(Received for publication, October 4, 1995; and in revised form, December 23, 1995)
From the
Influenza virus utilizes a unique mechanism for initiating the
transcription of viral mRNA. The viral transcriptase ribonucleoprotein
complex hydrolyzes host cell transcripts containing the cap 1 structure
(mGpppG(2`-OMe)-) to generate a capped primer for viral
mRNA transcription. Basic aspects of this viral endonuclease reaction
are elucidated in this study through the use of synthetic, radiolabeled
RNA substrates and substrate analogs containing the cap 1 structure.
Unlike most ribonucleases, this viral endonuclease is shown to catalyze
the hydrolysis of the scissile phosphodiester, resulting in
5`-phosphate- and 3`-hydroxyl-containing fragments. Nevertheless, the
2`-OH adjacent to the released ribosyl 3`-OH is shown to be important
for catalysis. In addition, while the endonuclease steady-state
turnover rate is measured to be 2 h
, phosphodiester
bond hydrolysis is not rate-limiting. The direct generation of a free
3`-OH and the subsequent slow release of this product are consistent
with the viral need for efficient use of the capped primer in
subsequent reactions of the influenza transcriptase complex.
Most viral and cellular mRNA molecules contain a methylated cap
structure at the 5`-end (for reviews see (1) and (2) ). The presence of a cap is important for mRNA maturation,
initiation of translation, and protection against degradation by RNases
present in the cell. The general structure of a capped RNA can be
designated as mG(5`)ppp(5`)Puo- (Puo, the penultimate base,
is typically a purine(1) ). The penultimate base of cap
1-containing mRNAs is 2`-O-methylated and can be designated
m
G(5`)ppp(5`)Puo(2`-OMe).
The first step in the synthesis of influenza virus mRNA is the binding of host cell nuclear mRNA having a cap 1 structure(3, 4, 5) by the PB2 protein of the viral transcriptase complex(6, 7, 8) . Subsequent cleavage of the capped RNA 10-15 bases from the penultimate nucleoside generates capped oligoribonucleotides that serve as primers in mRNA synthesis by the viral transcriptase(9, 10, 11, 12) .
Heretofore, the RNA endonuclease reaction of the influenza transcriptase complex has not been well defined, either in terms of the chemical structure of substrates and products or in the quantification of reaction kinetics. The difficulty has been the unavailability of homogeneous preparations of short substrates containing the cap 1 structure. Initial characterizations of the endonuclease have employed mRNAs isolated from natural sources (10) or prepared by in vitro ``run-off'' transcription, so that the preparations were heterogeneous in length or in cap structure. In the present study, the basic mechanism of the endonuclease reaction is defined through the use of chemically synthesized, short capped RNAs.
where p(t) is the amount of product formed at time t, a is the burst amplitude, k is the first order rate constant, and k
is
the steady-state turnover rate constant. The concentration of
endonuclease active sites was determined from the burst amplitude,
correcting for the difference between the burst and steady-state rates
by multiplying the burst amplitude by (k
+ k
)/k
.
Figure 1:
Sequencing gel (8% polyacrylamide)
electrophoretic analysis of the products from an endonuclease cleavage
reaction of P-labeled RNA-capped I. Incubation was from 3
to 60 min in the absence and presence of 40 µM GTP. The
major product results from specific hydrolysis at position 13. When GTP
was included, the cleavage product was extended by the
endonuclease-associated transcriptase activity by one or more GMP
residues. A small amount of nonspecific cleavage was obtained that was
not extended in the presence of GTP.
Figure 2:
Sequencing gel analysis of the
endonuclease cleavage products of the doubly labeled 20-base
mG
pppGmUUUUUAUUUUUAAUUUUC-
P-(3`-dA)-3`
analog of capped I. Lane 1 contains the 13-base product (c) of the endonuclease reaction with labeled capped I (b). Lane 2 is a control of doubly labeled analog of
capped I (a) with no reaction, whereas lane 4 contains the 13-base (c) and 6-base cordycepin-labeled (e) RNA cleavage products after incubation with the
endonuclease. Lanes 3 and 5 contain control synthetic
oligoribonucleotides 5`-AUUUUC-
P-(3`-dA)-3` (d)
and 5`-
P-AUUUUC(3`-dA)-3` (e),
respectively.
The ability of the
endonuclease to cleave modified capped RNAs, where the 2`-OH at the
preferred cleavage site was replaced by a hydrogen or a fluorine atom,
was investigated. Oligoribonucleotides I and II, having
either an unmodified adenosine or a 2`-deoxyadenosine located at
A, respectively, were incubated with ribonucleoprotein in
the presence or absence of one of the four nucleoside triphosphates.
The results are shown in Fig. 3. First, there was no detectable
cleavage at A
for the 2`-dA containing RNA, and the site
of cleavage was shifted from 3` to A
to the 3` side of
A
. In addition, the hydrolysis rate was approximately 10
times slower than that for capped I, and similar results were
obtained for the 2`-fluoro-containing oligoribonucleotide III (data not shown). The data presented in Fig. 3also show
that the specific cleavage products for both substrates (capped I and II) are extended when incubated in the presence of GTP
and not with any of the other three nucleoside triphosphates.
Figure 3:
Comparison of the cleavage of P-labeled capped I with an analog of capped I having a
deoxyadenosine residue at the site of cleavage (II). Reactions
contained no NTP (lanes 2 and 8) or 50 µM ATP (lanes 3 and 9), CTP (lanes 4 and 10), GTP (lanes 5 and 11), or UTP (lanes
6 and 12). The A
product (a)
resulting from capped I and the A
product (b)
from capped II were only extended by the endonuclease-associated
transcriptase activity when GTP was present. The presence of the
2`-deoxyadenosine nucleoside in oligoribonucleotide II was confirmed by
partial alkaline hydrolysis (compare lane 1, containing the
hydrolysis products of capped I, and lane 7)(24) . The
absence of base-catalyzed hydrolysis bands corresponds to the position
where the 2`-OH was replaced with a 2`-deoxysubstituted nucleoside (bracket c, lane 7). Two bands result from this
treatment, apparently due to the product having a 2`,3`-cyclic
phosphate or a 3`-phosphate terminus. The bands resulting from the
alkaline hydrolysis migrate faster through the gel as compared with the same length endonuclease-derived cleavage product due to the
presence of the 3`-phosphate group and possibly due to C-8/N-9 cleavage
within the cap base(1) .
Figure 4:
Reaction progress curve for the cleavage
of capped I by influenza endonuclease. Reactions were carried out as
described under ''Experimental Procedures.`` A,
protein concentrations were 2.0 (), 1.6 (
), and 1.2
(
) µg/ml. B, the reaction was quenched using the
standard protocol before being loaded on the gel (
).
Alternatively, aliquots were filtered through a nitrocellulose membrane
to remove enzyme and RNA-associated enzyme before being loaded
(
). The data were fit to .
Figure 5:
A, IC curve for
m
GpppGmUAUUAAUA(3`-dA)-3` (capped IV) against influenza
endonuclease activity using 400 pM m
GpppGmUUUUUAUUUUUAAUUUUC-3` I as substrate. The
inhibitor was preincubated with the enzyme for 30 min prior to
initiation of the reaction with capped I. The data were fit to the Hill
equation and resulted in an IC
of 83 ± 5
pM. B, equilibrium binding of capped IV to influenza
endonuclease. Binding reactions were carried out as described under
''Experimental Procedures.`` The data were fit to a
hyperbolic function using a nonlinear squares algorithm to yield the
following best fit parameters: amplitude = 52.0 ± 1.8
pM, K
= 170 ± 16
pM.
Equilibrium binding experiments were performed by
separation of free from bound capped IV using a nitrocellulose
filter retention assay. The equilibrium titration results are shown in Fig. 5B. The data were consistent with a single class
of noninteracting binding sites. Nonlinear least-squares fitting
returned values of K = 170 pM and
a saturation amplitude of 52 pM. Only 10% of the bound
P-capped IV was competed by up to 150 nM uncapped IV. In contrast, 95% was displaced by competition
with unlabeled capped IV. Thus, the binding to the endonuclease
was dependent on the presence of a 5`-cap and reflected direct binding
to the endonuclease active site. The effective endonuclease active site
concentration was derived from the value of the saturation amplitude.
The aim of this work was to elucidate the mechanism of influenza endonuclease. To carry out this study, a method was developed for the synthesis of short, sequence-specific cap 1-containing RNA molecules, which allowed for the site-specific incorporation of nucleoside analogs.
The data presented for the cleavage of capped II indicate that the 2`-OH at the cleavage site is important for either recognition or catalysis (Fig. 3). A large number of ribonucleases catalyze the cleavage of the phosphodiester bond between the 3` and 5` riboses of adjacent nucleosides by using the ribose 2`-OH as the nucleophile. This results in the formation of a 2`,3`-cyclic phosphate-terminated RNA, which slowly hydrolyzes to the 3`-phosphate(21) . In contrast, the final product of the influenza endonuclease reaction is an RNA with a free 3`-OH. This species is the required end product because it is utilized as a primer for the subsequent transcriptase reaction(10) . In addition, the data presented in Fig. 2indicate that the phosphate remains with the six-nucleotide 3`-cleavage fragment of I. Thus, the influenza endonuclease carries out the nucleophilic attack of the internucleotidic linkage with a group other than the 2`-OH, with the concomitant cleavage of the 3`-O-phosphorous bond. This is analogous to the mechanism proposed for the cleavage of RNA in DNA-RNA duplexes by Escherichia coli ribonuclease H(22) . When the endonuclease was incubated with another analog of capped I, where the nucleoside at the site of cleavage was replaced with a 2`-fluoro-modified ribose, no cleavage at this position was observed. In contrast, RNaseH is able to hydrolyze 2`-fluoro-substituted RNA, albeit at a reduced rate(22) . This suggests that the 2`-OH is required for influenza endonuclease, whereas it is not likely to be utilized as the nucleophile at the site of cleavage.
The cleavage of
capped 19-base I yielded a biphasic reaction progress curve. Fig. 4A illustrates that there was a burst in the
conversion of substrate to product at a rate of 21
h, followed by a much slower steady-state rate of 2
h
. The presence of a burst was consistent with the
rapid accumulation of enzyme-bound product (E
cap-13-mer)
followed by a slow breakdown of E
cap-13-mer to yield
free enzyme. The less than stoichiometric conversion of substrate to
product per enzyme determined from the burst amplitude was consistent
with the presence of some ribonucleoprotein that was able to bind
capped IV but was inactive catalytically.
Previous studies
have shown that influenza endonuclease was able to bind but was unable
to cleave capped RNA when divalent metal ion was absent from the
reaction(23) . The progress curves for reactions initiated with
capped I or Mg were compared to determine
whether productive binding of the capped RNA I was
rate-determining for the burst part of the reaction. No significant
difference in the reaction progress curves was observed, indicating
that the rate of substrate binding does not significantly affect the
rate of the burst phase. Thus, the rate-limiting step during the burst
phase might be due to a catalytic event or a rate-determining
conformational change.
The linear steady-state rate observed in the
biphasic reaction progress curve (Fig. 4A) was
consistent with the slow release of Ecap-13-base to
yield free enzyme. In order to confirm this, a method was developed
that removed all the E
cap-13-base at various time points
during a reaction by filtering an aliquot through a nitrocellulose
membrane. If release of the enzyme-bound product were rate-limiting,
then the data would originate near the origin and linearly parallel the
data obtained for the reaction with the burst. The results from this
experiment (Fig. 4B) clearly show that the data from
the two methods parallel each other. The slow phase of the reaction
with the rate of 2 h
was consistent with the
dissociation of the capped 13-base product from the enzyme. It is
reasonable that the product dissociates slowly from the enzyme since it
is the substrate for the ensuing transcriptase reaction. Also, it would
be deleterious to the virus if the endonuclease efficiently catalyzed
the cleavage of all the host cellular mRNA molecules, thus killing the
cell.
The slow release of the capped RNA product is consistent with the tight binding and potent inhibition by the product-analog, capped IV (Fig. 5). In fact, a recent report suggested that a mixture of short length capped RNAs inhibited the RNA polymerase activity of influenza ribonucleoprotein(23) . The capped 10-base IV that was selected for the current study was reported by Plotch and co-workers (10) to be the shortest influenza endonuclease-derived fragment from the cleavage of brome mosaic virus RNA 4. The tight binding of this product-analog also allowed for the determination of enzyme concentration and turnover number. It will be interesting to determine if shorter or linker-substituted capped RNA analogs also exhibit tight binding characteristics. Such compounds might prove useful as specific antiviral agents.
In summary, basic aspects of the influenza endonuclease reaction have now been elucidated with the use of chemically defined capped RNAs. In addition to establishing the bond hydrolyzed at the site of cleavage, endonuclease-catalyzed turnover was quantitatively determined.