Complementation and analysis of an NP mutant of influenza virus

97

Virus Research, 12 (1989) 97-112 Elsevier

VRR 00467

Complementation and analysis of an NP mutant of influenza virus Ruan Li *, Peter Palese and Mark Krystal Department

of Microbiology, Mount Sinai School of Medicine of CUNY, New York, NY 10029, U.S.A. (Accepted 17 October 1988)

Summary

Cell lines were constructed so as to express the influenza A virus nucleoprotein (NP) at levels approximating 5% of the total NP made throughout virus infection. Two types of cell lines were analyzed. One cell line (NP-5) expresses only the NP while another cell line was constructed which expresses the three viral polymerase proteins in addition to the NP (3PNP-4). Both cell lines were able to complement the growth of an NP mutant, ts56, at the non-permissive temperature. The 3PNP-4 cell line, constructed by transfecting a cell line already expressing the three polymerase proteins, continued to be able to complement viral PB2 mutants. In addition, sequence analysis was performed on the NP gene segment of A/WSN/33 and ts56 viruses. This analysis revealed that the mutant phenotype exhibited by ts56 at non-permissive temperature is due to a single serine to asparagine change (at codon 332) within the protein. NP mutant; Complementation;

Influenza virus

Introduction

The influenza A virus genome consists of eight single-strand segments of negative polarity, coding for at least ten polypeptides (Lamb, 1983). At the present time, at least four of these proteins have been implicated in the transcription and replication * Present address: Institute of Virology, China National Center of Preventative Medicine, Beijing, People’s Republic of China. Correspondence to: M. Krystal, Dept. of Microbiology, Mount Sinai School of Medicine of CUNY, Fifth Avenue and 100th Street, New York, NY 10029, U.S.A.

0168-1702/89/$03.50

0 1989 Elsevier Science Publishers B.V. (Biomedical Division)

98

processes of the virus. Of these four proteins, the three polymerase proteins (PB2, PBl, and PA) are present in catalytic amounts (Compans and Choppin, 1975) while the nucleoprotein (NP) is present in much larger amounts. Together with the virion RNA, these proteins comprise the ribonucleoprotein (RNP) complexes found within the virion and in infected cells (Inglis et al., 1976; Rochovansky, 1976). Cross-linking experiments have elucidated functions for the PB2 and PBl proteins during the primary transcription (mRNA synthesis) phase of the replication cycle (Ulmanen et al., 1981; Penn et al., 1982; Ulmanen and Krug, 1983). In addition, examination of temperature-sensitive mutants mapping to the PA and NP genes shows that these proteins play an important role during virus replication (Sugiura et al., 1972, 1975). Although no clear function has yet been ascribed to the PA protein, the NP protein has been implicated as being important in primary transcription (mRNA synthesis) and replication (cRNA and vRNA synthesis). Along with having a presumed structural function of conferring helical symmetry to the nucleocapsid, early data indicated that phosphorylation of NP stimulates viral transcription in vitro (Kamata and Watanabe, 1977). Also, recent data have suggested that the NP is necessary for template RNA synthesis, a prerequisite for replication (Scholtissek, 1978; Beaton and Krug, 1986). Previously, we had described the construction of cell lines which could complement the growth at non-permissive temperature of influenza virus ts mutants mapping to the PB2 or PA genes (Krystal et al., 1986). These cells (3P-cells) were constructed so as to express all three polymerase proteins, since cell lines which expressed only one of the polymerase proteins could not complement the growth of any mutant virus (Krystal et al., 1986). As a follow-up to these experiments, we were interested in seeing whether these cells can be used to study the interaction of various influenza viral proteins with the polymerase complex. If other viral proteins can be introduced into these 3P cells, then the complementation phenotype of these cells could be enhanced or inhibited depending upon how the newly expressed protein interacts with the polymerase complex. This could provide an in vivo system for studying the functional domains of various proteins involved in the replication of influenza virus. To this end, we have succeeded in introducing a high level of constitutive expression of NP into the cells expressing the three polymerase proteins as well as into native Cl27 cells. Although in previous work by other authors it was demonstrated that NP-expressing cell lines were unable to complement the growth of mutant virus (Ryan et al., 1986), these cells were able to complement the growth of an NP mutant, ts56. This mutant virus has recently been shown to be defective in template RNA synthesis in infected cells incubated at non-permissive temperature (Shapiro and Krug, 1988). Sequence analysis reported here reveals the defect to be due to a single point mutation. Materials and Methuds Plasmids, cell and viruses Plasmids pBMT3X and pSPR1 have been described 1986). Plasmid ~342-124 BPV is a derivative of plasmid

previously (Krystal et al., pdBPV-MMT neo(342-12)

99

(Law et al., 1983; gift of P. Howley). It was constructed by removing all bovine papilloma virus sequences through digestion with BumHI and religation. Transformed cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum. Cell line 3PNP-5 was also grown in the presence of 400 pg/nil Geneticin (GIBCO). Influenza virus A/WSN/33 and ts mutants tsl, ts15, ts53 and ts56 were grown in MDBK or MDCK cells as described (Sugiura et al., 1972, 1975; Krug et al., 1975). Isolation of transformed cells

For construction of cell line NP-5, Cl27 cells were transfected with plasmid pBMT3X/NP by the calcium phosphate precipitatioti procedure (Parker and Stark, 1979). One day after transfection, cells were split 1: 20 and 10 pm CdCl, was added to the media 4 h later. Media were changed every 3-4 days and foci picked at 10 days. Individual foci were cloned at limiting dilution and expanded for analysis. Cell line 3PNP-4 was constructed by transfecting 3F133 cells (Krystal et al., 1986) with 10 pg of a 20: 1 mixture of plasmids pBMT3X/NP and p342-12ABPV. One day after transfection, cells were split 1: 20 and 400 pg/ml Geneticin (GIBCO) was added. Media were changed every 4 days and foci were picked at 3 weeks. Individual foci were cloned by limiting dilution and expanded. In all experiments described, cells were split one or two days before use in DMEM media with 10% fetal calf serum without either CdCl, or Geneticin. Imm~o~uorescen~e analysis Cells were grown to subcon~uence

on 13 mm glass cover slips for 2 days. Cells were washed with PBS and fixed directly at - 20 o C with either a 1: 1 mixture of methanol : acetone or acetone. Cover slips were then incubated in a humidified chamber for 30 min at 37’C with polyclonal rabbit antisera made against A/PR/8/34 virus. Coverslips were washed three times in PBS and reacted with FITC conjugated goat-anti rabbit IgG (Cappel Biomedical, Inc.) for 30 min at 37 o C as before. Cover slips were washed three times in PBS, mounted in glycerol/PBS and examined in a Leitz microscope using epifluorescence. Metabolic labelling and immunoprecipitation

For metabolic labelling of expressed NP protein, cells were split one day prior to use. The next day su~onfluent monolayers in 35 mm2 dishes were washed twice with PBS and DMEM media minus methionine (gift of Dr. S. Chen-Kiang) was added for 30 min. In some dishes CdCI, was also added to 20 PM. One hour later, 200 ~1 of 3sS-labelled methionine (1100 C/mM) was added and incubation continued for 3 h. Cells were then washed twice with PBS and harvested in 200 ~1 of 1XRIPA buffer containing 0.15 M NaCl, 1% sodium deoxycholate, 1% Triton X-100,0.1% sodium dodecyl sulfate (SDS), 0.01 M T~s-hydr~hlo~de (pH 7.4) and 1 mM phenylmethylsulfonyl fluoride. Immunoprecipitation reactions were as described previously (Greenspan et al., 1985>. To assay for viral protein synthesis in infected cells, subconfluent monolayers in 35 mm dishes were infected with virus for 1 h at 33’ C. Prewarmed reinforced Eagle

100

medium was added, and dishes were shifted to 39.5 o C. At 6 h post-infeetion, dishes were washed 3 times with PBS and Hanks medium containing 200 pCi/ml 35Smethionine was added for 1 h. Media were removed and cells were harvested in lysis/loading buffer (45% glycerol, 325 mM BME, 15% SDS, 0.05 M Tris-H,PO, (pH 7.1), 0.025% bromo-phenol blue). All samples were examined by NaDod/SO,/ 7-14s PAGE as described (Ritchey et al., 1976; Young and Palese, 1979). Dot immunoassay

of cells

Transformed cells (5 X 10’) were seeded onto 35 mm’ dishes and allowed to incubate overnight. After two washes with PBS, cells were harvested in RIPA buffer. Two-fold dilutions were made in RIPA buffer and dot blotted with a vacuum filtration manifold (Schleicher & Schuell) using published procedures (Jahn et al., 1984). Infected cell protein was assayed by infecting Cl27 cells with ts+ (WSN) virus at an MOI of 10. Dishes were harvested every 2 h post-infection. Virus and cell debris released into the supernatant was recovered by centrifugation at 35 K for 1 h and added to the cell lysate. The dot immunoblot was reacted with a mixture of cell supematants of four anti-NP monoclonal antibodies (gift of Dr. J. Schulman) and developed using an ‘2sI-labelled rat alpha mouse kappa chain antibody (187.1; ATCC). CompIementation

assay

The growth complementation assay was performed as described (Krystal et al., 1986). Briefly, cells were infected at 33’ C for 1 h, washed 3 times with PBS, prewarmed medium without serum (untying 0.1% trypsin) was added and cells incubated overnight at 39.5”C. Media were then harvested and virus was titrated at permissive temperature to assay for total plaque forming units (pfu) produced. Cloning and se~encing

of NP genes

A/WSN/33 and ts56 were grown in 100 cm2 dishes as described (Suguira et al., 1972, 1975; Krug et al., 1975). Virus was purified by sucrose gradient centrifugation and virion RNA was isolated. Double-stranded cDNA was constructed and cloned using EcoRI linkers as described (Baez et al., 1980). DNAs were subcloned into Ml3 and sequenced by the chain termination protocol using the modified T7 polymerase, Sequenase (US Biologicals, Cleveland, OH).

Results Co~st~ction

of ceils expressing high Ieueis

of NP

A cDNA clone for the NP gene of A/PR/8/34 virus (Young et al., 1983) was inserted into the EcoRl site of vector pSPR1 (Krystal et al., 1986). In this vector, Sal11 sites are present on both sides of the EcoRl site. The cDNA was then excised with Sal11 and inserted into the XhoI site of vector pBMT3X downstream of the mouse metallothionein promoter. The selectable marker in this vector is the human metallothionein-1A (MT) gene which confers to Cl27 cells resistance to normally

Fig. 1. Expression of NP in transformed cell lines. Cells were grown on glass coverslips, fixed and developed in ~~o~uor~~ assays as described above. A. 3P-133 cell lines transfected with the pBMT3X/NP and p342-12ABPV expression vectors, selected with G418 and subcloned. B. Cl27 cells transfected with the pBMT3X/NP and selected with CdC12.

toxic levels of heavy metals (Krystal et al., 1986; Wright et al., 1986). Transformed Cl27 cells selected with 10 PM CdCl, were examined by immunofluorescence for expression of NP. Intense fluorescence was observed in all cells examined (Fig. 1A). In addition, 3P-133 cells were used as recipient for the NP vector (Krystal et al., 1986). This C127-derived cell line already expresses the three influenza virus polymerase proteins and can complement the growth of ts mutants mapping to the PB2 gene. However, as 3P-133 cells already contain three derivatives of pBMT3X and are already resistant to toxic levels of CdCI,, the human MT gene could not be used as a selectable marker. Therefore, when 3P-133 cells were transfected with the NP vector a separate plasmid containing the bacterial neomycin resistance gene (p342-12ABP~ was used for co-transfection and selection for G418 (Genetici~) resistance was applied. When first analyzed by immunofluorescence, only lo-40% of the cells in any G418 selected population exhibited positive NP expression. However, upon subcloning, certain clonal populations expressed NP in all cells examined (Fig. 1B). It should be noted that although the antisera used in this experiment were raised against whole virus, it does not detect positive staining for

102

the polymerase proteins expressed in 3P-133 cells (the parental cell line; not shown). Presumably, this is due to the low titer of antibody to the polymerase proteins within the polyclonal antibody preparation and the low expression level of the polymerase proteins in 3P-133 cells (Krystal et al., 1986). Therefore, the positive fluorescence seen in these cells is the result of high level expression of the NP protein. In both types of cell lines, constitutively expressed NP was localized mainly in the nucleus, as had previously been shown (Ryan et al., 1986). Cell lines which displayed the most intense fluorescence pattern were chosen for further study. The chosen cell lines using either 3P-133 and Cl27 as parents were designated 3PNP-4 and NP-5, respectively. Analysis of cell lines All expression vectors present in these cells contain the influenza virus gene placed behind the promoter for the mouse metallothionein gene. Therefore, these proteins should be inducible with heavy metals (Dumam and Palmiter, 1981; Mayo et al., 1982; Pavlakis and Hamer, 1983; Braam-Markson et al., 1985). However, previous complementation experiments performed with mutant-infected 3P-133 cells first induced by heavy metals gave identical levels of complementation as in cells which were not induced prior to infection (Li and Krystal, unpublished data). Therefore, it was of interest to determine if the nucleoprotein can actually be induced to express higher levels of protein in the presence of CdCl,. Cell lines 3PNP-4 and NP-5 were carried for two weeks prior to metabolic labelling in the absence of known inducers. Metabolic labelling with 35S-methionine was carried out as described above. Cell lysates were immunoprecipitated with a mixture of monoclonal antibodies and precipitates were run on gradient SDS-PAGE gels (Fig 2). Lanes 1, 3 and 5 are C127, 3PNP-4 and NP-5 cells, respectively, labelled in the absence of CdCl,. Lanes 2, 4 and 6 are 3PNP-4, NP-5 and Cl27 cells labelled in the presence of 20 PM CdCl,. The band corresponding to the NP protein is highlighted by the arrow in Fig. 2. This band is specific for 3PNP-4 and NP-5 cells and co-migrates with authentic NP protein synthesized in A/PR/8/34 virus infected cells (lane 7). As can be seen by comparing lanes 2 versus 3 and 4 versus 5, there is no apparent induction of NP protein in the presence of CdCl, in 3PNP-4 or NP-5 cells. Therefore, all further experiments were carried out without CdCl, induction. Experiments were then done to determine the steady state level of NP produced in these cells. Cells were were grown to subconfluence and immunoblotted with anti-NP monoclonal antibodies. In addition, Cl27 cells were infected with A/WSN/33 virus and an equivalent number of cells were harvested at each time point. In order to estimate the total amount of NP produced in these virus-infected cells, supematant was also harvested, released virus and cell debris were pelleted and added to the cell lysate at each time point. Samples were then immunoblotted in 2-fold dilutions and developed with a monoclonal antibody mixture as described. The results are illustrated in Fig. 3 for the 3PNP-4 cell line. In virus-infected Cl27 cells, the level of NP peaks at around 10 h postinfection and remains fairly constant up to the last time point at 24 h. From comparison of this and other blots, the steady state level of NP protein in 3PNP-4 cells corresponds to approximately 5% of

103

Fig. 2. NP expression is not inducible by heavy metals. NP-expressing cell lines were labelled with 35S-Met in the presence or absence of CdCl,. Cell lysates were then analyzed by immunoprecipitation and NaDodSO,/‘I-14% PAGE. Lane(s) 1, Cl27 cells-Cd; 2,3PNP-4 cells - Cd; 3,3PNP-4 cells + Cd; 4, NP-5 cells- Cd; 5, NP-5 cells +Cd; 6, Cl27 cells+ Cd; 7, Non-immunoprecipitated control of PRS-infected Cl27 cells. The viral NP is indicated by arrow.

the total amount of NP protein produced during infection. This extremely high level of expression is in sharp contrast to the low level of expression seen with the polymerase proteins in 3F133 cells (Krystal et al., 1986). The steady state level of NP protein in NP-5 cells is slightly less than that found in 3PNP-4 cells (not shown). Functional analysis of expressed proteins In an effort to examine the feasibility of this transfection approach for studying the interactions of various influenza virus proteins, expressed proteins need to be rechecked for activity. It is conceivable that the addition of NP expressing vectors to these cells may have caused the loss of expression of one or more of the polymerase

104

1

2

3

456

76

3 PNP-

Cl Cl

27

2 7 WSN 2h 6h 8h 10h 12h 14h 24h

Fig. 3. Quantitation of steady state level of expressed NP. Equivalent amounts of 3PNP-4, Cl27 and WSN-infected Cl27 cells harvested at various times were immunoblotted in 2-fold dilutions (slots 1-8) and developed with a mixture of monoclonal antibodies against the NP. The cell lines or the time of post-infection harvesting is indicated beside each set of dilutions.

proteins. Therefore, the 3PNP-4 cell line was tested in a protein synthesis complementation assay using ts mutants mapping to the PB2, PBl, PA and NP gene. Figure 4 examines cell protein synthesis 6 h post-infection at the non-permissive temperatures in Cl27 and 3PNP-4 virus infected cells. As can be seen in lanes 3 to 7, little virus specific protein is expressed in mutant infected Cl27 cells. In lane 4, there is a slight amount of viral protein visible, owing to the leakiness of this lot of ts53 mutant. However, significant levels of viral protein synthesis can be seen in 3PNP-4 cells infected with tsl (PB2 mutant-lane 9), ts53 (PA mutant-lane 10) and ts56 (NP mutant-lane 12). Therefore, 3PNP-4 cells continue to express functional polymerase proteins which are active in protein synthesis complementation assays. In addition, 3PNP-4 cells express functional NP, which can complement defects in the replication of ts56, a mutant which maps to the NP gene (Sugiura et al., 1975). As might be expected, NP-5 cells were able to complement ts56 virus in this assay, but had no effect on the other ts mutants (not shown).

Growth complementation of ts mutants The parental 3P-133 cell lines was previously shown to be able to complement the growth of the PB2 mutant tsl at the non-permissive temperature. The levels of complementation obtained in virus-infected 3P-133 cells are from 2 to 3 logs higher than in virus-infected control cells (Krystal et al., 1986). Therefore, growth comple-

105

Fig. 4. NaDodS0,/7-14% PAGE analysis of mutant-infected Cl27 or 3PNP-4 cells. Infected cells were incubated at 39.5’C and labeled as described above. The cell line and mutant virus genotype are indicated at the top of each lane. The viruses used were: M, mock infected; + , A/WSN/33; PB2, tsl; PA, ts53; PBI, ts15; NP, ts56. Influenza virus-encoded proteins are labelled.

106 TABLE

1

Complementation Cell line

of TS mutants Titer (pfu/ml)

in transformed

cell lines

a

ts+

tsl (PB2)

ts53 (PA)

tslS(PB1)

ts56(NP)

Cl27 3P-133

1.2x108 6.1 x 10’

1.6~10~ 7.3 x 105

1.1 x 104 5.1 x 104

2.5 x lo3 2.7 x 10’

3PNP-4

3.5 x 10’

(4.6) 8.9 x 104

NP-5

3.0 x 10’

(456) 1.8 x lo6 (1125) ND

55x103 3.8X103 (0.69) 6.4~10~

(8.1) ND

(1.2) ND

a Average of 2-4 experiments. cells. ND, not determined.

Values in parentheses

represent

ratios

(1.1) LOX105 (40) 3.2 x lo4 (12.8)

of titer in transformed

vs. Cl27

mentation experiments were performed in 3PNP-4 and NP-5 cells to determine if the presence of NP had any effect on complementation phenotypes previously obtained with 3P-133 cells and whether these cells could complement the growth of ts56, an NP mutant. In these experiments, C127, 3P-133, 3PNP-4 and NP-5 cells were infected with virus and incubated overnight at 39.5” C. Supernatants were harvested and virus was titrated at 33’ C in order to measure the total amount of virus produced. Table 1 shows the results from a number of separate experiments. With respect to the three polymerase mutants, cell line 3PNP-4 exhibits a similar if not slightly better (a-fold) complementation phenotype than the parental 3P-133 cell line. Therefore, high level NP expression had little effect upon the expression and function of the three polymerase proteins in these cells. In addition, 3PNP-4 cells infected with the NP mutant ts56 produce infectious titers which are 40-fold higher than those of virus-infected Cl27 or 3P-133 cells. Cell line NP-5 was also able to complement the growth of ts56, although virus titers averaged only 13-fold higher than those in control cells. Thus, in this system, high level expression of NP in the presence or absence of the polymerase proteins was sufficient to complement the growth of ts56 virus. However, the expressed proteins could not rescue the ts phenotype, as plaque titers of the complemented virus at 39.5 o C exhibited 3-4 logs lower titer than the 33 o C values (not shown).

Sequence analysis of parental and mutant NP genes Recently, Shapiro and Krug (1988) analyzed the biochemical defect present in ts56 infected cells. Using temperature shift experiments, the authors showed that at 39.5 o C (non-permissive temperature) ts56-infected cells failed to synthesize template RNA (cRNA). As a first approach to map functional domains of the NP protein we determined the nucleotide sequence of the parental WSN and of the ts56 NP gene segments (Fig. 5). The deduced NP protein of A/WSN/33 virus, as expected, shows a high degree of identity with previously known NP sequences from human viruses (Winter and

GAA GAT GE TCT TTC CAG GGG CT% GGA GTC EDVSFQGRGVFELSDEKATSPIVPSF 1500 GAC AT.2 MT MT GAA GGA T-CT TAT TTC TTC D ” s N E G S Y F F CTA

TTC

GM

CTC

GGA GAC AAT G D N

l-02

GAC GM

AI\G

GCA GAG GAG TAC R E E Y

GCA ACG AGC CCG ATC

GAC AAT D N

TM

AGA AM

GTG CCC TCC

TTT

1550 ATA CCC TIG

ITT

l

CT

Fig. 5. Nucleotide and amino acid sequence of the NP gene segment of WSN and ts56 virus. The entire nucleotide sequence and the single letter amino acid sequence of WSN virus are shown. The single G to A base change at nucleotide 986 causing a Ser to Asn mutation in the NP mutant is highlighted in the box.

Fields, 1981; Huddleston and Brownlee, 1982; Buckler-White and Murphy, 1986). Although the WSN protein differs from those of the A/PR/8/34 and A/NT/68 viruses at 20 and 32 residues, respectively, many of the amino acid changes are of a conservative nature. The sequence of the mutant NP gene from ts56 shows complete identity with that of the WSN gene except for a single G to A transition at nucleotide 986. This results in a conservative Ser to Asn amino acid change in the deduced protein sequence. The nucleotide change was observed in the cDNA clones and was later rechecked on viral RNA by direct RNA sequencing using the primer extension method. In addition, partial sequencing of a revertant of ts56 which was isolated by multiple high dilution passage at 39.5 o C showed that nucleotide 986 was

108

changed back to a guanine residue. Therefore, the mutant the result of a single serine to asparagine change.

phenotype

of ts56 virus is

Discussion Previously, we had shown that functional polymerase proteins of influenza virus can be constitutively expressed in cell culture, but in order to obtain growth complementation of virus mutants, all three polymerases needed to be expressed together (Krystal et al., 1986). The present study was undertaken for a number of reasons. The feasibility of using these cells as a model system to study the interaction of the polymerase complex with various influenza polypeptides was tested through the addition of an NP expression vector to 3P-133 cells. Also, this is a first step in developing systems in which one can examine the varied functions of the NP protein. The NP has been shown to be required for cRNA synthesis, the first step in the replicative phase of virus growth (Scholtissek, 1978; Beaton and Krug, 1986). Also, analysis of two ts’ mutants of A/HK/68 virus showed that one (ts2C) was defective in vRNA synthesis (but exhibited normal cRNA synthesis) while the other virus (ts463) contained an NP which is apparently a structural mutant (Thierry and Danos, 1982). Thus, influenza virus NP may play important roles in each step of the replication cycle. Cell lines were made which express the NP protein alone or in cells already expressing the three polymerase proteins (Fig. 1). Although the NP gene was placed downstream of the mouse MT-l promoter, induction by heavy metal did not result in higher level expression of the protein (Fig. 2). As one of the goals of this study was to examine whether the further introduction of expression vectors into 3P-133 cells would be practicable for the study of interactions of proteins, certain criteria must be met. As long as the newly expressed protein did not, as a normal function, inhibit the action of the polymerase, the introduction of another viral polypeptide should not affect the complementation phenotype of these cells when infected with polymerase mutants. The reason the NP gene was chosen is that as a normal part of the RNP particle, we believed its presence in 3P-133 cells would not harm the complementation of polymerase mutants. It is clear from the data that if anything, the addition of NP has improved the complementation characteristics exhibited by 3P-133. Cell line 3PNP-4 continues to complement tsl and ts53 in terms of protein synthesis and in controlled experiments, exhibits 2-fold higher growth complementation values when infected with tsl. This cell line now can also complement the growth of ts56 virus, resulting in an average titer 40-fold higher than ts56 infected Cl27 cells. Therefore, we feel this system can be used to study other viral polypeptides, such as Ml, M2, NSl, and NS2. It is also of interest to note that both 3PNP-4 and NP-5 cells were able to complement the growth of ts56. Previously, Ryan et al. (1986) expressed functional NP in CV-1 cells but could not obtain growth complementation of A/HK/68 NP mutants. This difference could be reflected by the fact that expression level in their

109

cells was lower than what we obtained (1% of total NP produced versus SW), or that they used different ts mutants for their analysis. Sequence analysis of the parental and mutant NP proteins revealed that a single nucleotide change resulted in the acquisition of the ts phenotype in ts56. The G to A transition at nucleotide 986 results in a serine to asparagine change at amino acid 314 of the NP protein. This change is upstream of the putative nuclear accumulation signal previously identified in the NP (Davey et al., 1985). As the serine to asparagine change is the only one found in the ts56 NP it probably is responsible for the defect in template RNA synthesis associated with mutant-infected cells at non-permissive temperature (Shapiro and Krug, 1988). This serine residue is conserved in all known influenza A virus NP’s (Winter and Fields, 1981; Huddleston and Brownlee, 1982; Bucker-W~te and Murphy, 1986). It is interesting that a serine to asparagine change results in the ts phenotype as this amino acid change is thought to be highly conservative. A possibility may be that the parental serine is modified by phosphorylation, thus making a Ser-Asn change more drastic. In this vein, it has been shown that the NP of WSN virus specifically contains one phosphorylated serine residue (Privalsky and Penhoet, 1981). Studies are underway to examine this question. Finally, Mandler and Scholtissek (1988) recently analyzed a ts mutant of A/FPV which mapped to the NP gene (ts81) and found a single alanine to threonine change at amino acid 332. Interestingly, biochemical analysis of this mutant also shows it to be defective in template RNA synthesis (Scholtissek, 1978). Thus, the domain of the NP around amino acids 314-332 may define a region necessary for the replication phase of the virus.

Acknowledgements

The authors would like to thank Mariana Nacht for her excellent technical assistance. This work was supported by Public Health Service grants Al-11823, AI-18998 (P.P.) and AI-26663 (M.K.) from the National Institute of Health. M.K. also received support from the New York Lung Association. Preliminary results were presented at the Fifth Stony Brook Symposium on ‘New Perspectives on the Molecular Biology of RNA (Krystal, M. and Palese, P. In: M. Inouye and B. Dudock (Eds.), Molecular biology of RNA: new perspectives, p. 212. Academic Press, New York).

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20 July 1988; revision

received

17 October

1988)