﻿Vol. 45, No. 1
JOURNAL OF VIROLOGY, Jan. 1983, p. 434-438
0022-538X/83/010434-05$02.00/0
The Influenza Virus Nucleoprotein Synthesized from Cloned
DNA in a Simian Virus 40 Vector Is Detected in the Nucleus
BOR-CHIAN LIN AND CHING-JUH LAI*
Molecular Viral Biology Section, Laboratory ofInfectious Diseases, National Institute ofAllergy and
Infectious Diseases, Bethesda, Maryland 20205
Received 28 June 1982/Accepted 8 September 1982
We obtained DNA sequences coding for the nucleoprotein (NP) of an influenza
A virus by reverse transcription of virion RNA with synthetic oligonucleotide
primers. Terminal sequence analysis showed that the cloned gene contained a full-
length copy of the virion RNA segment. The NP-specific DNA was inserted into
the late region of a simian virus 40 vector, and the DNA recombinant was
propagated in the presence of an early simian virus 40 temperature-sensitive
mutant helper. Infection of African green monkey kidney cells with the recombi-
nant produced a polypeptide immunoprecipitable with NP-specific antisera. The
polypeptide product had a molecular weight of 56,000, identical to that of the
nucleoprotein of influenza virus as estimated on polyacrylamide gels. The
putative NP was detected in the nucleus of infected primate cells by an
immunofluorescence assay. This nuclear localization of NP from recombinant
DNA was similar to that seen during influenza virus infection.
Influenza virus nucleoprotein (NP), serologi-
cally characterized as a type-specific antigen
that distinguishes type A, B, and C viruses,
constitutes a major internal component of the
virion (18). NP interacts with viral genomic
RNA to form helical ribonucleoprotein complex-
es (5, 7, 15). These complexes, in association
with the three viral polymerase proteins, are
transcriptionally active, generating viral mRNA
species (17). Monocolonal antibodies specific to
NP affect mRNA transcription in vitro, and at
least three nonoverlapping antigenic determi-
nants on the viral polypeptide have been demon-
strated (23). Influenza virus temperature-sensi-
tive mutants defective in the NP gene fail to
synthesize mRNA or virion RNA in infected
cells at restrictive temperature (11, 20, 21).
These data suggest that NP is directly or indi-
rectly involved in transcription and replication.
Recent evidence indicates that the nucleus is the
site of influenza mRNA transcription and that
NP is primarily present in the nucleus ofinfected
cells (3, 6, 9, 10). Studies on another negative-
strand virus, vesicular stomatitis virus, suggest
that its NP may play a role in the regulation of
viral replication through a specific binding to
vesicular stomatitis virus RNA (1); however,
analogous experiments have not been carried
out with influenza virus. Many important ques-
tions concerning the structural features and in-
volvement of NP in transcription remain to be
elucidated. In this report, we describe cloning of
the full-length NP gene and characterization of
the viral polypeptide produced in African green
monkey kidney (AGMK) cells during infection
with the cloned gene inserted into a simian virus
40 (SV40) vector.
Virion RNA was prepared from the egg-grown
virus as described previously (12) and used to
prepare double-stranded DNA for cloning. As
shown in Fig. 1, the first cDNA strand was
synthesized by reverse transcription of RNA by
using a synthetic dodecanucleotide primer
(AGCAAAAGCAGG) complementary to the
conserved 3' terminus. After removal of the
virion RNA from the hybrids (0.2 N NaOH at
70°C for 30 min), the cDNA strand was convert-
ed to double-stranded DNA by using another
dodecanucleotide primer (AGTAGAAACAAG)
which corresponds to the 5'-common sequence
of virion RNA (12, 16, 26). The DNA duplexes
were separated on polyacrylamide gel, and the
fifth largest DNA segment was isolated for char-
acterization of the NP gene by restriction en-
zyme digestion and for cloning in the Escherich-
ia coli system. The NP gene was inserted into
the late region between the HpaII and BamHI
sites of SV40 in an expression vector with a
BamHI linker. In this vector, the HpaII site of
SV40 was converted to a BamHI site that could
be used for insertion, and the TaqI site had been
converted into an XbaI site for joining with
pBR322; an XbaI site had been introduced previ-
ously into pBR322 (4) (kindly given by P.
Gruss). The cloned NP DNA was further char-
acterized by digestion with restriction enzymes.
434
NOTES 435
(-) vRNA -
I Synthetic primer
Reverse transcriptase
(+) cDNA -
Synthetic primer
Reverse transcriptase
ds DNA -
| Bam HI linker
N
Ligase
NP DNA
Bam Hi Bam Hl
(Hpa 11
Ori S40 )
Xba I ;Xba I
322
Ori -
Xba I
Ori
SV40-NP RECOMBINANT DNA
FIG. 1. Scheme for cloning influenza virus NP
DNA and construction of SV40-NP recombinant.
The map positions of restriction enzyme sites,
including PstI, BglII, and PvuI, corresponded
with those of NP genes from other influenza
viral strains (8, 22, 25). To ascertain that our NP
clone contained a full-length copy of the vRNA
segment, we sequenced the terminal nucleotides
of the DNA segment. At both termini, the con-
served nucleotide sequences of virion RNA
were completely represented (Fig. 2) (16). Simi-
lar to the NP sequence of A/PRI8/34(HlN1) and
A/NT/60/68(H3N2), the ATG initiation codon on
the sense strand was located at positions 46 to 48
and was followed by an identical coding se-
quence of eight amino acids in the region se-
quenced. At the 3' end of the sense strand,
identical sequences were also present in the
cloned gene, and a termination codon was there-
fore inferred from other NP DNA sequences
described.
To express NP polypeptide, we used the SV40
vector of Gruss et al. (4) and constructed an
SV40-NP recombinant that contained an NP
DNA insert in the sense orientation relative to
the late transcription of SV40. The SV40 moiety
lacked the late region between the HpaII and
BamHI cleavage sites; therefore, all the late
RNA splice sequences were absent in this vec-
tor. The SV40-NP recombinant yielded a pre-
dicted fragment of 1262 base pairs after PstI
digestion at the unique PstI site in the NP DNA
and in the pBR322-SV40 vector. Linear DNA of
SV40-NP was prepared from the plasmid after
XbaI digestion and was subsequently used for
infection of AGMK cells in the presence of an
early temperature-sensitive mutant of SV40
(tsA) (Fig. 1). The construction of SV40-NP
recombinant virus was essentially identical to
the procedure described previously (19).
AGMK cells infected with the SV40-NP
recombinant were analyzed for the production
of the NP polypeptide. Two separate approach-
es were employed: one with polyacrylamide gel
electrophoresis to analyze the molecular size of
the product and the other with an indirect immu-
nofluorescence assay to detect the intracellular
location of the polypeptide. In the first experi-
ment, confluent AGMK cells (2 x 105) were
infected with the recombinant for 72 h and
labeled with [35S]methionine (20 ,uCi; specific
activity, 1,300 Ci/mmol) for 5 h. Cell lysates
were subsequently prepared in 400 ,ul of RIPA
buffer (0.3 M NaCI, 1% deoxycholate, 1% Tri-
ton, 0.1% sodium dodecyl sulfate, 0.1 M Tris
[pH 7.4], 1 mM phenylmethylsulfonyl fluoride,
5,000 U of trasylol). Immunoprecipitation was
carried out with 10 ,ul (70 ng) of a mixture of
Met Ala Ser GIn G ly Thr Lys Arg...
5'AGCAAAAGCAGGGTAAATAATCACTCACTGAGTGACATCAAAATC T_GI GCG TCC CAA GGC ACC AAA CGG...
TCGTTTTCGTCCCATTTATTAGTGAGTGACTCACTGTAGTTTTAG TAC CGC AGG GTT CCG TGG TTT GCC...
BaHI Bg! 11 Bg! 11Pvu
Hpa 1 Psi Hpa 111
..Ph e Gly A sp A sn AlIa GlIu GlIu T yr A sp As n
...TTC GGA GAC AAT GCG GAG GAG TAC GAC AAT TAA GGAAAAATACCCTTGTTTCTACT3
...AAG CCT CTG TTA CGC CTC CTC ATG CTG TTA ATT CCTTTTTATGGGAACAAAGATGA
FIG. 2. Terminal nucleotide sequences of cloned full-length NP DNA. The cloned influenza virus NP DNA
was mapped by restriction enzyme digestion, and terminal nucleotides were sequenced by the method of Maxam
and Gilbert (14). Shown in the linear map are several endonuclease cleavage sites. Both oligonucleotide primers
completely represented in the cloned gene are underlined. These sequences are identical to the previously
published data from which the predicted amino acid sequences are indicated.
VOL. 45, 1983
436 NOTES
three monoclonal antibodies specific to three
distinct epitopes of NP (5/1, 3/1, and 7/3; kindly
provided by K. van Wyke) and a 10-,ul slurry of
protein A-Sepharose in phosphate-buffered sa-
line. The immunoprecipitates were analyzed on
sodium dodecyl sulfate-polyacrylamide gels
(Fig. 3). All three independent isolates of SV40-
NP recombinant yielded a labeled band of56,000
daltons, equivalent in size to the NP produced
during influenza virus infection. No such labeled
polypeptide was found in SV40-infected cells
(data not shown). Other minor bands were not
related to the NP recombinant, since they were
also present in the uninfected cell control. The
putative NP represented the only polypeptide
product of the cloned NP insert in the SV40
expression vector. Our cloned NP gene ap-
peared to contain a complete coding sequence,
and translation of NP transcript occurred at the
initiation codon at nucleotide positions 46 to 48
of the NP gene, since NP synthesized from
SV40-NP recombinant DNA-infected cells was
identical in size to that produced during influen-
za virus infection.
Although there is no direct functional assay
currently available for influenza virus NP, many
earlier studies indicated that NP is found inside
the nuclei of infected cells (2, 13, 24). To obtain
further information concerning the functional
activity of NP from SV40-NP recombinant, we
tested whether the NP product is also detected
in the nuclei of infected cells. Monolayers of
AGMK cells were infected with SV40 alone,
with influenza virus A/Udorn/72(H3N2), or with
the SV40-NP recombinant for a period pre-
scribed. Infected cells were fixed with acetone,
reacted with the monoclonal antibody mixture to
increase the sensitivity of detection, and stained
with a fluorescein conjugate of goat anti-mouse
serum. As a control, no immunofluorescence
was detected in SV40-infected cells. On the
other hand, as early as 1.5 h after infection with
influenza virus, cells showed intense nuclear
fluorescence, indicating that NP was predomi-
nantly detected in the nucleus; little or no NP
was found in the cytoplasm (Fig. 4A). The
appearance of NP during infection with the
SV40-NP recombinant was followed kinetically.
At 36 to 60 h, NP was largely detected in the
nucleus, and the pattern of nuclear fluorescence
was identical to that seen during influenza virus
infection (Fig. 4B). Despite a low multiplicity of
infection (1% of cells infected), detection of NP
in the nucleus was observed repeatedly. Howev-
er, later after infection (72 to 96 h) with the
recombinant, immunofluorescent staining of NP
became diffuse and was detected in the cyto-
plasm as well. One interpretation is that the
accumulation of NP in the cytoplasm is a result
of cellular dysfunction, since SV40 infection is
cytopathic at this late stage. These results indi-
cate that the NP product from the cloned gene
exhibits the same nuclear localization observed
during influenza virus infection. Since no other
influenza viral polypeptides or virion RNA seg-
ments are present in the infected cell, the specif-
ic influx into the nucleus is likely a functional
ANTI-NP SERUM
flu" "flu" SV40-NP C
U-.
' .4:
70-75K - .- i. -
. .
56K -
(NP)
28K-
26K-
FIG. 3. Polyacrylamide gel analysis of influenza
virus nucleoprotein. Lysates were prepared from
[35S]methionine-labeled AGMK cells previously in-
fected with influenza virus A/Udorn(H3N2) ("flu") or
three separate isolates of SV40-NP recombinant virus.
Uninfected cell control (C) lysate was prepared in a
similar manner. Immunoprecipitation was carried out
with a mixture of three monoclonal antisera specific
for NP (5/1, 3/1, and 7/5), and the labeled precipitate
was analyzed on sodium dodecyl sulfate-15% poly-
acrylamide gels (acrylamide:bisacrylamide = 30:1).
Molecular size markers of influenza viral proteins are
indicated.
J. VIROL.
NOTES 437
FIG. 4. Indirect immunofluorescence assay for detection of intracellular nucleoprotein. (A) AGMK cells
infected with influenza virus. (B) AGMK cells infected with the SV40-NP recombinant. Infected cell monolayers
were fixed with acetone and incubated with a mixture of three monoclonal antibodies specific to NP and a
fluorescein conjugate of goat anti-mouse serum. Infected cell nuclei are intensely stained. The nucleoli of
uninfected cells were brightened by staining with ethidium bromide to locate the nuclear contour.
VOL. 45, 1983
438 NOTES
property of NP. The specific detection of the NP
polypeptide in the nucleus is markedly different
from the previous observations concerning influ-
enza virus hemagglutinin that accumulates at the
cell surface (19). In summary, the present study
permits the following conclusions. First, when
inserted into an SV40 expression vector, the
cloned full-length NP DNA produced an immu-
noprecipitable polypeptide identical in molecu-
lar size to influenza virus NP. Second, the
putative NP detected in the nucleus early after
recombinant DNA infection was analogous to
the NP produced during influenza virus infec-
tion, suggesting that the NP product exhibits at
least one of its many functional characteristics.
We thank R. Chanock for his valuable suggestion and K.
van Wyke for her generous supply of monoclonal antibodies.
We are also grateful to S. Kruger and J. A. Berndt for their
expert assistance and to L. Jordan for typing the manuscript.
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