The Trasnscription Herpes Cacu

1973;33:1402-1416.
Cancer Res

Bernard Roizman and Niza Frenkel

Productive Infection and in Human Cervical Cancer Tissue
The Transcription and State of Herpes Simplex Virus DNA in

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[CANCER RESEARCH 33. 1402 1416. June 1973]
The Transcription and State of Herpes Simplex Virus DNA in
Productive Infection and in Human Cervical Cancer Tissue1
Bernard Roizman2 and Niza Frenkel
Departments of Microbiology [R. K.. .\. F.] and Biophysics [B. /?.]. The University of Chicago. Chicago, Illinois 60637
SUMMARY
We are reporting on the transcriptional program of
herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) in
productive infection in human epidermoid (HEp-2) cells
and on the transcription and the state of HSV-2 DNA
in a human cervical tumor. Our results may be summa
rized as follows.
In productive infection, a total of 48% of HSV-1 DNA
is transcribed. Analysis of the transcripts shows two kinds
of controls. "Off-on" control of transcription is evident
from the fact that early, before the onset of viral DNA
synthesis, the transcripts arise from 44% of the DNA
whereas late in infection the transcripts present in the in
fected cell arise from 48% of the DNA. The extent of tran
scription of the HSV-1 DNA early in infection is not af
fected by cycloheximide, an inhibitor of protein synthesis.
Control of RNA abundance is evident from the observation
that the transcripts present both early and late in infec
tion form two classes differing in molar ratios. The abun
dant RNA is complementary to 14 and 19% of viral DNA
early and late in infection, respectively, whereas the scarce
RNA is complementary to 30 and 28%, respectively, of
the DNA at the same time intervals. Several lines of evi
dence suggest that the abundant RNA specifies structural
proteins of the virus.
The transcriptional program of HSV-2 DNA differs
from that of HSV-1 DNA in two respects. First, the amount
of DNA transcribed early and late in infection corresponds
to 21 and 50%, respectively. Second, while the viral RNA
present late in infection also forms two classes differing in
abundance, that present early in infection forms only one
abundance class. Cycloheximide does affect transcription
of HSV-2 DNA in that in the presence of the drug 45% of
viral DNA is transcribed by 2 hr postinfection indicating
that at least one "off-on" control of transcription is medi
ated by protein synthesis.
HSV-1 and HSV-2 DNA's share in common approxi
mately 50% of their sequences with good matching of base
pairs. Analysis of the transcription of the DNA sequences
' Presented at the American Cancer Society Conference on Herpes-
virus and Cervical Cancer, December 8 to 10. 1972, Key Biscayne. Fla.
These studies were aided by grants CA-08494 from the National Cancer
Institute. Grant VC 103H from the American Cancer Society, and Grant
GB 27356 from the National Science Foundation.
2Presented by.
3Postdoctoral Trainee supported by a grant from the Whitehall Foun
dation to Dr. Roi/man.
shared in common indicates that they are about evenly
distributed among the templates for abundant and scarce
RNA. However, the common sequences form 71% of the
total sequences specifying abundant RNA and only 39%
of the sequences specifying scarce RNA.
A cervical tumor free of virus or viral antigen was
analyzed by DNA-RNA hybridization techniques for the
presence of viral RNA transcripts. The cervical tumor
contained viral RNA transcripts complementary to 5%
of HSV-2 DNA, and preliminary studies show that they
correspond to both early and late transcripts.
Analysis of the cervical DNA for the presence of HSV-2
DNA sequences led to three conclusions, i.e., first, only
a fragment representing 40% of HSV-2 DNA was present;
second, the fragment was present at concentrations of 1
mole/mole of cell DNA and, third, at least parts of this
fragment are covalently linked to host DNA.
INTRODUCTION
The Problem
HSV-14 and HSV-2 are biologically and immunologically
related and are among the most common viruses infecting
man (25, 40). Although both viruses are transmitted
by close personal contact, they usually infect different body
sites (25). Numerous studies indicate that HSV-2 is poten
tially if not actually oncogenic. Briefly, the association of
HSV-2 with human cervical cancer is based on viral and
seroepidemiological studies and on the presence of viral
antigen in exfoliated cells from patients with cervical dys-
plasias and cancers (25. 31, 41). The oncogenic potential
of the virus also emerges from reports of production of
tumors in hamsters and mice (25, 27) and from the experi
ments of Duff and Rapp (6) which resulted in the trans
formation of baby hamster fibroblasts into highly malig
nant, metastatic cells. HSV-1, albeit genetically related,
appears to be much less oncogenic in experimental studies.
The above recital of facts and observations leads to 2 obvi
ous questions. The 1st question is whether the reproductive
cycles of HSV-1 and HSV-2 differ in some specific prop
erty which might account for the apparently greater
oncogenic potential of HSV-2. The 2nd question relates
The abbreviations used are: HSV-1. herpes simplex virus type I;
HSV-2. herpes simplex virus type 2: HEp-2. human epidermoid carci
noma.
1402 CANCER RESEARCH VOL. 33
Research.
Transcription of Herpes Simplex Virus DNA
specifically to the association of HSV-2 to human cervical
cancer. If indeed this virus causes the tumor, it could be
predicted that the tumor would contain viral DNA and at
least some products specified by the virus. This report
summarizes our attempts to deal with these questions.
The Background
Pertinent to this report are several facts and observations
regarding the virus and the infected cell. Since much of
this information is published it may be summarized as
follows.
HSV-1 and HSV-2 consists of proteins, lipids, poly-
a.nines, and DNA. These structural elements are arranged
into 3 architectural components, i.e., a DNA core, a capsid,
and an envelope (39). The HSV-1 virion has been studied
most thoroughly to date and consists of at least 24 proteins
numbered consecutively 1 through 24 and ranging in size
from 275,000 to 25,000 dallons in molecular weight (44).
The envelope of the virion is derived from a modified
nuclear membrane. In the electron microscope it appears
as a trilaminar membrane with short spikes protruding
from its surface. The envelope contains spermidine (15)
and lipids, presumably derived from the host, and Pro
teins 7, 8, 11, 12, 13, 14, 15, 16, 17, and 18, all of which are
glycosylated (18).
Three kinds of capsids have been analyzed to date (16).
Capsid Forms A and B are found in the nucleus of the in
fected cell. Form A lacks the core and consists of 4 pro
teins (Proteins 5. 19, 23, and 24) arranged in 2 layers. The
outer layer consists of 162 subunits or capsomeres arranged
in the form of an icosahedron. Form B contains the DNA
core and consists of 6 proteins (Proteins 5, 19, 22A, 23,
and 24). Of the proteins present in Form B but not in
Form A, Protein 22A appears to be situated on the sur
face of the capsid leaving only Protein 21 as the candidate
for the core proteins. Capsid Form C is obtained by deter
gent treatment of the envelope virus. It contains sperm-
ine, trace amounts of virus glycoproteins, and Proteins
1 to 4, 5, 19, 21, 23, and 24. Analysis of the structural pro
teins of HSV-1 and their assembly indicates that in the
course of envelopment the B capsid binds Proteins 1 to 4
and at the same time Protein 22A is cleaved to yield Pro
tein 22. The detergents treatment removes Protein 22 but
leaves Proteins 1 to 4 and the rest of the capsid proteins
still attached to the capsid.
The core of the virion consists of the DNA spooled on a
cylindrical body probably consisting of proteins. The
spooled DNA has the shape of a toroid with the cylindri
cal mass body passing through its hole (12).
HSV-1 and HSV-2 DNA's differ in base composition
(2, 17, 19). At least 95%of the sequences contained in HSV-
1 DNA are unique (8). By contrast, as reported here, HSV-
2 DNA appears to have repetitive sequences amounting to
about 16% of its sequences. The DNA's of both HSV-1
and HSV-2 are linear double-stranded molecules [M.W.
(99 5) x IO6daltons] (17) and both contain alkali-labile
bonds at unique sites (9). The nature of the alkali-labile
bonds is not known. Analysis of the DNA extracted from
nuclei of infected cells after labeling with thymidine-3H
for intervals ranging from 3 to 120 min suggests that HSV-
DNA immediately after synthesis contains numerous alkali-
labile bonds which are repaired and/or ligated after syn
thesis. The alkali-labile bonds in viral DNA contained in
virions might be the consequence of incomplete repair at
unique sites (9). Hybridization studies indicate that the
HSV-1 and HSV-2 DNA's share in common 47% of their
sequences with good (85%) matching of base pairs (20).
Analyses of the kinetic complexity of HSV-1 DNA show
that 1 viral DNA molecule (M.W. 10" daltons) contains
all the genetic information necessary for the multiplica
tion of this virus (8).
The biosynthesis of viral structural components is com
partmentalized. Viral DNA is made and transcribed in
the nucleus. Viral proteins are made in the cytoplasm. Per
tinent to this report are several features of the transcrip
tion of the DNA. First, at least a portion of the viral DNA
is transcribed in the form of high-molecular-weight RNA
which contains the same sequences but is larger than the
RNA found on cytoplasmic polyribosomes. These findings
imply that the RNA transcripts are "cleaved" at some
point between synthesis and function on polyribosomes
(35, 45). Second, at least a fraction of viral RNA tran
scripts is adenylated. The adenylation of the RNA is a post-
transcriptional event taking place during a 15- to 20-min
interval between synthesis of the RNA in the nucleus and
its appearance in the cytoplasm (2).
The consequence of productive infection is inevitably
cell death (34). As has been stated before (32, 34), this
conclusion implies that if the infected cell is to survive the
expression of the virus in these cells must be less than that
in productively infected ones.
MATERI ALS AND METHODS
Cells and Virus
HSV-1 and HSV-2 prototypes used in these studies were
prepared in HEp-2 cells. The procedures for maintenance
and infection of these cells have been described elsewhere
(38). An isolate from a facial infection passaged at low
multiplicity a limited number of times and designated at
the F strain served as a prototype of HSV-1. An isolate
from a genital infection passaged in a similar fashion and
designated as the G strain served as a prototype of HSV-2.
In addition, we also tested strain 174 of HSV-2 isolated by
Dr. Maurizio Terni at the University of Ferrara, Ferrara.
Italy. The G and 174 strains cannot be differentiated with
respect to any of the properties relevant to the results pre
sented here. The general properties of HSV-1 and HSV-2
prototypes have been reviewed elsewhere (40).
Preparation of Viral DNA
The procedures for in vivo labeling, extraction, and the
characteristics of herpes simplex DNA were published
JUNE 1973 1403
Research.
elsewhere (8, 9, 19, 20). The procedure for preparation of
in vi-ro-labeledviral DNA was that of Nonayama and
Pagano (28) as described in detail elsewhere (11).
Extraction of RNA from I nfected Cells
The RNA was extracted by the method of Lee et al.
(21) to ensure recovery of adenylated virus-specific RNA
(2).
The Cervical Tumor
The cervical tumor, weighing approximately 70 g, was
surgically removed from an untreated patient and was
furnished by Dr. AndrNahmias from Emory University.
Approximately 80% of the tumor mass consisted of large,
well-differentiated cancer cells. It was extensively tested
at Emory University for infectious virus and viral antigens
with negative results (11). The tumor RNA and DNA were
extracted as previously described (11).
Experimental Design
The studies described in this paper deal with analyses of
the kinetics of hybridization of purified labeled viral DNA
with excess unlabeled RNA from productively infected
HEp-2 cells and with excess unlabeled DNA and RNA
extracted from a cervical tumor.
The DNA-RNA hybridizations involved incubation in
liquid of small amounts of in v/vo-labeled DNA with excess
RNA in 0.05 MTris (pH 8.05)-0.07 MNaCl as described in
detail elsewhere (10). The amounts of DNA-RNA hybrids
were determined following digestion of the hybridization
mixture with nucleases (Neurospora crossa nuclease alone
or after treatment with shark liver endonuclease) highly
specific for single-stranded DNA. The objectives of the
analyses were to determine: (a) the total amount of viral
DNA transcribed; (6) the number of classes of viral RNA
differing in abundance: (c) the molar ratios of the RNA
differing in abundance; and (d) the amount of DNA tem
plate transcribed transcribed at different times in the re
productive cycle. The results obtained by our techniques
readily permit calculation of these parameters. Specifically,
let D + R -D - R be the reaction of single-stranded DNA
fragments with homologous RNA; further, let the concen
tration of DNA be such that the reassociation of DNA is
negligible. It follows that:
Dt/D, = e
-k R,-l
(B)
dD/dt = k-R-D
(A)
where / is the length of hybridization, D is the molar con
centration of single-stranded DNA at time /, R is the molar
concentration of single-stranded RNA, and k is the rate
constant. In high RNA excess, where the concentration of
RNA in hybrid is small compared to that of the single-
stranded RNA left, R can be assumed constant and equals
R0 (the imput RNA concentration). Equation A upon in
tegration yields:
where Dt is the concentration of single-stranded DNA at
time / and >0 is the initial concentration of single-stranded
DNA.
Equation B assumes that all the DNA is transcribed to
yield RNA with a single abundance R0. The equation can
be applied to the more general case with n classes of RNA
each appearing in molar concentration Rn. Such reaction
can be actually visualized as the sum total of the independ
ent reactions of each RNA class with the DNA homolo
gous to it. For each such class the fraction of the DNA
remaining single stranded is:
Dln/Don =
(C)
However, Don equals D0-an where an is the fraction of
the total viral DNA serving as a template for this RNA
class, and >0 is the total DNA input. If follows therefore
that:
Dtn/D0-an = e
_ -kRl
(D)
The observed fraction of single-stranded DNA would then
be:
>,//> = (Dtl/D0) +
+ (Dtn/D0) + 1 - (a, +
+ a)
where 1 - (a, + 4- a) is the fraction of the DNA
that is not transcribed and will therefore remain single
stranded throughout the hybridization. Hence:
The calculation of the parameters and R requires the
numerical value of the hybridization rate constants in
addition to the technique for quantitative separation of
DNA in duplex from single-stranded DNA described
earlier in the text. When /?,/?, the concentration of
RNA in Equation E, are expressed in moles RNA per
liter, the numerical values of the rate constants are in
dependent of the complexity (sum of unique sequences) of
the RNA species represented in the different terms of the
equation. If we assume that the base composition of the
various RNA species do not vary appreciably, the nu
merical values of rate constant kt- -kappearing in the
different terms of the equation should be about the same.
The rate constant, k, defined by the equation
dD/dt = -kD2
where D is the concentration of DNA expressed as moles
DNA per liter was calculated from the reassociation
kinetics of the same batch of DNA as the one used in the
DNA-RNA hybridizations on the assumption that the hy
bridization rate constant for RNA-DNA is the same as
that of DNA-DNA reassociation. The reassociation was
monitored by digesting the hybridization mixture with
nucleases as described earlier in the text. The experi
mentally determined value of k for the DNA-DNA hy-
Research.
bridization in 0.07 M monovalent cation concentration
and for sonically disrupted DNA fragments with an
average sedimentation constant of 5 S was found to be
1.12 x IO5-liters-mor1-sec'1 (10).
The rate constant for DNA-DNA reassociation might
be different from that for DNA-RNA hybridization. In
deed Melli et al. (24) on the basis of hybridization of excess
Escherichia coli DNA to complementary RNA prepared
in vitro, calculated that the observed rate constants differ
by a factor of 0.43, but the applicability of this quotient
to our analyses is not entirely clear. Gelderman et al. (13)
also assumed that the DNA-DNA and DNA-RNA hy
bridization constants are the same. However, the absolute
numerical value of the hybridization rate constant is re
quired for calculations of the absolute concentrations of
the herpesvirus RNA in the cell. It is not required for the
calculation of the relative concentrations of the RNA
species or for the determination of the size of template
from which they are transcribed.
The purpose of the DNA-DNA hybridizations was to
determine the amounts and size of the DNA in the cervi
cal tumor. Except where stated otherwise, all hybridiza
tions were done at 79in 1.06 M NaCl-0.05 M Tris (pH
8.05). It is convenient to discuss the theoretical basis of
the DNA-DNA hybridization later in the text.
RESULTS
The Transcriptional Program of I I SV-I in Productively
I nfected Cells
The Program. Analyses of viral RNA transcripts in
productively infected cells were done on RNA extracted
at 2 and 8 hr postinfection. The time intervals were se
lected on the basis of observations that viral DNA syn
thesis begins roughly at 3 hr postinfection (33, 37) and that
infectious progeny appears at 6 hr postinfection and is
present in appreciable levels (33). The 2-hr RNA should
therefore contain the bulk of the RNA made before viral
DNA synthesis begins whereas the 8-hr RNA should con
tain all of the species made late in infection and required
for the synthesis of infectious progeny.
The results of the hybridization tests for HSV-1 RNA
are shown in Chart 1 and may be summarized as follows.
The transcripts present in RNA extracted from HSV-1-
infected cells at 2 and 8 hr postinfection were comple-
mentary to 44 and 48% of the total HSV-1 DNA, re
spectively (Table 1). Assuming that the RNA arises
from asymmetrical transcription of viral DNA, it follows
2hr
200 400 600 800 1000 1500
K0t (m ole s nuc le otide s -s e c -lite r-' )
2000
Chart 1. Hybridization of HSV-1 DNA with excess RNA extracted
from HEp-2 cells at 2 and 8 hr postinfection. Since variable amounts of
RNA were hybridized for different time intervals as described in the
text, the data are presented as a plot of the fraction of single-stranded
DNA as a function of the input concentration of RNA (Ra) in moles
nucleotides per liter times the time of hybridization (/). O, experimen
tally determined points. The line is a computer plot calculated ac
cording to Equation E and fitted by nonlinear least-squares method. The
computer analysis involved the determination of parameters <* and R
in Equation E in which the number of classes of RNA (n) was assumed
to be I, 2, 3, 4, etc. The fit for n = 2 was better than that for n = I.
When n was greater than 2, the results were meaningless. Thus for the
hybridization of RNA extracted at 2 hr postinfection, for n = 3, a, was
less than 0.001 whereas a and os retained the same values as o,
and a2 in the case of n = 2. The same result was obtained with RNA ex
tracted from 8-hr-infected cells.
Table I
Analysis of the transcripts of HSV-1 and HSV-2 DNA in productively infected HEp-2 cells at 2 and 8 hr postinfection
VirusHSV-1HSV-2(postinfection)(hr)2828Fraction
of viral DNA
transcribedi0.140.190.210.3120.300.280.000.19Total0.440.480.210.50R,(nmoles/1)7.97.10.512.23RNA abundanceR,(nmoles/l)0.0580.1830.00.28RJR,136.240.37.96
'Moles of viral RNA fragments. M.W. 0.99 x IO- dallons.
JUNE 1973 1405
Research.
that 88% of the total genome was already transcribed by
2 hr postinfection.
Analysis of the kinetics of hybridization of HSV-1
transcripts indicates the presence of at least 2 classes of
RNA differing in abundance (Table 1). At 2 hr postinfec-
tion the abundant class was complementary to 14%of viral
DNA and was present in molar concentrations 136-fold
greater than the scarce class. At 8 hr postinfection the
abundant class was complementary to 19%of viral DNA
and was present in molar concentrations 40-fold greater
than the scarce class.
The Requirement for Protein Synthesis. The difference
between the amount of HSV-1 DNA transcribed at 2 and
8 hr postinfection is so small as to raise the question
whether the RNA extracted from 2-hr infected cells is
indeed early RNA, i.e., RNA made before the onset of
viral DNA synthesis. The problem arises from the fact
that determinations of the onset of DNA synthesis are
imprecise: the experimental determination hinges on the
ability to detect trace amounts of viral DNA. The alterna
tives open to us were to test the transcripts made in the
presence of inhibitors of either DNA or protein synthesis.
The latter alternative is based on observations that viral
DNA synthesis requires prior protein synthesis and is
not initiated in cells treated with inhibitors of protein
synthesis from the time of exposure of virus to cells (37).
Of the 2 alternatives we choose the inhibition of protein
synthesis largely because in our hands most inhibitors of
DNA synthesis reduce but do not completely inhibit
DNA synthesis, whereas the inhibition of protein synthe
sis appears to be almost complete.
In 1 series of experiments HEp-2 cells were infected
with HSV-1 in the presence of 100 /ig of cycloheximide
per ml of medium and maintained in the presence of the
drug for the entire duration of the experiment. The RNA
was extracted at 2 hr postinfection and hybridized with
viral DNA. The results of the hybridization tests showed
that the amount of HSV-1 DNA transcribed by 2 hr
postinfection in the presence of cycloheximide was 44%,
i.e., exactly the same as in untreated cells. We conclude
from these experiments that (a) the transcription of at
least 44% of HSV-1 DNA is independent of protein syn
thesis and (b) the transcripts accumulating during the 1st
2 hr postexposure of cells to virus consists of transcripts
made before the onset of viral DNA synthesis.
The I dentity of Abundant RNA Sequences Early and
Late in I nfection. The presence of 2 distinct abundance
classes in 2-hr RNA raised the question whether the
nucleotide sequences in the most abundant class at 2 hr
are identical to those in the most abundant class present
at 8 hr postinfection. To answer this question, we per
formed (10) a series of abundance competition tests. This
type of analysis is designed to answer the general ques
tion of whether any class of RNA with a given abundance
contains the same nucleotide sequences as any other class
of RNA with the same or different abundance. As shown
in Table 2, 2 sets of hybridization tests were done. In the
control set the 2- and 8-hr RNA were each hybridized to
single-stranded DNA at a concentration (R0) and time
(/) such that all of the single-stranded DNA fragments
complementary to the most abundant RNA were driven
into DNA-RNA hybrids. In the abundance competition
set, the 8- and 2-hr RNA's were mixed. However, the
concentration of each RNA species was adjusted so that
for a common time of incubation tc the product R0-tc was
exactly the same as the product R0-t for each of the
RNA's in the control set.
Table 2 shows the amount of single-stranded DNA ex
pected to be driven into DNA-RNA hybrid if the most
abundant sequences at 2 and 8 hr were identical and if they
were different. The data clearly demonstrate that the se
quences in the most abundant 2-hr RNA are also present
in the most abundant RNA at 8 hr postinfection, i.e., the
most abundant RNA at 2 hr is a subset of the abundant
class of late RNA.
The Nature of the Proteins Specified by the Abundant
Table 2
Abundance competition between 2-hr most abundant species and 8-hr RNA
Compe
tition
experi
ment1RNAsource2
hr alone
8 hr alone
8 hr alone
8hr +R0t71.6
72.5
144.9
72.5Observed
% DNA
inhybrid18.8
24.8
28.8Predicted
%All
abundant sequences in
2-hr RNA are present in
competing 8-hr RNADNA
in hybridMost
abundant
RNA different
competingspecies
in 2-hr
from those in
8-hr RNA
2hr
2 hr alone
8 hr alone
8 hr alone
8 hr +
2hr
71.6
71.6
289.9
362.4
289.9
71.6
22.6
18.8
32.6
35.6
35.6
24.8 28.8 43.6
32.6 35.6 51.4
"Expressed as moles nucleotides-sec/liter. Two-hr RNA, 8-hr RNA, or both were incubated with labeled DNA to Ral speci
fied. Data from the work of Frenkel and Roi/man (10).
Research.
HSV-1 RNA Present in Productively Infected Cells in
High Abundance. One hypothesis concerning the function
of viral RNA present in high abundance is that it speci
fies structural proteins. This hypothesis is based not only
on the expectation that the most abundant RNA should
specify the most abundant viral proteins but also on the es
timated size of the DNA template required to specify the
amino acid sequence of viral structural proteins. As re
ported elsewhere in detail (18, 40, 44), the herpesvirion
consists of 24 structural proteins. On the basis of the-
aggregate molecular weights of the proteins, it was pre
viously estimated that 23.5% of viral DNA was required
to specify their amino acid sequences. This figure is an
overestimate since one-half of the proteins are glycosyl-
ated and the polysaccharide moieties, while contributing
to the molecular weight, should be excluded from the
calculation. Nevertheless even this amount of DNA tem
plate (23.5%) is in good agreement with the DNA template
for the abundant RNA (19%). A direct test for the in
formation content of the abundant viral RNA would re
quire in vitro synthesis of proteins using that RNA as
messenger. The experimental question that we would
like to ask is what proportion of the 24 viral structural
proteins is made early after infection particularly since, as
we have shown earlier in the text, a subset of high-abun
dance RNA corresponding to 14% of the DNA is already
present at that time.
In these experiments 4 x 10scells were labeled between
0.5 and 2 hr postexposure to virus with 10 ^Ci of 14C-
labeled amino acid mixture (reconstituted protein hydroly-
sate, New England Nuclear, Boston. Mass.) per ml of
Eagle's minimal essential medium lacking amino acids. At
the end of the pulse the cells were washed and replenished
with Mixture 199 supplemented with 1% calf serum.
Replenishment with medium containing unlabeled amino
acids precludes further incorporation of labeled amino
acids into proteins (7, 43). An infected cell culture la
beled continuously beginning 4 hr postinfection served as
control. Both the pulsed and the control cultures were
harvested 20 hr postinfection. The virus was purified,
solubilized, and subjected to electrophoresis on polyacryla-
mide gels. The absorbance profiles of the autoradiogram
are shown in Chart 2. The data show the following, (a)
Structural proteins are already made between 0.5 and 2 hr
postinfection; however, only Proteins 5 through 24 be
come labeled at that time, (b) All viral proteins are la
beled and therefore synthesized after 4 hr postinfection.
(c) On the basis of the molecular weights of the proteins,
we calculated that as much as 68% of the estimated DNA
template for structural proteins is expressed between 0.5
and 2 hr postinfection. This isin comparatively good agree
ment with the proportion of the DNA template for abun
dant RNA transcribed at that time (74%).
There is independent evidence that synthesis of most
structural proteins is an early function. Thus in addition
to the evidence presented in this paper that most viral
structural proteins are made between 0.5 and 2 hr post-
infection, it has also been shown by us and by others (14,
29, 34) that inhibitors of DNA synthesis do not block the
0.5-2 hr

4 20 h.
10 30 50 70
Di stance (mm)
90
Chart 2. Absorbance tracing of autoradiograms of structural proteins
in purified HSV-I virions subjected to electrophoresis in polyacrylamide
gels. Top, proteins labeled between 0.5 to 2 hr postinfection: bottom.
4 to 20 hr postinfection. The purification of the virus, the polyacrylamide
gel electrophoresis of the proteins, and the autoradiography were done
according to the procedures of Spear and Roi/.man (44).
synthesis of viral proteins, the assembly of proteins into
capsids, or the appearance of the structural components
of the virus on the surface of the cells. In this respect,
there is a substantial difference between the transcriptional
program of herpesvirus and that of other DNA viruses
infecting animal cells.
If our suspicions concerning the function of abundant
RNA are correct, it follows that the scarce RNA specifies
nonstructural proteins, i.e., enzymes involved in the syn
thesis and processing of viral structural components
(DNA, proteins, glycoproteins, lipids, and polyamines)
and in the modification of the host that involves inhibition
of host DNA and protein synthesis, alteration of the
synthesis, processing and transport of host RNA and of
the structure and function of cellular membranes.
Most of the viral functions listed here are known to be
early functions, expressed independently of viral DNA
synthesis (33-35).
On the basis of comparisons between total 2- and 8-hr
RNA, it would seem that the difference between early
and late functions consists of transcription of a small per
centage of DNA template at high abundance. The function
of this RNA could be to specify maturation proteins, etc.
We cannot exclude the possibility that the temporal pro
gramming of viral functions is also determined at the level
of processing and transport of the RNA from nucleus to
the cytoplasm of the infected cell.
The Transcriptional Program of HSV-2 in Productively
Infected HEp-2 Cells
The Program. The design of these experiments was
similar to those used in analyses of the transcriptional
JUNE 1973 1407
Research.
program of HSV-1. The results (Chart I; Table I) show
that the RNA transcripts present in HSV-2-infected cells
at 2 and 8 hr postinfection were complementary to 21
and 50% of HSV-2 DNA, respectively. Thus, unlike the
RNA extracted from HSV-1 infected cells, the early
HSV-2 transcripts were complementary to only 21% of
viral DNA. Analysis of the kinetics of hybridization of
the HSV-2 RNA transcripts present early in infection in
dicates that they constitute only 1 abundance class. On
the other hand, the RNA transcripts present in the cell at
8 hr after infection formed 2 classes of abundance dif
fering only 8-fold in molar ratios and were complementary
to 31 and 19% of the viral DNA, respectively. In other
experiments, we found that the difference in the extent
of HSV-1 and HSV-2 DNA transcribed by 2 hr postin-
fection is obliterated in cells treated with 100 /gof
cycloheximide per ml of medium. Thus RNA extracted
from treated cells at 2 hr postinfection hybridized with
45% of viral DNA compared with RNA from untreated
cells, which hybridized with 21% of viral DNA.
The Transcription of DNA Sequences Shared in Com
mon by HSV-1 and HSV-2. A previous report from our
laboratory showed that HSV-1 and HSV-2 DNA's share in
common at least 47% of their base sequences with good
matching of base pairs (19). It was of interest to de
termine (a) the extent of transcription of the common se
quences and (b) the distribution of the templates for
abundant and scarce viral RNA species among unique
and common DNA sequences. In these experiments RNA
extracted from 8-hr HSV-1-infected cells was hybrid
ized with labeled HSV-2 DNA. The results may be sum
marized as follows.
Comparisons of the hybridization of RNA extracted at
8 hr from HSV-1-infected cells with homologous and
heterologous DNA (Chart 3) indicate that HSV-1 RNA
is complementary to 24% of the HSV-2 DNA, i.e., the
fraction of HSV-1 and HSV-2 DNA's that are transcribed
is homologous to the extent of 50%. Since nearly 50%
of the DNA, i.e., the equivalent of 1 strand, is transcribed,
it follows that the extent of homology between HSV-1
and HSV-2 DNA's is 50%, i.e., the same value as that ob
tained from DNA-DNA hybridizations (20).
The calculation of the distribution of DNA templates
for abundant and scarce viral RNA species between the
unique and common DNA sequences of HSV-1 and
HSV-2 DNA's is based on 2 considerations. First, RNA
transcribed off common sequences must necessarily have
the same molar ratios vis--visthe complementary re
gions of both HSV-1 and HSV-2 DNA's. Second, the
thermal elution profiles of sheared HSV-1 DNA-HSV-2
DNA heterohybrids show a very uniform matching of
base pairs even though it is somewhat lower (85%) than
those of homohybirds. We conclude from this finding
that the matching of base pairs is uniform for all DNA-
DNA heterohybrids that the hybridization rate constant
for all of the DNA-RNA heterohybrids should be uni
form. On the basis of these considerations we calculated
(Table 3, Legend) that 13% of HSV-DNA is comple
mentary to a corresponding amount of HSV-1 DNA
1.00
UHSV-2
DNA
5 0.50
600 1200
Rgt (mol es nucl eoti des -sec-l i ter" ' )
Chart 3. Hybridization of HSV-2 DNA with excess RNA extracted
from cells 8 hr postinfection with HSV-1. The procedure for the hybrid
ization, etc., were the same as described in the legend to Chart I except
that the hybridization was carried out at a temperature 33belothe
Tm of the HSV-2 DNA. , computer plot for homologous DNA-RNA
hybridization shown in Chart I. , computer plot lor the heterologous
hybridization calculated according to Equation E except that the values of
/?, and R2 were taken to be the same as those for 8-hr RNA reactive
in the homologous DNA-RNA hybridization (Chart I).
Table 3
Complementary of HSV-l-HSV-2 DNA's serving as templates for
abundant and scarce RNA species
Hybridization was done at incubation temperature of 62(30below
the Tmof HSV-2 DNA).
Fraction of totalDNA
hybridizing
with 8-hr HSV-1
RNA,jTotalHSV-1DNA192848HSV-2DNA131124HSV-2/
HSV-10.710.390.50
"Region of DNA serving as template or reacting with RNA of high
abundance.
6 Region of DNA serving as template or reacting with RNA of low
abundance.
serving as template for RNA of high abundance and that
11% is complementary to the template specifying RNA
of low abundance. The data also show that the region of
the HSV-1 DNA serving as a template for RNA of high
abundance contains a larger fraction of sequences comple
mentary to HSV-2 DNA (71%) than does the template
for the low-abundance RNA (39%).
The Transcriptional Program of HSV-2 in Cervical
Tumor Cells
The hybridization of labeled DNA with unlabeled RNA
extracted from productively infected cells was designed to
Research.
preclude the reassociation of DNA. On the other hand,
because the amounts of RNA recovered from the tumor
were small, the formation of DNA-DNA hybrids could
not be prevented. For precise determination of the amount
of DNA-RNA hybrid, the labeled DNA was also hy
bridized with equal concentrations of RNA extracted
from uninfected HEp-2 cells. The net amount of labeled
DNA-tumor RNA hybrid determined by subtracting the
percentage of hybrid formed in the presence of uninfected
RNA from that formed in the presence of cervical tumor
RNA is shown in Chart 4 along with the transcriptional
program of HSV-2 in productively infected cells. The fol
lowing points, however, bear on these results.
Two features of the experimental design excluded the
possibility that the 5% difference at the plateau level
(Chart 4) between the amount of hybrid formed in the
presence of cervical tumor RNA and in the presence of
HEp-2 RNA are due to DNA-DNA hybridization or to
experimental errors. First, identical net DNA-RNA hybrid
(5% above control) was obtained with 3 different prepara
tions of purified in v/vo-labeled DNA differing widely in
specific activity and therefore incubated at different DNA
concentrations (C0 ) to reach the same Rat values.
Second, the same net amount of DNA-RNA hybrid
formed when R0t and DNA concentration were kept
constant, but the durations of hybridization (?) and RNA
concentration (R0) were varied in a fashion designed to
vary the DNA concentration and therefore the amount of
viral DNA-RNA hybrid formed. We conclude therefore
that the net amount of DNA-RNA hybrid obtained in
these experiments was independent of the DNA prepara
tion of the DNA C0t and dependent only on the R0t to
which the hybridizations were carried.
1.00 w l*wv ^"^^^B*O
ia90^.E.?
0.80-'5
0.70-s 'S
0.60-O
Wk\o\0_lv"-
0^,
Ce rvical tumor*2hr
Infe cte dHEp-2 ce lls j>.^_8hrjnfe cte d
HEp-2 ce lls 1000
2000 30007000
Rgt (mole s nucle otide s -s e c-lite r-')
Chart 4. Hybridization of labeled HSV-2 DNA with excess RNA ex
tracted from 2- and 8-hr-infected HEp-2 cells and from the cervical tu
mor. O. 3. 2- and 8-hr-infected cells, respectively. The lines are computer
plots calculated according to Equation E and fitted by a nonlinear
least-squares method as described in the legend to chart I. The net amount
of DNA hybridizing with cervical tumor RNA was calculated by sub
tracting the amount of DNA-DNA hybrid formed in the presence of
HEp-2 RNA from the amount of hybrid (DNA-DNA and DNA-
RNA) formed in the presence of cervical tumor RNA. Data from the
work of Frenkel et al. ( 11).
Additional and more direct evidence that the difference
between hybrid formed in the presence of cervical tumor
RNA and that formed in the presence of uninfected HEp-2
RNA was due to DNA-RNA hybrid emerged from an
analysis of the hybrid formed in the presence of cervical
tumor RNA. Briefly 4.6 /ug of DNA with a specific
activity of 12,000 cpm/Vg were hybridized with cervical
tumor RNA to a R0t of 1000. The same amount of
DNA was hybridized with an identical concentration of
uninfected HEp-2 RNA for the same interval. The
amount of hybrid formed in the presence of uninfected
HRp-2 RNA indicated that of the total DNA hybrid
formed in the presence of cervical tumor RNA (45% of
input), 88% was in DNA-DNA hybrid and 12% was in
DNA-RNA hybrid. The hybrid formed in the presence
of cervical tumor RNA was digested with N. crossa
nuclease, denatured with 0.3 MNaOH for 10 hr to hydro-
lyze the RNA, and then dialyzed against 0.07 M NaCl-
0.05 M Tris (pH 8.05). The DNA recovered after alkali
treatment was then rehybridized with RNA extracted
from cervical tumor and from uninfected HEp-2 cells. The
results are shown in Table 4. Since this DNA was en
riched with viral DNA sequences transcribed in the cer
vical tumor it could be predicted Table 2, (legend) that
the difference between the amount of hybrid formed in the
presence of cervical tumor RNA and in the presence of
HEp-2 RNA should be 14.6%. The actual difference was
14.4%. We conclude that the amount of viral DNA trans
cribed in the tumor is 5%, i.e., considerably less than in
productively infected cells.
In this and earlier (10, 36) publications we have re
ported on differences in the early and late transcriptional
programs of HSV-1 and HSV-2 and on the transcription
of DNA sequences shared in common between HSV-1 and
HSV-2. It was of interest to determine therefore whether
the DNA template transcribed in the tumor represents a
subset of templates transcribed early or late and whether
it is shared in common between HSV-1 and HSV-2.
Preliminary experiments indicate that a fraction of the
viral DNA template transcribed in the cervical tumor cells
is also transcribed early in productive infection of HEp-2
cells with HSV-2 and that approximately one-half of the
viral DNA template transcribed in the cervical tumor be
longs to the set of sequences which are shared in common
between HSV-1 and HSV-2 (20, 36).
The Amount and Geneti c Compl exi ty of the DNA Se
quences Pr esent i n the Cer vi cal Tumor and Compl e
mentar y to HSV-2 DNA
In the preceding section we have shown that the cervical
tumor contains viral RNA transcripts complementary to 5%
of viral DNA. It was of interest therefore to determine the
amount of viral DNA present in the cervical tumor and its
genetic complexity. The design of the experiments described
in this section was to compare the rates of reassociation of
in v/-A-0-labeledHSV-2 DNA in the presence of variable
amounts of cervical tumor and HEp-2 DNA. Two series of
experiments were done. In the 1st series, labeled HSV-2
JUNE 1973
1409
Research.
Table 4
Rehybridzalion of DNA hybrid formed in the presence of cervical tumor RNA
The experimental details are given in the text. The calculations are based on the following. The DNA in
the hybrid obtained by hybridization of m v/vo-labeled DNA with cervical RNA consists of 88.4%DNA-DNA
hybrid and 11.6%DNA-RNA hybrid. The calculation is based on the amount of DNA in hybrid formed in the
presence of HEp-2 RNA. In the 2nd hybridization carried out to about the same R0t as the first, but to a
different DNA C0l because of decreased DNA concentration, it could be expected that all 11.6%of DNA that
hybridized to cervical tumor RNA would end up in DNA-RNA hybrid. In addition, the DNA originally in
the DNA-DNA hybrid would end up in part (12.3 of 89%) in DNA-DNA hybrid and in part (5 of 89%) in
DNA-RNA hybrid. The predicted total DNA hybridizing in the presence of cervical tumor should therefore
be 14.6%greater than that hybridizing in the presence of HEp-2 RNA. Data from the paper of Frenkel et al.
(M).
Source of RNA1st
hybridization
Cervical tumor
Uninfected HEp-2DNA
RNA %of
C0f Rat" DNA in hybrid,
. 1000 45.0
1000 39.8%
of DNA in hybrid
above controlExpected
Observed5.0
5.2
2nd hybridization
Cervical tumor
Uninfected HEp-2
0.21
1100 26.7
In moles nucleotides sec-liter ' and for 0.07 MNaCl.
14.6 14.4
DNA was hybridized with a single concentration of cervical
tumor DNA and with an equivalent amount of HEp-2 DNA
for variable time intervals. In the 2nd series of experiments,
cervical tumor DNA and HEp-2 DNA were mixed in var
iable proportions so that the final concentration of DNA
was constant. These were then mixed with labeled DNA
and allowed to hybridize for variable lengths of time. It
is convenient to present the results in the framework of
the kinetics governing DNA-DNA reassociations. Ingenerai
the reassociation of DNA follows 2nd-order reaction
kinetics and is predicted by the relationship of Britten
(4)
D,/D0 -
(F)
where D, /) is the fraction of single-stranded DNA re
maining after hybridization for time /, C0 is the concen
tration of DNA in moles nucleotide/liter and K is a constant
A plot of D0/D, against / should yield a straight line with
an intercept of 1 and with a slope proportional to C0. The
line representing the reassociation of DNA without repetitive
sequences should have a slope of 1.
The results of our hybridization studies are shown in
Chart 5 in the form of a series of plots of D0/Dt against
/ for each concentration of cervical tumor DNA tested. The
in v/iro-labeled DNA in the presence of HEp-2 DNA alone
hybridizes at a constant rate consistent with the kinetic com
plexity of HSV DNA (8) except that, unlike HSV-1 DNA,
HSV-2 DNA has a small amount of repetitive sequences
which cause the intercept (Chart 5) to be 1.198. This ap
pears to be a general characteristic of HSV-2 DNA since
similar kinetics were obtained with DNA preparations la
beled in vivo of 2 different HSV-2 strains. The salient fea
ture of our results is that the points for the labeled DNA,
hybridized in the presence of cervical tumor DNA, form
initially a straight line with the same intercept but later
level off and ultimately appear to form a line parallel or
M
200
1.8
1.6
U
U
L
100
ti me(hr
200
Chart 5. Hybridization of in vi/ro-labeled DNA in the presence of
DNA from cervical tumor cells and from uninfected HEp-2 cells. A,
HSV-2 DNA-'H (0.046 ^g/ml) was hybridized in the presence of
HEp-2 DNA (3.07 mg/ml) ()or cervical tumor DNA (3.26 mg/ml)
(O) for the time intervals shown. The plot for HSV-2 DNA in
the presence of the cervical tumor DNA begins to deviate from the initial
rate after 40.2% of the DNA was in the hybrid form. The plot for HSV-2
DNA in the presence of DNA continues to increase linearly until at least
488 hr at which point 65.7% of the DNA was in hybrid form, indicating
that the in w/ro-lubeled DNA reassociates normally with 2nd-order
kinetics. B. HSV-2 DNA-3H (0.012 ^g/ml) was hybridized in the
presence of cervical tumor DNA in concentrations of 5 mg/ml (D).
3.26 mg/ml (y). 1.74 mg/ml (A). 1.09 mg/ml (O) or HEp-2 DNA
,O). The final concentration of DNA in the hybridization was adjusted
with HEp-2 DNA to the highest concentration of tumor DNA in order
to minimize the variation in viscosity of the hybridization mixture. Data
from the work of Krenkel et al. ( 11).
intercepting the line representing the hybridization of the
labeled DNA in the presence of HEp-2 DNA alone. The re
sults imply that only a portion of the viral DNA is present
Research.
in the tumor cells and that after this portion of the DNA
was exhausted the hybridization of labeled DNA decelerated
to that observed in the control hybridization, i.e., in the
presence of HEp-2 DNA alone.
The amount of viral DNA in the tumor may be calcu
lated by substituting in the general equation for DNA-DNA
hybridizations the observed 1.198 intercept and by rewriting
it as
[(Do/0,) - 1.1981-r1 - KC0
Since the viral DNA in the hybridization test comes from
2 sources, i.e., the labeled DNA and that fraction of the
cervical tumor DNA which is viral, the equation becomes
[(/>/,) - 1.198]-r1 = KC + KfC0c
where / is the ratio of moles nucleotides of viral DNA to
moles nucleotides of cellular DNA in the cervical tumor
and Co and Cac are the concentrations of in v/'/ro-labeled
viral DNA and cervical tumor DNA, respectively, in
the hybridization mixture. / may be calculated by di
viding the slope of a plot of [(>/>,)1.198]-r1
against C0Cby the value of K calculated from the inter
cept KCa ". The plot based on initial, ascending points in
Chart 5 B yielded a straight line; the value of/calculated
from this plot (Chart 6) is (1.02 0.17) x IO'5 The
calculation of the number of viral DNA molecules per
cell hinges on the content of DNA in human cells. This
value ranges from 6 x 10"6 /gper diploid cell (22) to
17 x 10~6 tg/heteroploid HeLa cell (23), which or
iginated from a cervical tumor. On the basis of the molec
ular weight of HSV-2 DNA [(99 5) x IO6] and the
correction factor (0.8) for the fraction of tumor cells in
the tumor tissue, we calculated that the number of viral
DNA molecules per tumor cell ranges from 1 (heteroploid
cell) to 3.5 (diploid cell).
The genetic Complexity of the DNA fragment present
in the tumor emerges from the region of the plot at which
the hybridization begins to deviate from the straight lines
representing the initial rates. The data summarized in
Table 5 indicate that the tumor contains a unique frag
ment constituting approximately 39%, i.e., 39 x IO6
daltons, of the HSV-2 DNA molecule.
The State of the DNA Sequences of the Cervical Tumor
Complementary to HSV-2 DNA
The purpose of this series of experiments was to de
termine whether viral DNA and host DNA's are co-
valently linked. The choice of experimental technique was
based on the observation that HSV-1 and HSV-2 contain
alkali-labile bonds (9, 19). Since the nature of these bonds
is not clear, we chose an alternative to the classical tech
nique of looking for virus-specific sequences in high-
molecular-weight DNA obtained by sedimentation of tu
mor DNA in alkaline density gradients. The alternative
was to look for viral DNA sequences in tumor cell DNA
reassociated under conditions designed to exclude by a
wide margin the reassociation of the viral DNA.
1.5
(G) ^
1-0
(H)
0.5
IO2-Ci;
15
Chart. 6. Calculation of the ratio of HSV-2 DNA to cervical DNA
in the cervical tumor. The data in the initial, linear portions of the plots
shown in Chart 5 B were plotted as [(DJD,) - 1.198]-1 ' versus
the concentration of the cervical tumor DNA in the hybridization mix
ture. The line is a computer plot fitted by a linear regression. The ratio
(/) of viral DNA to tumor cell DNA was calculated from Equation
H. The calculated intercept for the line is (4.5 0.5)-10 7-sec~ '
Since C0' is 4.11 x 10"' mole nucleotide/liter and the calculated values
for K S.D. is (10.97 1.07) liter-moles nucleotides-'sec~' and the
slope of the line S.D. is (1.12 - 0.08)-10 ', the value of/is (1.02
0.17). IO"5. The values for the intercept and the slope are significant
within 95% confidence levels. The apparent scatter of the points in the
plot is due to the extreme sensitivity of this type of plot. Data from the
work of Frenkel et al. (11).
Table 5
Genetic complexity of HSV-2 DNA in the cervical tumor
The points used in these calculations are the 1st 2 points in the region
where the curve deviates from the initial rate. Data from the work of
Frenkel et al. (11).
Concentration (mg/ml) of
cervical tumor DNA in Time i
hybridization mixture (hr)Chan
5 A3.26
43.483.2Chart
SB5.00
46.378.13.26
78.1119.11.74
96119.1Av.
%of viral genome in tumor5o//>,9
ID.67.82.61.67.61.65.56.62%
of
DNA in
hybrid40.
1644.9938.0440.0437.8739.2635.9338.1339.30
The experiments consisted of 3 steps. The first involved
partial reassociation of the cervical tumor DNA; it was
designed to permit recovery of reassociated sequences
and viral DNA covalently linked to these sequences but
to exclude viral DNA that is not integrated, i.e., not
JUNE 1973 1411
Research.
Bernard Roiiman and Niza Frenkel
covalently bound to host DNA. The cervical tumor DNA
in concentrations of 3.26 mg of buffer per ml containing
0.07 M NaCl-0.05 M Tris (pH 8.05) was denatured by
heating at 110for 10 min and then allowed to reassociate
at 53for 1.6 hr. The calculations were based on the
following considerations, (a) The equivalent (i.e., at 0.18
M Na+ concentration and for fragments 400 nucleo-
tides long,) C0t of 50 viral DNA is 0.4 mole nucleotide-
sec-liter ' (8). At C0t of 10, the DNA is completely
reassociated. On the assumption that cervical tumor DNA
contains 10 moles of viral DNA per mole of cellular DNA
and that viral DNA strands are intact, i.e., i .6 x 10s
nucleotides long and not integrated, it could be calculated
that viral DNA would require a minimum of 1600 hr com
pletely to reassociate but that little or no reassociation
would take place if the DNA were allowed to hybridize
for 1.6 hr. i.e., to an equivalent C01 of 0.01. The reassocia-
tion of viral sequences was expected to be lower since the
hybridization was done at 41below the Tm of HSV-2
DNA. (b) In calculating the C01 to which the DNA was
reassociated, we assumed that the average size of the
denatured cervical tumor DNA consisted of fragments
with an average molecular weight of 5 x IO7 dallons,
i.e., the same as that of intact single strands of viral DNA.
The calculated equivalent C0 /, i.e., after correction for
size [from the assumed 1.6 x IO5 nucleotides long to 400
nucleotides long (46)] and for molarity of the monovalent
cations [from 0.07 M Na+ to 0.18 M Na+ (5)] in the hy
bridization buffer, was 91.8. Human cell DNA reassociated
to this C0i consists largely of repetitive sequences (3, 42).
The average size of the DNA was probably smaller and
therefore the equivalent C0 / to which the reassociation was
carried out is probably lower. The 2nd step in the exper
imental sequence was to fractionate the DNA on hydroxy-
apatite. The design of the fractionation experiment was
based on the following considerations. HSV-2 DNA con
tains 69 guanine plus cytosine moles per 100 ml. In a re
construction experiment in which nonsheared HSV-2 DNA
in a concentration equivalent to that of 10 DNA mole
cules per DNA cell equivalent was allowed to reassociate
for 1.6 hr and was then fractionated on hydroxyapatite,
the viral DNA that remained single stranded was quanti
tatively eluted from hydroxyapatite columns with 0.19
M phosphate but not with 0.12 M phosphate. Conse
quently, after the partial reassociation, the cervical tu
mor DNA was diluted with sodium phosphate buffer to a
final concentration of 0.12 Mphosphate buffer and put on
hydroxyapatite in a water-jacketed column heated to
50.The absorbed DNA was eluted first with 0.19 and
subsequently with 0.4 vi phosphate buffer. The DNA
contained in the 0.12 Meffluent and that eluted with 0.19
and 0.4 M phosphate buffer were concentrated and di-
alyzed against the hybridization buffer. The results of
the reassociation and fractionation were as follows. Of the
total, 63% was recovered in the 0.12 M effluent and was
therefore single stranded, 26% eluted in 0.19 M phos
phate, and 11%eluted in 0.4 Mphosphate.
In the 3rd step of the experimental sequence the re-
associated cervical tumor DNA eluted with 0.4 M phos-
,,0"
IO
20 30 40
tim e (hr )
Chart 7. Initial rates of hybridization of in v/'fro-labeled HSV-2 DNA
in the presence of a fast-reassociating fraction of tumor cell DNA. HSV-2
DNA-3H (0.009 iig/ml) was hybridized in 0.05 M Tris-HCl (pH 8.05)-
1.06 M NaCl in the presence of sonically disrupted tumor DNA (0.42
mg/ml) reassociated and eluted from hydroxyapatite as described in the
text (O) and in the presence of sonically disrupted unfractionated cervi
cal tumor DNA (0.42 mg/ml) ().Data from the work of Frenkel el al.
(ID.
phate buffer was hybridized with in v//ro-labeled DNA.
The design of the experiment was limited by the amount
of the cervical tumor DNA recovered in the 0.4 M phos
phate buffer fraction. Briefly, in v/'/ro-labeled viral DNA
was mixed with the reassociated cervical tumor DNA and
with exactly the same amount of unfractionated cervical
tumor DNA and hybridized to relatively low C0/ levels.
The results were as follows, (a) At low C01 levels shown
in Chart 7 the amount of hybrid formed in the presence
of reassociated tumor cell DNA was consistently higher
than in the presence of cervical tumor DNA indicating
both the presence and some enrichment of viral DNA se
quences in the reassociated DNA. (b) At 400 hr (not
shown) the amount of the hybrid formed in the presence
of reassociated cervical DNA was the same as that formed
in the presence of unfractionated cervical DNA, indicating
no enrichment. The enriched sequences arise from at
most 20% of total HSV-2 DNA.
DI SCUSSI ON
The DNA' s of HSV-1 and HSV-2
As summarized under "Introduction," the HSV-1 and
HSV-2 DNA's cannot be differentiated with respect to size
[(99 + 5) x IO6daltons] or the presence and distribution
of alkali-labile bonds (9, 19). They differ, however, in
base composition [67 and 69 guanine plus cytosine moles
per 100 ml for HSV-1 and HSV-2 (17, 19)] and, perhaps
more strikingly, in genetic complexity. Thus we pre
viously reported that HSV-1 DNA sequences are at least
95% unique (8), and indeed a plot of reassociation of
HSV-1 DNA according to Equation (F) yields a line
with the intercept at 1 indicating absence of appreciable
repetitive sequence. On the other hand, the reassociation
of HSV-2 DNA plotted in a similar fashion yielded an
Research.
intercept of 1.189 indicating that approximately 16% of
HSV-2 DNA consists of repetitive sequences. The sig
nificance of this observation particularly in regard to the 1
bilogical difference between HSV-1 and HSV-2 of interest
to us here (the oncogenic potential of HSV-2 as compared
to that of HSV-2) is not clear. It would be interesting to
determine not only what genetic information is repetitive
in HSV-2 DNA but also what information present in
HSV-1 DNA has been replaced by the repetitive se
quences in HSV-2 DNA.
The Transcriptional Programs of HSV-1 and HSV-2
in Productively I nfected Cells
The transcriptional programs of HSV-1 and HSV-2 in
productively infected cells have been analyzed from 4
points of view. These are (a) the fraction of viral DNA
complementary to viral RNA transcripts present in the
infected cells at 2 and 8 hr postinfection, i.e., pre- and
postviral DNA synthesis; (b) the effect of protein synthesis
on the extent of viral DNA transcribed early in infection;
(c) the number of classes of viral RNA transcripts dif
fering in abundance and the size of the corresponding
viral DNA templates from which they are transcribed;
and (d) the distribution of DNA sequences shared in com
mon by HSV-1 and HSV-2 DNA's among the templates
for abundant and scarce RNA's, respectively.
As summarized in Table 6, the transcriptional programs
of HSV-1 and HSV-2 share in common 2 features. Both
exhibit "off-on" controls of transcription and controls
of RNA abundance, described in a previous paper of
this series (10). They differ, however, in detail and more
specifically in 4 major respects as follows: (a) the extent
of DNA transcribed at 2 hr postexposure of cells to virus,
i.e., prior to the onset of DNA synthesis; (b) the com
position of RNA transcripts and, more specifically, the
number of classes of RNA templates differing in molar
ratios; (c) the size of the templates specifying abundant
RNA; and d the extent of DNA transcribed in 2-hr in
fected cells treated with cycloheximide. Several points
should be made in connection with these data.
HSV-2 DNA is transcribed to only 21% early in in
fection. The RNA made early forms only 1 abundance
class. Late in infection, the RNA is transcribed from 50%
of the DNA and forms from 2 classes, abundant and
scarce, arising from 31 and 19%of the DNA, respectively.
By contrast, both the abundant and scarce RNA's are
made early in cells infected with HSV-1. It would seem,
therefore, than an "off-on" control of transcription present
in HSV-2 DNA is either muted or expressed much earlier
in HSV-1-infected cells. The significance of these findings
emerges from the observation that among the HSV-1 tem
plates transcribed early are those specifying at least 20 of
the 24 structural proteins. If the oncogenic potential of
herpes viruses is the consequence of an expression of an
"early" function in the absence of expression of functions
concerned with the synthesis and assembly of the virus and
cell death, the virus expressing the least functions other
than those concerned with cell transformation is more likely
Table 6
The transcripiional programs of HS V-1 and HSV-2 in productively
infected HEp-2 cells
Features of the program
HSV-1 HSV-2
Early (2 hr postinfection)
i of DNA transcribed 44 21
No. of RNA classes differing in abun- 2 I
dance
%of DNA specifying abundant class 14 21
%of DNA specifying scarce class 30
) of DNA transcribed in the presence of 44 45
cycloheximide
Late (8 hr postinfection)
) of DNA transcribed 48 50
No. of RNA classes differing in abun- 2 2
dance
%of DNA specifying abundant class 19 31
%of DNA specifying scarce class 28 19
to be oncogenic. From this point of view HSV-2 might be
in certain situations potentially more oncogenic than
HSV-1.
In the absence of protein synthesis inhibited by cy
cloheximide, the HSV-2 DNA early in infection is trans
cribed to 45%, i.e., to the same level as HSV-1 DNA.
These observations suggest that the transcription of 24%
of HSV-2 DNA (the difference between 45% transcribed
in the treated cells and 21% transcribed in the untreated
cells) is regulated by a negative control which involves
proteins made after infection. A more trivial hypothesis is
that 24% of HSV-2 DNA is transcribed but the transcripts
are rapidly degraded. Parenthetically, the effects of cyclo
heximide noted in our studies differ materially from those
deduced by Rakusanova et al. (30) who concluded that the
RNA made in the presence of cycloheximide was comple
mentary to a smaller template than that made "early." We
cannot account for the differences in our respective conclu
sions except to point out that our techniques involving
hybridization in liquid of excess unlabeled RNA with labeled
DNA is free of errors due to ribonucleotide pool size and
more amenable to analytical treatment than hybridization
tests involving labeled RNA in liquid to DNA bound to
filter, particularly since the kinetics of such hybridizations
is not known.
HSV-DNA's may contain more than 1 site for "off-on"
control of transcription. This conclusion is based on the
fact that early in infection HSV-1 is transcribed to only
44%, whereas late in infection 48% of the DNA is trans
cribed. The difference is significant on the basis of 3 ob
servations, i.e., the finding is reproducible, several struc
tural proteins of the HSV-1 are made only late in infection
(10), and in the presence of cycloheximide both HSV-1
and HSV-2 are transcribed to approximately 44 to 45%.
Nothing is yet known concerning this "off-on" control of
transcription.
The abundant HSV-1 RNA extracted late in infection
is transcribed off 19% of HSV-1 DNA. Several lines of
evidence cited elsewhere in detail (10) led us to suggest
that the abundant RNA specifies structural proteins.
The conclusions were in part based on the size of the DNA
template required for structural proteins, their abundance
JUNE 1973 1413
Research.
relative to nonstructural proteins, and analyses of the
structural proteins made at different times in the repro
ductive cycle. By contrast, the late abundant HSV-2 RNA
is complementary to 31% of HSV-2 DNA. Since HSV-1
and HSV-2 DNA are closely related, it is not likely
that HSV-1 structural proteins require 50% more DNA
template (31% of total) than the HSV-1 structural pro
teins (19% of total). One possible explanation of this
discrepancy arises from the observation that HSV-1 DNA
consists largely (at least 95%) of unique DNA sequences
(18). By contrast, both the G and 174 prototypes of
HSV-2 isolated and propagated a continent apart con
tained repetitive sequences, amounting to 16% of the
DNA (Ref. 11; N. Frenkel and B. Roizman, unpublished
studies). The nature of the functions specified by the
repetitive sequences are not known; they could be a subset
of the sequences specifying the abundant RNA late in
infection.
As summarized in this paper, earlier studies (10) have
demonstrated the presence in the infected cells of RNA
transcripts differing in abundance. This could be pre
dicted on the basis of the fact that structural proteins
are made in much larger amounts than nonstructural pro
teins. The obvious and very relevant question is mechanism
by which RNA abundance is regulated. Preliminary stud
ies in our laboratory (S. Silverstein, S. L. Bachenheimer,
N. Frenkel, and B. Roizman, manuscript in preparation)
indicate that abundant DNA species in both nuclei and
polyribosomes are adenylated whereas the scarce species
which are also present in polyribosomes are not adenylated.
The data presented in this paper show that the region
of HSV-1 DNA serving as a template for RNA of high
abundance contains a larger fraction (71%) of sequences
complementary to HSV-2 DNA than the template for
the low-abundance RNA (39%). The significance of
this finding arises from the nature of functions specified
by the DNA templates for early and late RNA. Specif
ically, earlier in the text we listed several lines of evidence
that suggested that the HSV-1 template for abundant
RNA specifies the structural proteins of the virus. In
this paper we have shown that, although the shared se
quences are divided among the templates for abundant and
scarce RNA, those contained in the template for abundant
RNA constitute 71% of that set. If our interpretation of
the function of the abundant RNA species is correct, it
would follow that, in the course of evolution of HSV-1
and HSV-2 from a common ancestor, the sequences coding
structural proteins diverged less, on the average, than
those specifying nonstructural proteins.
The Significance of the Virus-specific RNA Sequences
in the Cervical Tumor
The tumor contains transcripts arising from 5% of viral
DNA. This amount of DNA is considerably less than
that transcribed in productively infected HEp-2 cells either
early (21%) or late (50%) in infection and by a wide margin
less than that required to specify structural proteins (10,
44). Two points bear on these data. The first arises from
the fact that HSV-2 like all other herpesviruses normally
kills the productively infected cell (34). Most of the cy-
tolytic functions of the virus, i.e., inhibition of host DNA
and protein synthesis and alteration of host RNA metab
olism, etc., are early functions. Clearly, if the infected host
is to survive, the functions expressed by the virus should be
fewer than those expressed early in productive infection.
As we pointed out in the text, we do not know what func
tions are expressed in the tumor or whether this amount
of transcription invariably accompanies the presence of
viral DNA in tumor cells.
Characteristics of Viral DNA Present in the Tumor
DNA-DNA hybridizations indicate that only 39% of the
viral genome is present in the tumor cells. The findings
are significant from 2 points of view. First, since only a
fragment of the DNA is present in the tumor tissue in
ratios of 1 to a maximum of 3.5 fragments per cell, it
seems reasonable to conclude that the fragment arose as a
consequence of a single event of infection. The 2nd point
concerns the significance of the finding of a fragment
rather than of an entire genome. As indicated elsewhere,
infection of human cells cultivated in vitro with HSV-2
is invariably productive (34) and in acute infection the virus
readily multiplies in cervical tissues. A nonproduction in
fection could arise in 2 ways, as a consequence of infection
by a defective virus or as a result of infection of a non-
permissive cell with competent virus. While we cannot ex
clude the possibility that the cervix contains nonpermis
sive cells which preclude the expression of many viral
functions and survive, an attractive alternative is that non
productive infections in which the viral genome is per
petuated without cell destruction arises also from infec
tion with particles carrying defective genomes. The trans
formation of susceptible cells in vitro (6) by HSV-2 was
accomplished only with UV-irradiated virus and, to our
knowledge, infectious virus cannot be isolated routinely
and with ease from cervical tumor tissues or cell cultures.
The Association of Viral DNA Sequences with Repetitive
Sequences of Cervical Tumor DNA
As indicated in the text, these experiments were de
signed to determine whether viral DNA present in the tu
mor is covalently linked to host DNA. The results indicate
that the viral sequences are linked to host DNA in the
proximity of host repetitive sequences. The amounts of
viral DNA sequences recovered in association with the
repetitive sequences appear to be smaller than those cal
culated to be present in unfractionated cervical tumor
DNA. A trivial explanation of the data is that viral se
quences are fragmented and that only some of these se
quences are covalently linked to host repetitive sequences.
A more likely explanation is that (a) fragmentation of
tumor cell DNA did occur, particularly during heat de-
naturation of the DNA at 115for 7 minutes; (b) viral
sequences that remained linked to repetitive sequences
Research.
were those near to the ends of the viral DNA molecule;
and (c) the probability of viral sequences remaining co-
valently linked to host DNA decreased in proportion to
their distance from the ends. We cannot presently discrim
inate between these 2 possibilities, but the data do argue
that at least some of the viral sequences are covalently
linked to repetitive sequences of host DNA.
The Relationship between I I SV-2 and Cervical Tumors
This is the 1st demonstration of viral DNA, viral
RNA, and of covalent linkage of viral DNA to cell
DNA in a cervical tumor. These data would be predicted
if the virus causes the tumor but in themselves do not
exclude the possibility that the presence of the viral
genome is unrelated to the transforming event that led to
the tumor. As suggested elsewhere (11), while more tu
mors both natural and experimental should be analyzed,
direct proof of the causal relation may ultimately emerge
only from analysis of populations protected from acquir
ing infection with HSV-2 (26).
Acknowledgments
The studies on the cervical tumor were done in collaboration with Dr.
AndrNahmias at Emory University who contributed the tumors and
analyzed them for the presence of infectious virus and viral antigens
and with Dr. Enzo Cassai who extracted the nucleic acids. We wish to
acknowledge the participation of Dr. Saul Silverstein in the experiments
on the effect of cycloheximide on the transcription of viral DNA and
for the preparation of the N. crossa nuclease.
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Research.

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