J. gen. Virol. (1984), 65, 1225 1228. Printedin Great Britain
1225
Key words: HSV/shut-off/protein synthesis/mRNA
Early Virion-associated Suppression of Cellular Protein Synthesis by
Herpes Simplex Virus is Accompanied by Inactivation of mRNA
By M. L. F E N W I C K *
AND M. M. M c M E N A M I N
The Dunn School of Pathology, University o f Oxford, South Parks Road, Oxford OX1 3RE,
U.K.
(Accepted 5 April 1984)
SUMMARY
Vero cells infected with herpes simplex virus in the presence of actinomycin or
cycloheximide, or with u.v.-inactivated virus, suffered a rapid loss of functional
m R N A as determined by translation in vitro. It is suggested that a virion-associated
factor causes a structural change in the m R N A of the host cell.
Some strains of herpes simplex virus (HSV) carry in their virions a factor that causes, or
initiates, the suppression of cellular protein synthesis, a process known as early, primary or
virion-associated shut-off, to distinguish it from a distinct phenomenon known as delayed,
secondary or expression-dependent shut-off which requires the synthesis of viral protein
(Nishioka & Silverstein, 1978; Fenwick & Walker, 1978; Fenwick & Clark, 1982a; Read &
Frenkel, 1983). Delayed shut-off involves the degradation of cellular m R N A as identified by
hybridization to a specific D N A probe but early shut-offdoes not (Nishioka & Silverstein, 1978 ;
Inglis, 1982). Inglis & Newton (1981) have shown by translation of extracted m R N A in vitro that
cellular m R N A is inactivated after infection with HSV. We were interested to know whether
inactivation occurs during early shut-off, while the m R N A remains in a hybridizable condition,
or only later, as a result of delayed shut-off.
Infection of Vero cells with strain F of HSV type 1 [HSV-I(F)] or strain G of HSV type 2
[HSV-2(G)] (Ejercito et al., 1968), labelling of proteins and electrophoresis in SDSpolyacrylamide gradient gels were done as previously described (Fenwick et al., 1978).
Extraction of total cytoplasmic RNA and its translation in a reticulocyte lysate were also
described previously (Fenwick & Clark, 1982b). Preliminary tests were carried out to determine
the concentration of RNA giving maximum incorporation of [35S]methionine. In subsequent
experiments this concentration of RNA or twice this concentration were used in duplicate
translations of each RNA sample. The results were the same in both cases.
HSV-2(G), which causes rapid early shut-off (Fenwick & Walker, 1978), was used to infect
duplicate cultures of cells under conditions in which the production of viral proteins was
prevented. One flask of each pair was washed and labelled with 14C-amino acids in the presence
of 2 ~g/ml actinomycin 2 to 2.5 h after infection. The second culture was lysed and the nuclei
removed by centrifugation. Cytoplasmic RNA was extracted and translated in the presence of
[3SS]methionine. Both preparations of labelled proteins were subjected to electrophoresis and
detected by autoradiography. The autoradiograms in Fig. 1 show that closely similar patterns
were obtained by translation in vivo (a) and in vitro (b). Whether the suppression of cellular
protein synthesis was caused by infection in the presence of cycloheximide or actinomycin or
with virus that had been previously inactivated by irradiation with u.v. light (Fig. 1a), in each
case it was accompanied by a substantial loss of functional cellular m R N A (Fig. 1 b). The only
viral proteins that were detected were the ~-polypeptides ICP 4 and ICP 0 which were made
after removal of cycloheximide (Fig. 1 a, lane 5 ; Fig. 1 b, lane 6).
The suppression of cellular protein synthesis by HSV-2 is more efficient in the presence of
actinomycin than with cycloheximide or with u.v.-inactivated virus (our unpublished
observations). This is consistent with a shut-off mechanism involving inactivation of a pool of
m R N A which is being relatively slowly replenished.
0022-1317/84/0000-6064 $02.00©
1984 SGM
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(a)
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1
Mock
+ Act
2
G
+ Act
3
4
UV-G
Mock
+ CX
5
(b)
G
+ CX
1
Blank
2
Mock
+ Act
3
G
+ Act
4
UV-G
5
6
Mock
+ CX
G
+ CX
Fig. 1. Autoradiograms of 7 to 20~ acrylamide gradient gels showing electrophoretically separated
polypeptides synthesized (a) in intact cells 2 to 2.5 h post-infection or (b) in vitro with cytoplasmic RNA
extracted at 2 h post-infection. Cells were mock-infected or infected with HSV-2(G) in the presence of
2 gg/ml actinomycin (Act) or 50 gg/ml cycloheximide (CX) or with virus that had been irradiated
with u.v. light (1.5 x 10- 4 J/mm 2 to virus suspended in 3 ml PBS/1 ~ glucose in a 9 cm diam. Petri dish;
UV-G). ct-polypeptides ICP 4 and ICP 0 are indicated in lanes 5 and 6.
HSV-1 (F) causes a slower early shut-off than does HSV-2(G) (Morse et al., 1978; Fenwick et
al., 1979). This difference is illustrated in Fig. 2(a) and Fig. 2(b) shows that it is correlated with
a partial inactivation of functional host m R N A by HSV-I(F), either u.v.-inactivated or in the
presence of cycloheximide (lanes 2, 3), as compared to the more marked effect of HSV-2(G)
(lanes 4, 5).
Early shut-off is accompanied by the disaggregation of polysomes (Sydiskis & Roizman, 1966;
Fenwick & Walker, 1978). Whether inactivation of m R N A precedes or follows dissociation of
the polysomes is uncertain. A modification near the 5' end of the m R N A to prevent initiation of
translation would not be enough to explain the breakdown of polysomes since the breakdown
also occurs in the presence of cycloheximide, which stops the ribosomes running off the m R N A
(Fenwick & Walker, 1978).
The HSV- 1 mutant tsB7 (Knipe et al., 1981) causes early shut-off at 34 °C but not at 39 °C. W e
have reported that early shut-off caused by u.v.-inactivated tsB7 at 34 °C could be reversed by
raising the temperature to 39 °C (Fenwick & Clark, 1982a). On the other hand, Read & Frenkel
(1983), using mutant vhs4 which is also temperature-sensitive in its early shut-off function, found
that if the temperature was raised to 39 °C after shut-off had occurred at 34 °C in the presence of
actinomycin the suppression was not reversed. Our subsequent (unpublished) experiments with
tsB7 have confirmed that after raising the temperature, the recovery of host protein synthesis
(which was nearly complete within 90 min at 39 °C) was prevented by adding actinomycin,
indicating that the recovery depended on the synthesis of new R N A and was not simply due to
the reversal of temperature-sensitive interaction.
N i s h i o k a & Silverstein (1978) showed that in F r i e n d erythroleukaemia cells early shut-off by
HSV-1 (F) was not accompanied by the disappearance of hybridizable globin m R N A . W h e t h e r
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Short communication
(a)
1
Mock
2
F
+ CX
3
UV-F
4
G
+ CX
5
(b)
UV-G
1
Mock
2
F
+ CX
3
4
UV-F
G
+ CX
5
UV-G
Fig. 2. Inactivation of cellular m R N A by HSV-I(F) and HSV-2(G) in the presence of cycloheximide or
after u.v. irradiation. (a) Proteins made in intact cells 2 to 2.5 h post-infection (7 to 2 0 ~ acrylamide
gradient gel); (b) proteins made in vitro with cytoplasmic R N A extracted 2 h post-infection (10~
acrylamide gel). ~-polypeptides ICP 4, 0, 22 and 27 are indicated to the right of the appropriate lanes.
this is also true of total Vero cell m R N A is not known. We can conclude, however, that the early
virion-associated shut-off of cellular protein synthesis, which is accompanied by the
disaggregation of polysomes, involves some alteration of host m R N A to the extent that it is no
longer functional in a translation system in vitro.
This work was supported in part by a grant from the Cancer Research Campaign to Professor H. Harris.
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(Received 19 January 1984)
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