV or show a strong temperature dependence for the production of infectious virus. It has been shown that within any particular adenovirus transformed cell line, the transformants show variation from clone to clone. However, a number of features are common to cells transformed by adenovirus type 5; namely the insertion of a fraction of the left hand end of the adenovirus genome and the expression of the T antigen (Graham et al. 1974; Sambrook et al. I974). Thus, with a battery of adenovirus transformed cells it should be possible to define how these particular adenovirus functions influence HSV replication. Our experiments with the transformed rat cell, line ~o7, show that virus-cell interactions can be altered by small shifts in temperature. In these cells, at 37 °C, some virus DNA replication and virus protein synthesis occurs, but the yield of infectious progeny was greatly reduced.

3o8

R. S T E N B E R G ~ D. S P E C T O R

A N D L. P I Z E R

Increasing the m.o.i, increased the quantity of virus DNA made but not the number of infectious progeny. Our observation that the HSV induced shut-off of cellular DNA and polypeptide synthesis appeared less efficient in comparison to that occurring in the R E F cells or the permissive human transformed line, suggests that inhibition of cellular macromolecular synthesis may be an important factor in efficient HSV replication. In an earlier paper we examined another aspect of host shut-off in the 1o7 cells, the influence of HSV infection on the metabolism of cellular RNA. Our results show that synthesis of ribosomal R N A was inhibited by HSV infection but that poly(A)-containing R N A was synthesized and transported to the cytoplasm in a normal fashion (Spector & Pizer, 1978). The synthesis and processing of adeno-RNA in the 1o7 cells is modified after HSV infection. It would be informative to carry out similar experiments on adeno-RNA metabolism at lower temperatures or with the permissive adeno-transformed cells (293-31 cells). The studies reported in this paper demonstrate that transformation by adenovirus changes the properties of the cells so that their ability to support the replication of HSV is modified. The modification may be stable and involves a cell-virus interaction that should be of some concern when one is investigating the effect of super-infection by HSV on the cellular processes. While these transformed cell lines provide model systems with considerable promise for unravelling the influence of HSV on complicated processes such as m R N A maturation, caution will be required in extending this model system to the non-transformed cell. These observations may be extended to a consideration of the various tissues within the whole animal. It is clear that the replication of HSV occurs to different extents within the different cell types of an infected animal and that the expression of the virus genome will be a ffected by the state of development and metabolic capacity of the infected organs. Whether endogenous viruses influence the fate of HSV infection and also influence the establishment and maintenance of latency, is worth consideration. This research was supported by Public Health Service grant DE-o2623 from the National Institute for Dental Research and grant NP-2o9 from the American Cancer Society. David Spector was supported by grant 2 TI AI-2o 3 from the National Institute of Allergy and Infectious Diseases. We thank Pia Nystrom for carrying out the work with the H T I 4 a and WI-38 cell lines, Richard Guilfoyle for performing the studies on the z93-31 cells shown in Fig. 4 and 6 and Walter Pfendner and Meredith Peake for excellent technical assistance. We would also like to acknowledge Dr Gary Cohen for providing guidance and support throughout these investigations and Dr Margaret Miovic for critically reviewing the manuscript.

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(Received 3o August I 9 7 8)