Bioscience Reports, Vol. 6, No. 11, 1986
Transcriptional Induction of Cellular Gene
Expression During Lytic Infection with
Herpes Simplex Virus
L. M. Kemp, 1 P. M. Brickell, 2'3 N. B. La Thangue 2 and
D. S. Latchman 1'4
Received November 27, 1986
KEY WORDS: Herpes simplex virus; cellular genes; transcriptional induction
Herpes simplex virus Type 2 causes a severe repression of host cell biosynthesis at a
number of levels. We show that despite this, non-viral cDNA clones derived from
cellular RNA species which accumulate to high levels after infection can be isolated
using differential screening techniques. By using nuclear run-off assays, we have shown
that this RNA accumulation is mediated by transcriptional induction of the
corresponding cellular genes.
INTRODUCTION
Lytic infection with Herpes simplex viruses (HSV) types 1 and 2 causes a well
characterised shut off of host-cell protein synthesis (Sydiskis and Roizman, 1968;
Fenwick and Walker, 1978; Inglis, 1982) which has a number of components,
including polysome disaggregation (Nishioka and Silverstein, 1978) and degradation
of cellular RNAs (Nishioka and Silverstein, 1977). Together, these effects produce a
drastic reduction in the levels of host messenger RNA's and proteins. Recent work in
our laboratory however, has led to the identification of a small number of cellular
proteins present at low levels prior to infection which accumulate in increased amounts
after infection (La Thangue et al., 1984). This finding indicates that the viral repression
Department of Zoology, University College London, Gower St, London WC1E 6BT.
2 CRC Eukaryotic Molecular Genetics Research Group, Department of Biochemistry, Imperial College
London SW7 2AZ.
Present address: Medical Molecular Biology Unit, Courtauld Institute of Biochemistry, The Middlesex
Hospital Medical School.
4 To whom correspondence should be addressed.
945
0144-8463/86/1100-0945505,00/09 1986PlenumPublishingCorporation
946
Kemp, Brickell, La Thangue and Latchman
of host function is not complete, and suggests the possibility that some cellular RNA
species may accumulate in viral infection in a manner paralleling the accumulation of
these cellular proteins.
We report here the isolation of cDNA clones derived from such RNA species and
the use of these clones to show that viral infection results in the transcriptional
activation of their corresponding genes.
MATERIALS AND METHODS
Library Construction and Screening
In order to prepare the eDNA library, sub-confluent cultures of BHK cells (clone
13: Macpherson and Stoker, 1962) were infected with HSV-2 strain 333 at a
multiplicity of infection of 20 pfu/cell and cytoplasmic polyadenylated RNA isolated
eight hours after infection by the method of Favolaro et al. (1980). Complementary
DNA was then synthesised using the method of Watson and Jackson (1985) and after
addition of EcoR1 linkers inserted into the bacteriophage vector lambda gtl0 as
described by Huynh et al. (1985). A library of approximately 2 • 10 s independent
recombinants was obtained. Replicate filters taken from the library were treated as
described by Benton and Davis (1977) prior to hybridisation with 32p labelled eDNA
prepared from the mRNA of HSV-2 infected BHK cells or exactly parallel cultures
which were mock-infected. For subsequent screening, phage stocks were spotted onto
a lawn of growing E. coli and after overnight incubation to allow lysis to occur,
replicate filters were taken and hybridised either with labelled eDNA or with HSV-2
DNA labelled by the method of Feinberg and Vogelstein (1983).
Northern Blotting
Northern blotting was carried out as previously described (Brickell et al., 1983)
using equal amounts (5 #g) of mRNA from uninfected or HSV-2 infected (eight hours
post-infection) BHK cells. Inserts purified from the eDNA clones by EcoR1 digestion
and gel electrophoresis were labelled by oligonucleotide labelling (Feinberg and
Vogelstein, 1983) and used as probes.
Nuclear Run-off Assays
Nuclei prepared from uninfected and infected cells were allowed to continue RNA
synthesis in the presence of 32p labelled GTP under the conditions described by Mason
et al. (1986). After purification of labelled RNA it was used to probe replicate dot blots
of DNA from up-regulated clones.
RESULTS AND DISCUSSION
A eDNA library was constructed using poly A + RNA prepared from BHK 21 cells
infected with HSV-2. An aliquot of the library (104 plaques) was plated out and
HSV Induction of Cellular Genes
947
duplicate filters screened with labelled cDNA prepared from the messenger RNA of
either HSV-2 infected BHK cells or exactly parallel cultures which were mock infected.
Plaques which react under these conditions can be characterised according to their
relative reaction with the two different RNAs (Table la), the relative reaction of a clone
being determined by the level of its specific RNA in the two different samples
(Crampton et al., 1980).
Table
1
(a) Primary screenof cDNA clonesfrom HSV-2infectedcellswith cDNA from uninfectedand infectedcells
Number
Class I
Present only in infectedcells
317
Class II
Elevated in infectedcompared to uninfected cells
20
Class IlI
Present at equal levels in infected and uninfectedcells
58
CIass IV
Elevated in uninfectedcompared to infectedcells
67
Class V
Present only in uninfectedcells
45
(b) Secondary screeningof 115 Class I cDNA clones
Class I
Class II
Class III-V (or failed to react)
94
10
11
As might be expected a considerable number of clones appear to be derived from
RNA species repressed upon infection either partially (class IV) or more extensively, to
a level where reaction can be detected only with the uninfected cell RNA (class V)~
More surprisingly however, a number of clones (class III) appear to be derived from
RNA species not subject to such repression. A small number of clones were found to
show weak hybridisation to uninfected cell RNA and a much stronger hybridisation to
the infected cell RNA (class II). Such behaviour would be expected of clones derived
from cellular RNA species which increase in abundance upon viral infection. However,
the much larger class of clones which react only with infected cell RNA (class I),
although likely to be predominantly derived from viral genes may also contain cellular
sequences accumulating upon viral infection but present before infection at levels
undetectable by plaque screening.
In order to confirm the induction of expression of class I and class II clones
observed in the primary screening, 115 class I clones and all class II clones were spotted
out onto a lawn of bacteria and after lysis had occurred, replicate filters were taken and
screened as before. Figure 1 shows the results of such a screening in which putative
induced clones of class I have been spotted out along with control clones from class III
which show no change in expression upon infection. This secondary screening not only
confirms the results of the original screening but because it is of greater sensitivity than
the initial screen resulted in the detection of low levels of hybridisation given by several
class I clones with uninfected cell RNA (Fig. 1, Table lb).
The reclassification of several such clones as class II and hence likely to be cellular
in origin suggested that other de novo induced cellular RNA species might still be
represented amongst the remaining class I clones. To test this possibility the class 1
clones were screened for homology to H SV-2 strain 333 DNA. Because of the reported
homology ofHSV-2 DNA to mammalian RNA species (Peden et al., 1982; Puga et al.,
1982; Jones et al., 1985) all class II clones were also included in this screen to confirm
948
Kemp, Brickell, La Thangue and Latchman
Fig. 1. Secondary screening of cDNA clones. Plaques from the
primary screen were plated out on a lawn of growing E, coli and
after overnight incubation replacate filters were screened with
cDNA prepared from cytoplasmic poly A + RNA of cells either
mock infected (A) or eight hours after infection with HSV-2 (B). All
clones were originally picked as class I with the exception of three
class III clones (indicated by horizontal arrows) included as
controls. One clone (indicated by the vertical arrow) was
reclassified as class II on the basis of its weak reaction with
uninfected cell cDNA in this experiment.
their cellular nature. The screening was originally carried out by plaque hybridisation
(Fig. 2), negative clones being subsequently rescreened by Southern blotting using
inserts purified from phage DNA. The results of this screen are summarised in Table 2.
Although, as expected, the majority of class I clones react positively with HSV DNA
and are therefore derived from virus sequences, a significant minority are negative in
this screen and therefore have no homology to virus DNA. These clones must therefore
be derived from cellular RNA species present prior to infection at levels undetectable
by plaque screening and induced upon infection. Screening of class II clones with HSV
DNA, gives the opposite result to that seen with the class I clones. Thus as might be
expected for clones showing some hybridisation to uninfected cell RNA, the majority
fail to react with HSV DNA and are therefore derived from cellular RNA species
present at detectable levels before infection and accumulating to a higher level upon
infection. A minority of class II clones did react with virus DNA. These are likely to be
Fig. 2. Screening of cDNA clones for homology to HSV-2 DNA.
Clones were plated as described in the legend to Fig. 1 and screened
with labelled HSV-2 DNA, Key: 1. Clone 7 (class I) 2. Clone 14 (class
I) 3. Clone 123 (class III) 4, Non recombinant 2gt 10.5. Clone 72 (class
II) 6. Clone 94 (class II).
HSV Induction of Cellular Genes
949
Table 2. Hybridisationof Class I and C1ass II clones with HSV-2 DNA
Class I (novel)
Class II (increased)
HSV positive
HSV negative
Total
66
16
29
27
95
43
Screening of the original library, indicated that it contained approximately4 ~o HSV-2
positive clones.
derived fi'om viral sequences having homology to transcribed cellular sequences and
therefore showing reaction with uninfected cell RNA, the greater reaction with infected
cell RNA being attributable to reaction with viral as well as cellular RNA.
To further study the RNAs recognised by the cDNA clones, Northern blotting
was performed with representative virus negative clones from each of the five classes
defined by plaque screening using RNA from infected and mock infected BHK cells.
This experiment (Fig. 3 panels a-e) confirmed the results of the plaque screening with
the specific RNA(s) detected by the clones being either increased (a and b), decreased (d
and e) or remaining the same upon infection (c). Of particular interest was the result
with a class II clone (Fig. 3b) which hybridises to several discreen RNA species present
at low levels before infection and at much higher levels after infection.
Fig. 3. Northern Blot of cytoplasmicpoly A+ RNA from HSV-2infected (eight hours post-infection,
track 1) or uninfected(track 2) BHK cells, probed with representativevirus negativeclonesfrom class I
(panel A), class II (panel B), class III (panel C), class IV (panelD) and class V (panel E). Arrowsindicate
the positions of ribosomal RNA markers.
In order to assess the level at which this accumulation was mediated, we carried
out nuclear run-off assays to directly assess the transcription rates of the cellular genes
corresponding to the class I and II clones. In such assays (Fig. 4) increased
transcription could be detected with both class I and class II clones despite an apparent
decrease in transcription detectable with the control (class III) clones.
The identification of cellular RNA species which accumulate upon infection
parallels our previous finding of cellular proteins which behave similarly. It is clear
therefore that the well-established repression of host cell function by HSV is not as
complete as has been assumed. Rather, against a background of such repression, a
small number of cellular RNAs and proteins accumulate. It has previously been shown
950
Kemp, Brickell, La Thangue and Latchman
Fig.& Nuclear run-off assay. Nuclei isolated from HSV-2
infected (eight hours post infection, panel A) or mockinfected (panel B) cells were incubated in vitro with labelled
nucleotide under the conditions described by Mason et al.
(1986). The labelled products were used to probe replicate
dot blots of DNA from 2gtl0 vector (1), clone 72 (class II,
2), clone 33 (class I, 3) and clone 123 (class III, 4).
that several heat shock proteins accumulate in HSV infected cells (La Thangue et al.,
1984; N. B. La Thangue and D. S. Latchman; unpublished observations). To test
whether any of our clones were derived from such heat-inducible genes, we screened
replicate filters taken from them with labelled cDNA prepared from the mRNA of
control (37~ or heat shocked (42~
The results of this study (data not shown)
indicated that n.one of the clones exhibited heat inducibility. In agreement with this we
have recently shown that the accumulation of the heat shock protein hsp90 we have
observed in HSV-2 infected cells (N. B. La Thangue and D. S. Latchman; unpublished
observations) is not paralleled by increasing levels of hsp90-specific RNA, suggesting
that this accumulation occurs through increased translation or protein stability in
infected cells. The accumulation of the RNA species identified here thus appears to be
specific to HSV infection rather than a response to generalised stress. The role of these
RNAs and the protein they presumably encode in the cell's response to viral infection is
unclear. In particular any role that differences in their induction may play in the onset
of latency (Baringer and Swoveland, 1973) or transformation (Galloway and
McDougall, 1983) by HSV is at present obscure.
In order to throw light on this problem we are currently investigating the levels of
the induced RNA species in cells transformed or latently infected by HSV. In addition
sequencing the cDNA clones corresponding to the induced RNAs should allow the
identification of the genes from which they are derived and possibly the functions of
their protein products.
ACKNOWLEDGEMENTS
We are grateful to the Cancer Research Campaign for supporting this work.
REFERENCES
Baringer, J. R. and Swoveland, P. (1973). N. Eno. J. Med. 228:648-650.
Benton, W. D. and Davis, R. W. (1977). Science 196:I80-182.
Bdckell, P. M., Latchman, D. S., Murphy, D., Willison, K. and Rigby, P. W. J. (1983). Nature 306:756-760.
Crampton, J., Humphries, S., Woods, D. and Williamson, R. (1980). Nucl. Acids Res. 8:6007~017.
HSV Induction of Cellular Genes
951
Favolaro, J., Treisman, R. and Kamen, R. (1980). Meth. Enzymol. 65:718-749.
Feinberg, A. P. and Vogelstein, B. (1983). Anatyt Biochem. 132:6-13.
Fenwick, M. L. and Walker, M. J. (1978). J, Gen. Virol. 41:37-51.
Galloway, D. A. and McDougall, J. K. (1983). Nature 302:21-24.
Huynh, T. V., Young, R. A. and Davis, R. W. (1985). In DNA Cloning Techniques, a Practical Approach
(Glover, D., ed.), IRL Press, Oxford, pp. 49-78.
Inglis, S. C. (1982). Mol. Cell Biol. 2:1644-1648.
Jones, T. R., Parks, C. L., Spector, J. D. and Hyman, R. W. (1985). Virolo#y 144:384-397.
La Thangue, N. B., Shriver, K., Dawson, C. and Chan. W. L. (1984). EMBO J. 3:267-277.
MacPherson, I. and Stoker, M. (1962). Virology 16:147-151.
Mason, I. J., Murphy, D., Munke, M., Franke, U., Elliot, R. W. and Hogan, B. M. L. (1986). EMBO J.
5:1831-1838.
Nishioka, Y. and Silverstein, S. (1977). Proc. Natl. Acad. Sci. USA 74:2370-2374.
Nishioka, Y. and Silverstein, S. (1978). J. Virol. 27:619-627.
Peden, K., Mounts, P. and Hayward, G. (1982). Cell 31:71-80.
Puga, A., Cantin, E. and Notkins, A. (1982). Cell 31:81-87.
Sydiskis, R. J. and Roizman, B. (1968). Virology 34:562-565.
Watson, C. J. and Jackson, J. (1985). In DNA Clonin9 Techniques, a Practical Approach (Glover, D., ed.),
IRL Press, Oxford, pp. 79-88.