(Received for publication, September 14, 1995)
From the
To examine the possible role of the vaccinia virus glutaredoxin
as a cofactor for viral ribonucleotide reductase, viral growth, DNA
synthesis, and dNTP pools were measured in infections of B-SC-40 monkey
kidney cells with wild type vaccinia virus and with mutants of vaccinia
that lacked a functional reductase or glutaredoxin. In infections of
untreated host cells, the lack of viral ribonucleotide reductase or
glutaredoxin had only small effects upon virus growth. When host cells
were pretreated with -amanitin, which blocks host RNA polymerase
II but not viral transcription, viral DNA synthesis was markedly
reduced in infections with either of the mutants when compared with
wild type infections. Relative to dNTP levels in wild type infections,
pools of dCTP, but not of the other dNTPs, were significantly reduced
in infections of amanitin-treated cells with either mutant. The
parallel depletion of dCTP in the two mutants suggests that the role of
glutaredoxin may be to function as a cofactor for viral ribonucleotide
reductase. The data suggest that both viral proteins become essential
for DNA replication only when levels of the corresponding host cell
proteins are depleted.
Poxviruses, such as vaccinia virus, replicate their DNA in the cytoplasm of host cells. This means that the viral genome must encode most, if not all, of the proteins required for DNA synthesis, since the host enzymes for replication are localized in the nucleus. Indeed, vaccinia virus has been shown to encode many of the enzymes involved in the synthesis of DNA and its deoxynucleotide precursors(1) .
Among such virally encoded enzymes is ribonucleotide reductase
(RNR), ()which provides the precursors for DNA synthesis by
the reduction of ribonucleotides to their corresponding
deoxynucleotides. To carry out this reaction, ribonucleotide reductase
presumably acts in conjunction with a hydrogen donor, which, in turn,
reduces the enzyme. Among the cofactors known for various
ribonucleotide reductases are the small thiol transferase proteins
called the glutaredoxins(2) .
It was recently shown that the vaccinia O2L open reading frame encodes a functional glutaredoxin, a 12-kDa protein that is expressed postreplicatively but which is packaged in the virions(3) . Although the protein is synthesized after DNA replication has begun and may well have other roles in the virus growth cycle, one cannot rule out the possibility that it is acting as a hydrogen donor for ribonucleotide reductase.
To assess the role of the viral glutaredoxin in relation to the
activity of the viral ribonucleotide reductase, viral growth, DNA
synthesis, and dNTP pools were compared in infections of B-SC-40 monkey
kidney cells with wild type vaccinia virus, and with two mutant
viruses, M1, with an inactive ribonucleotide reductase R1 subunit,
and vGRX
(gpt), a glutaredoxin mutant. M1
, which
has been shown to have no ribonucleotide reductase activity as measured
by conversion of tritiated CDP to dCDP, has been reported to replicate
at wild type levels, as measured by plaque-forming ability at 24 and 48
h postinfection(4) . This would suggest that in the absence of
functional viral RNR, viral replication could be sustained by a supply
of dNTPs produced by the host cell's ribonucleotide reductase. If
this is true, then the loss of a functional glutaredoxin might have no
discernible effect on viral replication, even if glutaredoxin was, in
fact, a required cofactor for the viral reductase. Thus, it is
important to measure DNA synthesis and dNTP pools in the absence of an
active host cell RNR. To achieve this, we have used
-amanitin
treatment of host cells for 18 h prior to infecting them with virus.
Amanitin has been shown to inhibit host RNA polymerase II but not viral
transcription(5, 6) . Therefore, we expect amanitin
pretreatment to deplete the cell of relatively short-lived mRNAs,
including those for RNR and glutaredoxin. In this report, we show that
in the absence of host cell transcription, the synthesis of dNTPs, as
well as of viral DNA, is greatly affected in mutant viruses for either
ribonucleotide reductase or glutaredoxin.
Vaccinia virus, WR strain (wild type), and mutant strains, M1
and vGRX
(gpt), were maintained as described
previously(7) . M1
was a generous gift from Dr. Dennis
Hruby, Oregon State University.
Figure 1: DNA blot analysis of recombinant virus genomes. Top, a segment of the vaccinia virus genome containing the 1.75-kbp grx (hatched) open reading frame with the plasmid pGRX(gpt) below. In the plasmid, the grx gene (hatched) is shown interrupted by the gpt gene (unhatched). The crossed lines are potential sites of crossover recombination. Middle, single (i) and ii)) and double (iii)) crossover genomes are depicted. The numbers indicate the sizes of predicted XbaI fragments that should hybridize to a grx DNA probe. Bottom, Southern blot autoradiograph showing the XbaI fragments that hybridized to grx probe. W, wild type vaccinia virus DNA; lanes 1-7, independent recombinant viruses after five successive plaque isolations in gpt selective medium. Arrows show the sizes of XbaI DNA fragments.
Immunoblot analysis was performed to confirm
the absence of grx expression. As shown in Fig. 2,
cells infected with wild type vaccinia virus or recombinant viruses 1,
3, 5, 6, and 7 produced an M 12,000 protein that
reacted with rabbit polyclonal antibody to glutaredoxin. By contrast,
cells infected with recombinant viruses 2 and 4 did not produce an
immunoreactive protein. Thus, recombinant viruses 2 and 4 had neither
an intact grx gene nor detectable expressed glutaredoxin and
were, therefore, used for further experiments. No difference was noted
in the size or appearance of wild type and mutant plaques that formed
on B-SC-1 cells, indicating that a functional grx was not
necessary for replication in tissue culture.
Figure 2:
Immunoblot analysis of cells infected with
recombinant viruses. B-SC-1 cells were infected with the same
recombinant viruses described in Fig. 1, and the lysates were
subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
The proteins were transferred to a nitrocellulose membrane, which was
then incubated with rabbit polyclonal antibody to recombinant
glutaredoxin followed by I-protein A. Lanes containing wild type and recombinant viruses correspond to Fig. 1. The sizes in kDa of protein markers are shown on the left. The arrow points to
glutaredoxin.
Figure 3:
Time course of virus growth. B-SC-40 cells
were infected with wild type (w.t.), M1, or
vGRX
(gpt) virus at 1 plaque forming unit (p.f.u.)/cell. At the indicated times postinfection infected
cells were harvested, and the titer of progeny virus was determined by
plaque titration. Titers are based on triplicate plaque counts, which
generally agreed within 10%.
Viral DNA synthesis in
infections of untreated cells with wild type, M1, and
vGRX
(gpt) virus is shown in Fig. 4A.
DNA accumulation increased steadily with time up to 8 h in all three
strains. The levels of DNA accumulation at this time in the mutant
viruses were 84 and 98% of wild type for M1
and
vGRX
(gpt), respectively. Fig. 4B shows viral DNA synthesis in infections of
-amanitin-treated
cells. DNA accumulation in infections with wild type virus were
comparable in amanitin-treated and untreated cells at all time points
measured. In contrast, infections of amanitin-treated cells with either
M1
or vGRX
(gpt) virus showed a marked reduction
in DNA synthesis. At 8 h, the DNA accumulations in M1
and
vGRX
(gpt) infections were 1 and 14%, respectively, of
the corresponding wild type levels.
Figure 4:
Viral DNA accumulation in infections of
B-SC-40 cells, untreated (A) or treated with 6 µg/ml
-amanitin (B). Infections with wild type (W.T.)
and mutant viruses were carried out as for growth measurements.
Infected cells were harvested at the indicated times after infection,
lysed, and assayed for viral DNA accumulation using a quantitative dot
blot hybridization method.
Figure 5:
Pool sizes of dNTPs in infections of
B-SC-40 cells, untreated (A) or treated with 6 µg/ml
-amanitin (B). Infections with wild type (w.t.)
and mutant viruses were carried out as for growth measurements.
Infected cells were harvested at 6 h postinfection, and extracts were
prepared. Pool sizes of the four dNTPs were measured by an enzymatic
method. Error bars represent standard deviations in triplicate
assays. In B, dCTP and dGTP were virtually undetectable in
infections by M1
and vGRX
(gpt) mutant viruses. M.I., mock infected.
Under conditions where host cell RNA polymerase II is
inactivated, it is possible to examine the effects of mutations in
viral genes that are not apparent in infections of untreated cells. The
experiments to measure growth of the mutant viruses (in terms of
plaque-forming ability) could not be done using amanitin-treated host
cells, as it has been shown that host cell functions are required for
viral assembly, though not for viral DNA
replication(12, 13) . Viral growth, measured in
infections of untreated cells, showed relatively small differences at
early time points and no difference after 12 h postinfection between
wild type and either of the mutant virus strains. For M1, this is
in agreement with earlier reports showing that M1
replicated to
levels comparable with that of wild type virus in infections of B-SC-40
cells, when measured at 24 and 48 h postinfection(4) . This is
somewhat unexpected, given the importance of ribonucleotide reductase
in supplying precursors for DNA replication. A simple explanation of
this observation could be that in infections with virus lacking a
functional reductase, the host cell's reductase might supply the
virus with sufficient dNTPs to sustain growth. The growth experiments
were done with subconfluent monolayers of B-SC-40 cells, which,
although not synchronized, would have at least a subset of the
population in active division and thus have functional ribonucleotide
reductase activity. In the case of the vGRX
(gpt)
mutant, it is not entirely surprising to see no difference in growth
compared with the wild type virus, since (a) it may not have
any direct role in the production of dNTPs and (b) even if it
did function as a cofactor for ribonucleotide reductase, a similar
argument could be advanced as for M1
, namely that a host cell
protein stands in for the missing viral glutaredoxin.
If host
proteins were, indeed, substituting for viral gene products, then this
should be apparent if host transcription was inhibited for long enough
to ensure that existing host reductase or glutaredoxin had been turned
over prior to virus infection. The experiments to measure DNA synthesis
and dNTP pools were designed with this in mind. A comparison of DNA
synthesis by the three strains of virus in untreated and
amanitin-treated cells showed that wild type DNA synthesis was
unaffected by the shutdown of host cell transcription, as might be
expected for a virus that has all of the machinery required for the
synthesis of DNA, as well as for its precursors. M1 and
vGRX
(gpt)virus, however, showed severely reduced
levels of DNA synthesis under conditions where host cell proteins were
presumably unavailable, while synthesizing close to wild type levels of
DNA in host cells that had not been amanitin-treated. While this
observation supports the idea that cellular proteins are recruited by
viruses mutant for reductase or glutaredoxin, it does not shed any
light on the question of whether the similar reductions in DNA
synthesis in the two mutants are because they function together in the
production of dNTPs. The classical way to answer this question would be
to make a double mutant virus that lacked both reductase and
glutaredoxin. The prediction would be that the double mutant would have
a phenotype no different from those of either of the single mutants if
they both act through the same pathway. However, it is very likely that
even if glutaredoxin were a cofactor for reductase, it also has other
cellular functions(14) . Thus, it would be difficult to analyze
such a mutant and separate the effects of the losses of the different
functions of glutaredoxin.
A simpler way to assess whether
glutaredoxin is involved in the production of dNTPs is to measure dNTP
pools, as we did, in an infection by a vGRX(gpt)
mutant and compare them with pools in infections with a reductase
mutant and with wild type. Such measurements showed that while dNTP
pools were considerably reduced in all amanitin-treated cells,
including those that were mock-infected, the M1
and
vGRX
(gpt) mutant infections showed a strikingly
similar depletion of dCTP pools as compared with wild type infections.
It is interesting that although wild type infections in
amanitin-treated cells showed a large reduction in the pool sizes of
all four nucleotides, there were apparently sufficient levels of dNTPs
nevertheless for viral DNA synthesis to occur at levels comparable with
those in untreated cells. The drastic reduction in DNA synthesis in the
two mutants, then, seems related to the almost total depletion of the
dCTP pools. The reason for this apparently selective depletion of dCTP
pools in both mutants is not clear, but its parallel occurrence in both
mutant infections indicates that the loss of reductase and the loss of
glutaredoxin causes a very similar effect, supporting the idea that
they may be working together in the production of dNTPs. It has been
noted that the viral ribonucleotide reductase has a 3-fold lower K
for CDP compared with the cellular
enzyme(8) . Thus, under conditions where substrate levels are
low, such as might be prevalent in the amanitin-treated cells, there
would be a depletion of dCTP in infections with viruses lacking a
functional reductase or reductase cofactor, while wild type virus would
still be capable of reducing CDP efficiently and producing sufficient
dCTP to sustain normal levels of DNA replication and growth.