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Journal of Virology, October 1999, p. 8095-8103, Vol. 73, No. 10
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Mutation of Neuraminidase Cysteine Residues Yields
Temperature-Sensitive Influenza Viruses
Christopher F.
Basler,
Adolfo
García-Sastre, and
Peter
Palese*
Department of Microbiology, Mount Sinai
School of Medicine, New York, New York 10029
Received 8 April 1999/Accepted 30 June 1999
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ABSTRACT |
The influenza virus neuraminidase (NA) is a tetrameric, virus
surface glycoprotein possessing receptor-destroying activity. This
enzyme facilitates viral release and is a target of anti-influenza virus drugs. The NA structure has been extensively studied, and the
locations of disulfide bonds within the NA monomers have been identified. Because mutation of cysteine residues in other systems has
resulted in temperature-sensitive (ts) proteins, we asked whether
mutation of cysteine residues in the influenza virus NA would yield ts
mutants. The ability to rationally design tight and stable ts mutations
could facilitate the creation of efficient helper viruses for influenza
virus reverse genetics experiments. We generated a series of
cysteine-to-glycine mutants in the influenza A/WSN/33 virus NA. These
were assayed for neuraminidase activity in a transient expression
system, and active mutants were rescued into infectious virus by using
established reverse genetics techniques. Mutation of two cysteines not
involved in intrasubunit disulfide bonds, C49 and C146, had modest
effects on enzymatic activity and on viral replication. Mutation of two
cysteines, C303 and C320, which participate in a single disulfide bond
located in the 
5L0,1 loop, produced ts enzymes. Additionally, the
C303G and C320G transfectant viruses were found to be attenuated and ts. Because both the C303G and C320G viruses exhibited stable ts
phenotypes, they were tested as helper viruses in reverse genetics experiments. Efficiently rescued were an N1 neuraminidase from an avian
H5N1 virus, an N2 neuraminidase from a human H3N2 virus, and an N7
neuraminidase from an H7N7 equine virus. Thus, these cysteine-to-glycine NA mutants allow the rescue of a variety of wild-type and mutant NAs into influenza virus.
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INTRODUCTION |
The neuraminidase (NA) of influenza
A and B viruses is an enzymatic, tetrameric, type II integral membrane
glycoprotein found on the surface of virions. The NA possesses
receptor-destroying neuraminidase (acylneuraminyl hydrolase; EC
3.2.1.18) activity and cleaves the 
-ketosidic linkage connecting
terminal sialic acid residues to adjacent D-galactose or
D-galactosamine residues (17). This activity
facilitates viral release from infected cells by removing sialic acid
residues from glycoproteins and glycolipids at the cell surface, which
are potential ligands for the viral hemagglutinin (HA). The presence of
these residues during viral budding otherwise results in virus-virus
aggregation and retention of viral particles at the cell surface. Thus,
when temperature-sensitive (ts) NA mutant viruses are grown at
nonpermissive temperature, or when influenza viruses are grown in the
presence of NA inhibitors, viral particles form aggregates at the cell
surface (40, 41).
Because NA activity is required for efficient viral release, drugs
which inhibit NA activity can be effective anti-influenza virus agents.
This was first demonstrated in the mid-1970s when the sialic acid
analogues DANA and FANA were shown to inhibit influenza virus
replication in vitro (36, 41). Recently, using the X-ray
crystal structure of NA, additional NA inhibitors have been designed
(39, 53). Two such compounds, zanamivir (GG167 or
4-guanidino-Neu5Ac2en) and GS4104, are in clinical use in humans (20, 21).
Other functions have also been attributed to NA. For example, the NA of
influenza A/WSN/33 (H1N1) (WSN) virus is required for the
neuropathogenicity of this strain in mice (29) and has been
postulated to be a virulence factor (16, 34). Also,
compatibility of new HA-NA combinations generated by reassortment may
influence the emergence of pandemic strains (46). Because NA
is a structural protein, it may contribute to the assembly of viral
particles (24, 55). NA may also contribute to virus-induced
apoptosis (38, 47). Additionally, avian influenza viruses
possess NAs with hemagglutinating activity of unknown significance
(25, 26, 52).
The influenza virus NA proteins have been extensively studied. Mutant
proteins have been generated and assayed in transient expression
systems; viruses possessing mutant NAs have been isolated and
characterized; and detailed X-ray crystal structures of NA head
structures have been solved for two of the nine influenza A NA types,
N2 and N9, as well as for the influenza B virus NA. Reverse genetics
technology has allowed the introduction of specific NA mutants into
influenza viruses (9, 12). Most of these reverse genetics
experiments have used the WSN virus NA, because of its relatively
efficient rescue system (10). However, other rescue systems
based on either a host range selection or antibody selection have been
described (25, 29, 30).
Structurally, all of the NA types have a common head structure. Each
monomer head consists of a propeller-like structure with six blades
each composed of a four-stranded, anti-parallel -sheet. The strands
of each -sheet are connected via short loops, and separate sheets
are also connected by intervening loops. Among the various NA types,
particular residues are absolutely conserved. These conserved residues
include amino acids in or near the active site, residues at interfaces
between -sheets and at reverse turns, and cysteine residues involved
in disulfide bonds (6).
A number of the conserved NA cysteines participate in intrasubunit
disulfide bonds, and most of these bonds link cysteines located within
strands of -sheets. Additionally, a disulfide bond forms between two
cysteines (C303 and C320 [WSN numbering]), located in an extended
loop, 5L0,1 (51). In addition, some of the nonconserved
cysteines participate in intersubunit disulfides which join monomers in
the mature tetrameric enzyme.
Disulfide bonds are thought to be important for maintaining protein
stability but may not be essential for proper protein folding
(8). Previous analyses of several viral proteins, including the influenza virus HA, the herpes simplex virus type 1 (HSV-1) gD, and
the HSV-1 TIF ( trans-inducing factor; VP16) proteins, indicated that mutation of cysteine residues, including those known to
participate in disulfide bonds, can alter protein stability and/or
function (3, 32, 48, 54). Additionally, studies with the
HSV-1 gD-1 and TIF proteins indicated that mutation of cysteine
residues may result in temperature-sensitive proteins (31,
44).
We were interested in determining whether ts influenza viruses could be
generated by mutation of NA cysteine residues. Such mutants might prove
useful as helper viruses in reverse genetics experiments. Successful
generation of ts NA mutants would also recommend this approach for
other viral proteins, either for the generation of novel helper viruses
or for the introduction of attenuating mutations into live vaccine
strains. Therefore, using the available influenza NA X-ray crystal
structures as a guide to the location of disulfide bonds
(51), we generated a series of cysteine-to-glycine mutations
within the WSN NA. These mutants were constructed such that each
disulfide bond would be disrupted. The resulting mutants were screened
for NA activity after transient expression at different temperatures.
We identified four individual cysteines which, when mutated, yielded
active enzymes. Two C-to-G mutants (C303G and C320G) were attenuated
and ts, while two C-to-G mutants (C49G and C146G) were similar to the
wild type. These four mutant NAs were introduced into transfectant
viruses via the established WSN NA rescue system. The C303G and C320G
transfectant viruses displayed tight ts growth properties and failed to
produce properly folded, stable NAs at the nonpermissive temperature. Additionally, these ts viruses have been used in an efficient reverse
genetics system to rescue an N1 from an avian H5N1 virus, an N2 from a
human H3N2 virus, and an N7 from an equine H7N7 virus.
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MATERIALS AND METHODS |
Cell lines and viruses.
COS-1 cells were maintained in
Dulbecco modified Eagle medium (DMEM)-10% fetal bovine serum (FBS).
MDBK cells were maintained in REM-10% FBS, and viruses were plaqued
on MDBK cells in REM. MDCK cells were maintained in minimal essential
medium-10% FBS, and viruses were plaqued on MDCK cells in DMEM-F-12,
supplemented with 5 µg of trypsin (Trypsin 1:250; Difco) per ml. Vero
cells were maintained in serum-free AIM-V medium. Stocks of WSN virus and the transfectant WSN NA mutants C49G, C146G, C303G, and C320G were
grown on MDBK cells. Stocks of WSN-HK virus and WSN-N1(HK/97), WSN-N2(LA/87), and WSN-N7(Cor/74) (see below for explanation of designations) transfectant viruses were grown in 10-day-old embryonated chicken eggs.
Generation and expression of mutant WSN NA proteins.
The NA
sequence from pT3NAm1, which encodes wild-type WSN NA protein, was
subcloned between the EcoRI and HindIII sites
of pGEM-11zf(
) such that NA expression could be driven by the T7 promoter. The resulting plasmid, pGEM-NAm1, was used as template for
site-directed mutagenesis. Mutagenesis reactions were performed by
using a Stratagene QuikChange site-directed mutagenesis kit. Primers
used in these reactions are listed in Table
1.
NAs were expressed via the vaccinia virus-T7 expression system
(
11) previously used to express other NA mutants
(
14).
Confluent 35-mm-diameter dishes of COS-1 cells were
infected with
the T7 RNA polymerase-expressing vaccinia virus, vTF7-3,
at 1
PFU/cell; 45 min after addition of virus, cells were transfected
with 5 µg of NA expression plasmid by using DOTAP liposomal
transfection
reagent (Boehringer Mannheim) and then grown at 33, 37, or
39.5°C
for 15
h.
NA assays.
COS-1 cells transfected with NA expression
plasmids were washed once in phosphate-buffered saline, scraped from
the dish, pelleted at 4°C, resuspended in lysis buffer (10 mM
Tris-HCl [pH 7.4], 1 M NaCl, 10 mM CaCl2, 2% Triton
X-100, 1 mM Pefabloc [Boehringer Mannheim]), and incubated on ice 30 min. The lysate was then microcentrifuged at 15,000 rpm for 30 min at
4°C. The pellets were removed, and the supernatants were assayed for
NA activity.
NA assays were performed at 30°C for 1 h. Reaction mixtures
contained 0.1 M potassium phosphate buffer (pH 6.0), 1 mM
CaCl
2,
and 0.5 mM
2'-(4-methylumbelliferyl)-
-
D-
N-acetylneuraminic
acid
(Mu-NANA). Reactions were terminated by addition of 2 ml of 0.5
M
glycine-NaOH (pH 10.4). Fluorescence, resulting from cleavage
of the
ketosidic bond, was measured using a Turner model 112 fluorometer.
Tenfold dilutions of each lysate were prepared and assayed in
order to
compare samples in a linear
range.
Cloning of N1, N2, and N7 NA genes.
The N1 from influenza
A/chicken/Hong Kong/27402/97 (H5N1) virus [N1(HK/97)], the N2 from
influenza A/Los Angeles/2/87 (H3N2) virus [N2(LA/87)], and the N7
from influenza A/equine/Cornell/16/74 (H7N7) virus [N7(Cor/74)] were
amplified by reverse transcription-PCR (RT-PCR) from viral RNA (vRNA).
The H5N1 vRNA was provided by Michael Perdue, and the H7N7 virus was
provided by Gary Whittaker. The primers (influenza virus sequences in
uppercase; other sequences, [restriction enzyme sites for cloning or
plasmid linearization; bacteriophage promoters] in lowercase) used to
amplify the NA genes were as follows: for N1,
5'gcgcaagcttgaagacgcagcaaaAGCAGGAGTTTAAAATG-3' and
5'-gcgctctagaattaaccctcactaaaAGTAGAAACAAGGAGTTTTTTG-3'; for N7,
5'gcgatcatcgatctcttcgAGCAAAAGCAGGGTAATTTTGAAATGAATCCTAATCAAAAACTC-3' and
5'gcgcctcgagaattaaccctcactaaaAGTAGAAACAAGGGTTTTTTTCGTTTTACG-3'; for N2, 5'-tctagaggacgcgggggAGCAAAAGCAGGAGTGAAGATG-3'
and 5'-gcgcaagctttaatacgactcactataAGCGAAAGCAGGAGT-3' (these primers possess noncoding sequences corresponding to those of influenza A/Bangkok/1/79 (H3N2) virus).
RNP transfections.
Rescue of the WSN NA mutants was
performed as described elsewhere (10) except that
transfectant viruses were grown at 33°C. Briefly, MDBK cells were
infected with WSN-HK virus (multiplicity of infection of 1). One hour
after addition of virus, cells were transfected with in
vitro-synthesized NA vRNAs transcribed in the presence of influenza A
virus polymerase. Following transfection, the cells were incubated at
33°C for 18 h. Supernatants were then harvested and plaqued at
33°C on MDBK cells. Resulting transfectant viruses were plaque
purified three times on MDBK cells and subsequently amplified on MDBK cells.
Rescue experiments for N1(HK/97), the N2(LA/97), and N7(Cor/74) viruses
were performed as follows. Vero cells, maintained
in serum-free medium,
were infected with C320G virus at 1 PFU/cell.
One hour after addition
of virus, the cells were transfected with
NA vRNAs synthesized in vitro
from linearized plasmids. These
vRNAs were transcribed in the presence
of influenza A virus polymerase
which, to inactivate endogenous vRNAs,
was previously UV irradiated
for 90 s at a distance of 10 cm,
using an 8W G3T5 GL-8 germicidal
UV bulb. Following transfection, cells
were grown in MEM-0.2%
bovine serum albumin-3 µg of trypsin per ml
at 33°C for 24 h.
Supernatants were then harvested and plaqued
on MDCK cells at
39.5°C. Additionally, 10-day-old embryonated chicken
eggs were
infected with 0.1 ml of supernatant from transfected cells
and
incubated at 39.5°C for 2 days. Rescued transfectant viruses were
plaque purified three times on MDCK cells at 39.5°C.
Analysis of transfectant vRNAs.
Wild-type WSN,
WSN-N1(HK/97), WSN-N2(LA/87), and WSN-N7(Cor/74) viruses were grown in
10-day-old embryonated eggs for 2 days at 37°C. Allantoic fluid
containing virus was harvested from the eggs and centrifuged at
3,000 × g for 15 min at 4°C in a tabletop centrifuge. Subsequently, the virus-containing supernatants were centrifuged at 10,000 rpm in an SW28 rotor for 30 min at 4°C, virus
from the supernatants was pelleted through a 30% sucrose cushion by
centrifugation at 25,000 rpm in an SW28 rotor for 90 min at 4°C, the
viral pellet was resuspended in Trizol Reagent (Gibco BRL), and vRNA
was extracted by following the manufacturer's protocol.
RT-PCR amplification of the vRNAs was performed with the Titan one-tube
RT-PCR system (Boehringer Mannheim). Reactions were
performed as
follows: 30 min at 60°C for one cycle; 2 min at 94°C
for one cycle;
30 s at 94°C, 30 s at 60°C, and 1 min 10 s at 68°C
for 35 cycles; 7 min at 68°C for one cycle. The primers used to
amplify the WSN N1 and N1(HK/97) were the same as those used to
clone
the N1(HK/97) segment (see above). The primers used to amplify
a
portion of N2(LA/87) were 5'-AATAGAAAGAAAATAACAGAGATAGTGTA-3'
and 5'-TGGTTCCCTGCCCAAGTGCAAATTGATAAC-3' and
correspond to nucleotides
171 to 200 and the complement of nucleotides
386 to 415 of the
influenza A/Shanghai/11/87 (H3N2) virus N2 sequence
(GenEMBL accession
no.
U42633). The primers used to amplify N7(Cor/74)
were the
same as those used to clone this segment (see
above).
Polyacrylamide gel analysis of vRNAs was performed with a 2.8%
polyacrylamide gel (acrylamide:bisacrylamide, 30:0.8) containing
7.7 M
urea and 1× Tris-borate-EDTA. Gels were run at 150 V for
1 h 50 min and silver stained (
10).
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RESULTS |
Generation and assay of NA mutants.
A series of single
cysteine-to-glycine mutations were generated in the WSN NA ectodomain
such that two nucleotide changes would be required for the glycine to
revert to a cysteine (Table 2). A
schematic diagram of locations of the WSN NA cysteines is shown in Fig.
1. Both cysteines not involved in
intramolecular disulfide bonds (C49 and C146) were individually
mutated. Additionally, each disulfide bond was individually disrupted.
To accomplish this, the amino-terminal member of each disulfide-bonded
cysteine pair was mutated. Additionally, two double cysteine-to-glycine mutants (C49G,C146G and C303G,C320G) were generated.
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FIG. 1.
Locations of cysteine residues of the NA of WSN virus.
In the schematic diagram of the WSN NA primary structure, locations of
all 19 cysteine residues are indicated (numbering according to Hiti and
Nayak [22]), and thin lines connecting cysteines
represent intrasubunit disulfide bonds. Domains of the protein are
indicated as follows: tail, the six-amino-acid cytoplasmic tail; T.M.,
the transmembrane domain; stalk, indicates the long, slender stalk
which extends from the virion surface; head, the large, globular domain
possessing enzymatic activity. Equivalent cysteines in the heads of the
WSN N1 and the influenza A/RI/5-/57 virus N2 (6) are as
follows (WSN/N2): 76/92, 109/124, 113/129, 169/183, 218/232, 223/237,
264/278, 266/280, 275/289, 303/318, 320/337, 277/291, 216/230, 402/417,
406/421, and 431/447. Equivalent cysteines form equivalent disulfide
bonds in the two proteins. However, the N2 contains an additional
intramolecular disulfide bond (C175-C193 [N2 numbering]) not found in
N1 NAs.
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Each mutant was expressed in vitro via a coupled in vitro
transcription-translation system to confirm that each clone could
be
expressed from the T7 RNA promoter (Fig.
2).
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FIG. 2.
In vitro translation of WSN NA cysteine-to-glycine
mutants. Wild-type (WT) WSN NA and the indicated mutant WSN NAs were
transcribed and translated in vitro in the presence of
[35S]methionine. (), no-DNA negative control reaction.
The products were run on a sodium dodecyl sulfate-10% polyacrylamide
gel, which was then exposed to film. The arrowheads indicate positions
of full-length, unmodified NA.
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The resulting mutant NA molecules were then expressed at 33, 37, or
39.5°C, using the vaccinia virus-T7 expression system.
The mutants
were assayed for activity at 30°C, using as substrate
the
low-molecular-weight sialic acid analog Mu-NANA; the results
are
summarized in Table
2. The relative activities reported in
Table
2 do
not necessarily reflect the specific activities of
the mutants, since
the amount produced of each mutant was not
precisely
quantified.
Mutation of either cysteine not involved in intramolecular disulfide
bonds, C49 or C146, had relatively little effect on neuraminidase
activity following expression at any temperature. C49G yielded
activity
levels similar to those of wild-type NA when expressed
at 33, 37, or
39.5°C. C146G was somewhat attenuated and slightly
ts. When expressed
at 33 or 37°C, C146G was approximately one-third
as active as
wild-type NA; however, when expressed at 39.5°C,
C146G displayed only
17.4% of the activity of wild-type NA. The
C49G,C146G double mutant
displayed defects only slightly more
severe than those of the single
C146G
mutant.
Mutation of any of the cysteines involved in intramolecular disulfide
bonds severely affected the production of functional
enzyme, regardless
of expression temperature. The only mutations
affecting cysteine
residues involved in intrasubunit disulfide
bonds which retained
activity targeted the same disulfide bond.
When either C303 or C320 was
mutated to glycine, NA activity was
significantly impaired after
expression at 33, 37, or 39.5°C.
Interestingly, both mutant enzymes
were ts, displaying 2% of wild-type
activity at 33°C and
approximately 10-fold-lower activity (0.2%)
at 39.5°C. The
C303G,C320G double mutant, however, exhibited no
NA activity. Of note,
both C303 and C320 lie within a single loop,
5L0,1, of the NA,
whereas the remainder of the disulfide bonds
found in the NA head
involve cysteines within
-sheets (
51).
Effects of cysteine-to-glycine mutations on viral growth.
To
study the role of NA cysteines in the context of an infectious virus,
the four active NA mutants were rescued into a WSN virus background by
using the established WSN NA rescue system. Rescue of the C49G and
C146G mutants was as efficient as rescue of wild-type WSN NA. Rescue of
the attenuated C303G and C320G mutants was approximately 10-fold less
efficient than rescue of wild-type NA. Upon rescue, both C303G and
C320G were found to form small plaques on MDBK cells at 33°C, while
C49G and C146G plaques were similar in size to wild-type WSN plaques
(data not shown).
The plaque-forming phenotype of the mutant viruses was further analyzed
on MDCK cells in the presence of trypsin, at 33 or
39.5°C. The
plaques formed by C49G and C146G at either 33 or 39.5°C
were similar
in size to those formed by wild-type WSN (data not
shown). However,
both C303G and C320G were found to form small
plaques at 33°C and no
plaques, even after 4 days, at 39.5°C.
In these experiments, the
efficiency of plaque formation of both
C303G and C320G viruses was more
than 3 logs lower at 39.5°C than
at 33°C.
To further assess the impact of the cysteine-to-glycine mutations on
viral replication, multicycle growth of the transfectant
viruses was
analyzed on MDCK cells at either 33 or 39.5°C (Fig.
3). At 33°C, the C49G and C146G viruses
grew similarly to wild-type
WSN virus, achieving titers of
10
8 PFU/ml. However, at 33°C both C303G and C320G were
attenuated
1 and 2 logs, respectively, compared with wild-type WSN
virus
(Fig.
3A). At 39.5°C, C49G again grew as well as or better than
wild-type WSN. C146G grew to a slightly lower titer, perhaps reflecting
the slightly ts phenotype seen in the transient expression experiments.
At 39.5°C, the ts phenotype of mutants C303G and C320G was apparent.
These viruses grew to titers 5 to 6 logs lower than wild-type
WSN
titers (Fig.
3B). These results demonstrate that there is
a good
correlation between the relative amount of NA activity
obtained in the
transient expression assay (Table
2) and the
extent of viral growth in
tissue culture (Fig.
3). These experiments
also indicate that the
mutant viruses C303G and C320G exhibit
tight ts phenotypes.
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FIG. 3.
Growth curves of transfectant viruses C49G, C146G,
C303G, and C320G. (A) Multicycle growth of the transfectant viruses at
33°C. (B) Multicycle growth of the transfectant viruses at 39.5°C.
MDCK cells were infected with wild-type WSN, C49G, C146G, C303G, or
C320G virus at a multiplicity of infection of 0.001. Plaque assays were
performed on MDCK cells at 33°C to determine the titer of the
collected samples.
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Clearly, C49 and C146 are not required for efficient function of the
WSN NA in the context of viral infection. However, disruption
of the
disulfide bond between C303 and C320 significantly impairs
NA function,
rendering the molecule ts. In the context of an infectious
virus, C303G
appears to be a more severe mutation despite the
similar levels of
activity seen in the NA proteins expressed from
plasmids. This may
reflect the use of the low-molecular-weight
Mu-NANA as substrate in the
in vitro NA assays. There may be differences
in the ability of the two
NAs to cleave sialic acid residues from
large glycoconjugates, but such
differences may not be seen with
a low-molecular-weight
substrate.
C303G and C320G fail to produce stable, properly folded NA at the
nonpermissive temperature.
To better understand the specific
defect in the C303G and C320G viruses, we examined viral protein
expression levels at 33 and 39.5°C. MDCK cells were infected with
WSN, C303G, or C320G virus, grown at 33 or 39.5°C for 12 h, and
then labeled with [35S]methionine and
[35S]cysteine for 1 h. After the label was chased
for 1 h with unlabeled medium to allow maturation of the labeled
proteins, lysates were prepared and analyzed by immunoprecipitation
with a mixture of two monoclonal antibodies that do not recognize
denatured WSN NA protein or with polyclonal antiserum raised against
whole WSN virus.
At 33°C, the C303G virus produced 6.9% and the C320G virus produced
9.6% as much immunoprecipitable NA as did wild-type WSN
virus. At
39.5°C, neither mutant virus produced detectable levels
of
immunoprecipitated NA (Fig.
4A). In
contrast, slightly more
HA, NP, and M1 were produced at 39.5°C than
at 33°C. Additionally,
mutants C303G and C320G produced HA, NP, and
M1 at levels similar
to those for wild-type WSN at both 33 and 39.5°C
(Fig.
4B).
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FIG. 4.
Immunoprecipitation assays indicating that C303G and
C320G NAs do not fold normally at 39.5°C. (A) Immunoprecipitation of
NA proteins. (B) Immunoprecipitation of viral structural proteins HA,
NP, and M1. Immunoprecipitations were performed on lysates of MDCK
cells infected with the indicated viruses and incubated at the
indicated temperatures. The cultures were labeled with
[35S]methionine-[35S]cysteine for 1 h
and chased with unlabeled medium for 1 h, beginning 12 h
postinfection. Immunoprecipitation of NA was performed with a mixture
of two monoclonal antibodies, 3C8 and 10C9 (43).
Immunoprecipitations of HA, NP, and M1 were performed with a polyclonal
anti-WSN virus antiserum, 8236.
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These results suggest that the C303G and C320G NA molecules are not
simply defective in enzymatic activity. Rather these NAs
likely possess
gross structural defects, perhaps due to the loss
of the C303-C320
disulfide bond. Thus, both C303G and C320G virus-infected
cells have
decreased amounts of stable, properly folded NA compared
with wild-type
virus, even at 33°C. At 39.5°C, stable, properly
folded NAs are
essentially absent from mutant-virus infected
cells.
A general reverse genetics system for the rescue of influenza A
virus NAs using C303G and C320G as helper viruses.
The tight ts
phenotype exhibited by the C303G and C320G viruses suggested that these
viruses might serve as efficient helper viruses in reverse genetics
experiments. Following RNP transfection of wild-type WSN NA, helper
virus-infected MDBK cells were incubated for 24 h at 33°C.
Supernatants for the RNP-transfected/infected cells were then passaged
on MDBK cells at 39.5°C to select against the helper virus. More than
104 transfectant viruses could be rescued by this method,
an efficiency that compares favorably with that of the established WSN
NA rescue system using WSN-HK as the helper virus (10). The
resulting viruses appeared to be transfectants and not revertants,
because no viruses were recovered from C303G and C320G virus-infected, mock-transfected controls (data not shown). These results suggested that C303G and C320G can function as efficient helper viruses. In
addition, the rescue of the ts phenotype by introduction of new NA
genes provides genetic confirmation that the ts lesions encoded by the
C303G and C320G viruses reside in the NA segment.
Rescue of N1, N2, and N7 NA genes into virus.
The
better-growing mutant virus, C320G, was then used in reverse genetics
experiments designed to rescue non-WSN NAs. Vero cells were transfected
in these experiments because of their relatively high transfection
efficiency and because they withstand the presence of trypsin in the
growth medium. These cells were infected with 1 PFU of C320G virus per
cell and RNP-transfected with in vitro-synthesized vRNAs corresponding
to the N1(HK/97), N2(LA/87), or N7(Cor/74) virus. Following incubation
of the RNP-transfected cells at 33°C for 24 h, the supernatants
were plaqued on MDCK cells at 39.5°C or passaged in 10-day-old,
embryonated chicken eggs at 39.5°C. Transfectant viruses were
obtained for the N1, N2, and N7 NAs [WSN-N1(HK/97), WSN-N2(LA/87), and
WSN-N7(Cor/74), respectively]; 60 WSN-N1(HK/97) transfectants, <15
WSN-N2(LA/87) transfectants, and 30 WSN-N7(Cor/74) transfectants were
obtained in a transfection. After passage at 39.5°C, no viruses were
obtained from the mock-transfected controls.
The transfectant viruses were plaque purified three times at 39.5°C
and amplified at 39.5°C in 10-day-old embryonated chicken
eggs. vRNA
was then extracted from these viruses and analyzed
by RT-PCR and by
polyacrylamide gel
analysis.
RT-PCR was performed with primers designed to amplify full-length N1
[WSN or N1(HK/97)] vRNA, a fragment of N2 vRNA, or full-length
N7
vRNA. The N1 primers yielded products of the expected sizes
of 1,453 and 1,445 bp (longer than the NA vRNA due to sequences
at the 5' and 3'
ends of the primers), for the WSN control and
the WSN-N1(HK/97)
transfectant virus, respectively. The N1 primers
did not yield a
product for the WSN-N2(LA/87) or WSN-N7(Cor/74)
virus (Fig.
5A). These data indicate that the
WSN-N2(LA/87) and
WSN-N7(Cor/74) viruses do not possess N1 NAs and
therefore are
not C320G revertants. Additionally, the WSN-N1(HK/97)
virus possessed
an N1 vRNA as expected. To confirm that the
WSN-N1(HK/97) virus
was not a revertant, the WSN and WSN-N1(HK/97)
RT-PCR products
were digested with
PstI. The WSN NA and
HK/97 N1 have different
PstI restriction patterns, with WSN
NA expected to yield bands
of 1,224 and 221 bp and the HK/97 N1
expected to yield bands of
944 and 509 bp. As seen in Fig.
5B, the
WSN-N1(HK/97) virus possesses
an NA with the
PstI
restriction pattern characteristic of the
HK/97 N1. The NA of the
WSN-N1 virus was therefore derived from
the transfected N1 segment.
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|
FIG. 5.
RT-PCR analysis of transfectant viruses WSN-N1(HK/97),
WSN-N2(LA/87), and WSN-N7(Cor/74). (A) RT-PCR was performed on vRNAs
obtained from the indicated viruses, using primers specific for the N1,
N2, or N7 vRNA. (B) Restriction enzyme analysis of the N1, N2, or N7
RT-PCR products. Products were left undigested () or were digested
with the indicated restriction endonuclease(s).
|
|
RT-PCR of the WSN-N2(LA/87) NA yielded a product of the expected 244 bp
(Fig.
5A). It was necessary to confirm that the rescued
N2 segment
corresponded to the transfected N2(LA/87) and not the
N2 of the X-31
virus from which the viral polymerase preparation
(used during creation
of the in vitro-synthesized N2 vRNA) was
generated. The WSN-N2(LA/87)
RT-PCR product should possess an
SpeI site 70 bp from one
end. The X-31 N2 RT-PCR product should
possess a
BstXI site
73 bp from one end but no
SpeI site. The
WSN-N2(LA/87)
RT-PCR product could be cleaved 70 bp from its end
by
SpeI
but was not cleaved by
BstXI, as expected for the N2(LA/87)
(Fig.
5B). The WSN-N2(LA/87) NA was therefore derived from the
transfected N2
segment.
RT-PCR of the WSN-N7(Cor/74) NA yielded a product of the expected 1,504 bp (longer than the NA due to sequences at the 5'
and 3' ends of the
primers) (Fig.
5A). The N7(Cor/74) RT-PCR product
was expected to
contain an
EcoRI site, while the WSN, N1(HK/97),
N2(LA/87),
and N2(X-31) vRNAs do not possess
EcoRI sites. The
N7(Cor/74) was indeed cleaved by
EcoRI, yielding the
expected
927- and 531-bp products, confirming that this virus possessed
the transfected N7 segment (Fig.
5B).
As a final confirmation that the transfectant viruses possessed the
transfected NA vRNAs in a stable form, the transfectants
were amplified
once again in 10-day-old embryonated chicken eggs,
and vRNAs were
prepared. These vRNAs were then analyzed by polyacrylamide
gel
electrophoresis (Fig.
6). The
WSN-N1(HK/97) virus possessed
an NA of the expected 1,401 nucleotides,
slightly smaller than
the 1,409-bp WSN NA. The WSN-N2(LA/87) and the
WSN-N7(Cor/74)
vRNAs migrated slower than the WSN NA vRNA, as expected.
These
results confirm that the ts C303G and C320G viruses serve as
efficient
helper viruses for the rescue of a variety of NA types.
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[in a new window]
|
FIG. 6.
RNA gel analysis of transfectant vRNAs. vRNAs are
labeled according to the gene products which they encode, and the
location of the 18S rRNA is indicated. Positions of NA vRNAs are
indicated by arrows. RNA samples were run on a 2.8% 7.7 M
urea-containing polyacrylamide gel and silver stained. Ps, polymerase
(PA, PB1, and PB2) segment; NEP, nuclear export
protein.
|
|
| |
DISCUSSION |
The cysteine-to-glycine mutants analyzed in this study can be
classified into three groups. The first group consists of enzymatically inactive mutants in which intramolecular disulfides formed between -sheets were disrupted (51). These mutants may be
defective due to loss of the disulfide bond, due to the loss of the
side chain of a single cysteine residue, or due to the presence of a
single, unpaired cysteine. By analogy with the N2 structure, the
disulfides C218-C223 and C264-C275 will link 3S1 to 3S2 and
4S1 and 4S2, respectively. These disulfide bonds are located near
the loops 3L0,1 and 4L0,1 which contain charged residues which
may be involved in catalysis or in maintaining the structure of the
active site (51). These disulfide bonds may therefore be
necessary to maintain the structure or function of the enzymatic active
site. Alternatively, loss of a cysteine side chain or the presence of
an unpaired cysteine in this region may perturb the structure of the
active site. It is also possible that the mutation of cysteines in
-sheets might cause defects in NA processing or stability.
Precedents do exist for single amino acid changes yielding both stable,
inactive NA mutants and degraded NA mutants (27, 28).
In contrast to the first group of NA mutants, mutation of the residues
which form the C303-C320 disulfide bond resulted in ts proteins.
Neuraminidase activity detected following expression at 33°C of
either of these mutants was reduced significantly (about 50-fold)
compared to wild-type NA, while both mutant proteins were almost
inactive at 39.5°C. When these mutant genes were rescued into virus,
both mutant viruses formed small plaques at 33°C and grew to titers 1 to 2 logs lower than those of wild-type WSN virus. At 39.5°C, no
plaques could be detected for either mutant virus. In liquid culture, a
5- to 6-log decrease in titer was observed at 39.5°C compared with
33°C. These results demonstrated that an NA which has only 2% of
wild-type activity (as measured with a low-molecular-weight substrate)
is able to support replication of an influenza virus. They also
demonstrate that mutation of the residues which form the C303-C320
disulfide bond yields a virus with a very tight ts phenotype.
Temperature-sensitive cysteine mutations have been seen in other
systems as well. These may appear because disulfide bonds are important
for stabilization of protein structure but are not usually required for
a protein to assume its proper configuration (8). However,
the ts phenotypes may also arise due to the properties of the side
chain of the substitute amino acid. When several cysteines of the HSV-1
gD protein were individually changed to serine, the resulting mutant
proteins were ts in their transport and/or processing. Additionally,
while the mutants Cys-2 and Cys-4 were able to complement a gD-minus
virus at 31.5°C, these two mutants were unable to complement the
gD-minus virus at 37°C (32). Likewise, when any of the
three cysteines of the HSV-1 TIF protein (VP16) were individually
changed to glycine, the resulting mutants were ts for binding to host cell proteins and for binding to the TIF response element. Double cysteine-to-glycine TIF mutations were also found to be ts in the
context of viral infection. These mutant viruses grew to a titer 3 to 5 logs lower than that of a wild-type virus at nonpermissive temperature
(44).
The third group of mutants were those affecting the two ectodomain
cysteines which do not participate in intrasubunit disulfide bonds (C49
and C146 [WSN numbering]). Mutation of C49 resulted in an enzyme
which functioned as well as wild-type NA at any temperature tested,
both in the transient expression system and in the context of an
infectious virus. This result was not surprising given that viruses
possessing deletions encompassing C49 grew as well as wild-type WSN
(33). Despite its nonessential nature, even at 39.5°C, C49
was found to participate in intersubunit disulfide bonding in the fowl
plague virus N1 (19). Similarly, intersubunit disulfide
bonds of the influenza A virus M2 protein are nonessential for protein
function (5, 23). Mutation of C146 resulted in an enzyme
which was about one-third as active as wild-type NA at 33°C and about
one-fifth as active as wild-type NA at 39.5°C. However, this modest
decrease in NA activity had little impact on replication of viruses
possessing this mutation. The slightly ts nature of this enzyme likely
reflects some disturbance in NA structure which is enhanced at higher
temperatures. Although it was suggested that this residue might
participate in intersubunit disulfide bonds in N1 molecules
(51), no biochemical evidence for the participation of this
cysteine in a subunit-subunit link was found (19). When a
C49G,C146G double mutant was tested, it behaved similarly to the C146G
mutant. Given these data, it is clear that intersubunit disulfide bonds
are not required for the formation of stable, functioning influenza
virus NAs, even at elevated temperatures.
The C303G and C320G mutants described here display interesting and
useful properties (42, 49). In particular, both mutants are
extremely tight. At the nonpermissive temperature, they displayed a
greater than 3-log decrease in efficiency of plaque formation and a 5- to 6-log decrease in titer after multicycle growth in liquid culture.
The frequency at which revertant or suppressor mutations of C303G or
C320G arise must be explored further, however. Mutations in HA, which
result in decreased affinity for sialic acid, can confer resistance to
NA inhibitors (1, 18, 35). It remains possible that similar
HA mutations which suppress the ts phenotype of C303G and C320G will arise.
The C303G and C320G mutants have also been shown to be useful as helper
viruses in reverse genetics experiments, permitting the rescue of WSN
NA, an avian virus N1, a human virus N2, and an equine virus N7. The
ability to efficiently rescue non-WSN NAs should facilitate future
studies of NA structure and function. Possible examples include the
rescue of wild-type and mutant forms of NAs from clinically important
viruses. The avian N1 that we rescued is 99% identical to the N1 of an
H5N1 human isolate which caused a fatal infection in a child
(50), and the N2 that we rescued is from a human H3N2 virus.
Additionally, since we can also rescue an N7, it should now be possible
to rescue other NA types which have interesting properties, such as
hemagglutination. Also, cytotoxic T-cell epitopes have been expressed
from viruses possessing the WSN NA segment (4, 13, 43), and
these constructs have been useful in generating effective cytotoxic
T-lymphocyte responses against other pathogens and against tumor cells
(15, 37, 45).
In summary, we have demonstrated that ts influenza viruses can be
generated by mutation of specific cysteine residues within the viral
NA. These ts viruses displayed tight and stable ts phenotypes which
permitted their use as helper viruses in reverse genetics experiments.
| |
ACKNOWLEDGMENTS |
This work was partially supported by grants from the National
Institutes of Health to A.G.-S. and P.P. and an NIH NRSA postdoctoral fellowship to C.F.B.
We thank Louis Nguyenvu for excellent technical assistance and Hongyong
Zheng for helpful discussions.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, Mount Sinai School of Medicine, One Gustave L. Levy
Place, New York, NY 10029. Phone: (212) 241-7318. Fax: (212) 722-3634. E-mail: ppalese{at}smtplink.mssm.edu.
| |
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Journal of Virology, October 1999, p. 8095-8103, Vol. 73, No. 10
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
