Isolation and properties of a temperature-sensitive mutant (ts 412) of an influenza A virus recombinant with a is lesion in the gene coding for the nonstructural protein

VIROLOGY

110, 16-25 (1981)

Isolation and Properties of a Temperature-Sensitive Mutant (ts 412) of an Influenza A Virus Recombinant with a is Lesion in the Gene Coding for the Nonstructural Protein

INGO KOENNECKE, 1 C. BRUCE BOSCHEK, AND CHRISTOPH SCHOLTISSEKZ Institutfur Virologie, Justus-Liebig-Universitat Giessen, 6800 Giessen, Germany Accepted October 7, 1980

After mutagenizing a recombinant between fowl plague virus (FPV) and virus N a temperature-sensitive (ts) mutant (ts 412) was isolated which carries a defect in the gene coding for the nonstructural (NS) proteins . This mutant has been characterized in biological terms . In spite of the fact that the NS proteins are known to be early proteins, the lesion of is 412 is a late defect : At the nonpermissive temperature all viral products are synthesized in normal yields except for hemagglutinin and M protein which are produced in relatively low amounts . No particles are formed at 40° . Electron-dense inclusions were found under these conditions in the cytoplasm of chick embryo cells infected with is 412 . These structures may be accumulations of one of the NS proteins .

ing RNA segments, and to functions by Ritchey and Palese (1977) . The only gene, for which no is mutant was available, was that coding for the NS proteins . In our laboratory is mutants could be assigned to six different RNA segments and corresponding functions . Again a mutant with a lesion in segment 8 (gene coding for the NS proteins) was missing (Scholtissek et al ., 1974, 1976 ; Scholtissek and Bowles, 1975). Recently Almond et al . (1977, 1979) isolated two is mutants with a defect in the gene coding for the NS proteins . The biological properties of these mutants have been described recently (Wolstenholme et al ., 1980) . In an effort to prepare influenza virus recombinants with RNA segments of three different parent strains, we mutagenized a recombinant between fowl plague virus (FPV) and virus N, which carries RNA segments 2 (transport protein gene) and 8 (gene coding for the NS proteins) of the latter parent, while all other segments were derived from FPV (Scholtisseket al ., 1976) . Among the ts-mutants obtained with this recombinant one was found to carry a is lesion solely in segment 8 . The present

INTRODUCTION

Influenza viruses have a segmented genome consisting of eight distinct singlestranded RNA segments (for reviews see Palese, 1977; Scholtissek, 1978) . Each RNA segment codes for one virus-specific protein except for the smallest one which carries the information of the two nonstructural (NS) proteins (Inglis et al ., 1979 ; Lamb and Choppin, 1979) . Temperature-sensitive (ts) mutants of influenza A viruses were isolated first by Simpson and Hirst (1968) and since that time by many other groups (for reviews see Suguira, 1975; Palese, 1977 ; Scholtissek, 1978) . Hirst (1973) found is mutants belonging to eight recombination groups . However, these mutations were not assigned to specific RNA segments, and were not characterized extensively in biological terms. Suguira et al . (1972, 1975) were able to place their is mutants into seven recombination groups which were assigned to correspond' Present address : WHO Collaborating Center for Influenza, Department of Health, Education and Welfare, Public Health Service, Center for Disease Control, Atlanta, GA 30333 . % To whom reprint requests should be addressed . 0042-6822/81/O50016-10$02 .00/0 Copyright C 1981 by Academic Press, Inc . All rights of reproduction in any farm raxerved .

16



INFLUENZA A VIRUS RECOMBINANT MUTANT WITH GENE LESION

communication will describe the characterization of this is mutant in genetic as well as in biological terms . MATERIALS AND METHODS

Viruses . The following influenza A viruses were used: fowl plague virus (A/FPV/ Rostock/34, Hav1N1) ; virus N (A/chicken/ Germany/N/49, Hav2Neql) ; equi 2 (A) equine/Miami/1163, Heq2Neq2) ; FM1 (A/ FM/1/47, H1N1) ; and the Singapore strain (A/Singapore/1/57, H2N2) . For the isolation of temperature-sensitive (ts) mutants a recombinant between FPV and virus N (90/N3) was investigated, which carries RNA segments 2 (transport protein gene), and 8 (NS protein gene) of virus N and the other segments of FPV (Scholtissek et al ., 1976) . This recombinant forms plaques on chick embryo cells which are identical in size with those of FPV . It multiplies in embryonated chicken eggs and on primary chick fibroblasts to the same titer as FPV . The following known is mutants of FPV were used for recombination with the newly isolated is mutants : is 3 carries a lesion in RNA segment 1 (polymerase 1 gene) ; is 90 has a lesion in segment 2 (transport protein gene); is 263 has a lesion in segment 3 (polymerase 2 gene) ; is 227 has a lesion in segment 4 (hemagglutinin gene) ; is 19 has a lesion in segment 5 (nucleoprotein gene) ; is 113 has a lesion in segment 6 (neuramidase gene (Scholtissek et al., 1974, 1976 ; Scholtissek and Bowles, 1975 ; Harms et al., 1978) . Isolation of temperature-sensitive mutants . The procedure for the isolation of is mutants by mutagenesis with 5-fluorouracil was essentially the same as published by Simpson and Hirst (1968) and described in detail by Scholtissek et al . (1974) . Recombination experiments . For recombination, primary chick embryo cells were either singly or doubly infected with a multiplicity of 10 to 50 PFU of each parent virus at 40°. Eight hours after infection cells were broken up by three repeated cycles of freezing and thawing in the supernatant medium . Plaque tests on chick embryo cells were performed at 33° or 40° in an incubator located in a warm room (40°) . The

17

recombination frequencies were calculated according to the formula published by Suguira et al . (1972) . Each test was repeated at least twice . For details see Scholtissek and Bowles (1975) . For the isolation of recombinants between the is mutant in question and other influenza A prototype strains chick embryo cells were doubly infected at 40° as described above . Plaque formers obtained at 40° were purified three times by a plaqueto-plaque test . Finally stocks were grown by injecting plaques into embryonated chicken eggs (Scholtissek et al ., 1976) . Hybridization of individual 32P-labeled vRNA segments with nonlabeled eRNA . Labeling of vRNA with 32 P, the isolation of the vRNA segments by polyaeAylamide gel electrophoresis in the presence of 6 M urea, and the hydridization technique using nonlabeled cRNA have been described recently (Scholtissek et al ., 1976, 1978) . Determination of influenza virus vRNA and cRNA . The procedure has been described in detail by Scholtissek and Butt (1970) . Total cellular RNA was pulselabeled by [3H]uridine starting 4 .5 hr after infection under various temperature conditions . Six hours after infection total RNA was isolated and aliquots were hybridized with a surplus of either nonlabeled influenza virus vRNA or eRNA . After treatment with pancreatic RNase the remaining acidprecipitable radioactivity determines the relative amounts of either eRNA or vRNA synthesized between 4 .5 and 6 hr after infection. Noninfected control cells were run in parallel . The corresponding values were substracted . Determination of the viral RNA polymerase activity . Aliquots of cytoplasmic extracts isolated 5 hr after infection from 2 x 10 7 cells were incubated at 32° with [3 H]GTP and cofactors for 10 or 20 min, respectively (Scholtissek and Rott, 1969) . The label found after acid precipitation is incorporated exclusively into viral eRNA (Scholtissek, 1969) . Values of control extracts were substracted . Hemagglutination and neuraminidase tests . These tests were performed by standard procedures (Davenport et al., 1960 ; Drzeniek et al ., 1966) .



18

KOENNECKE, BOSCHEK, AND SCHOLTISSEK

Quantitative hemadsorption test . Six hours after infection the cells (6 x 10 6) were washed twice with 5 ml PBS . Thereafter the layer was covered with 1 ml of 1% chick erythrocytes . After 20 min at 4° the cells were washed twice with 5 ml PBS and scrapped off with 1 ml H 2O. After hemolysis and centrifugation the optical density of the supernatant was measured at 575 nm . Noninfected control cells were treated in the same way . This value was substracted (Nakamura and Homma, 1974) . Polyacrylamide gel electrophoresis of labeled proteins . Proteins were labeled by

[35 S]methionine for 10 min (10 pCi for 90/ N3- and is 412-infected cells, 6 x 10 6). The pulse was started at different times after infection . Cells were incubated either at 33° or 40° . Cell extracts were prepared and electrophoresed on 15 or 25% polyacrylamide urea slab gels according to Bosch et al . (1978) . Electron microscopy . Cell monolayers were fixed in situ by adding glutaraldehyde to a concentration of 2% to the growth medium . After 15 min at 20° the medium was replaced with cold 2% glutaraldehyde in cacodylate buffer, pH 7 .2, and the cells postfixed for 30 min at 4° . The monolayer was then treated for 30 min with 1% OsO, in cacodylate buffer followed by 1% tannic acid for 30 min according to the method of Simionescu and Simionescu (1976) . After rinsing with 1% Na2SO4 for 5 min the cells were treated overnight with aqueous 1% uranyl acetate, dehydrated in ethanol, and finally embedded in Epon . After polymerization the plastic dish was broken away and the embedded monolayer cut into small blocks which were glued together face to face to provide two parallel tracks of cells . These double blocks were then clamped in a vise-type microtome chuck and were cut perpendicular to the growth substrate on a Reichert Ultracut ultramicrotome using a diamond knife . Silver-grey to grey sections were treated with aqueous uranyl acetate and lead citrate and were observed and photographed at 80 kV using a Zeiss EM 10 electron microscope. Materials . The materials used for polyacrylamide gel electrophoresis were obtained from BioRad Laboratories (Rich-

TABLE 1 RECOMBINATION BETWEEN THE STANDARD MUTANTS OF FPV AND TS 412"

is

Ts mutants of FPV

Recombination frequency

3

19

90

113

227

263

14

12

5

21

18

11

The recombination frequencies were calculated according to the formula published by Sugiura et al., 1972 .

mond, Calif.), 12 P-labeled orthophosphate (carrier-free) and ['SS]methionine (7 .75 Ci/ mmol) were obtained from the Radiochemical Centre, Amersham, England . RESULTS

Isolation and Assignment of TemperatureSensitive Mutants to Recombination Groups

Of the is mutants isolated 30 exhibited differences in plaque titers of 5 to 6 log units when incubated at 33° or 40°, respectively . All were genetically stable enough for recombination experiments . Chick embryo cells were doubly infected with these is mutants together with is mutants which had known defects . All mutants except one can be assigned to the six known recombination groups of FPV . Mutant is 412, which recombines with all six standard is mutants (Table 1), appeared to carry a is lesion in either the gene coding for the M protein or for the NS protein . These two genes are not covered by the six standard is mutants . Recombinants between is 412 and Other Prototype Influenza A Strains

The localization of a is lesion in a specific RNA segment can be achieved by double infection of a suitable host cell at the nonpermissive temperature (40°) by the is mutant together with another influenza strain which does not form plaques on that host . All plaque formers obtained under these conditions must have exchanged at



19

INFLUENZA A VIRUS RECOMBINANT MUTANT WITH GENE LESION

a is lesion solely in segment 8 which is known to code for the NS proteins (Harms et al ., 1978; Inglis et al ., 1979 ; Lamb and Choppin, 1979) .

TABLE 2 GENE CONSTELLATION OF RECOMBINANTS OF

412"

RNA segments Strain

1

2

3

4

5

6

7

8

FPV

P

P

P

P

P

P

P

P

90/N3

P

N

P

P

P

P

P

N

A A A A A

A A A A A

A A A A A

P P P P P

A A A A A

P P P P P

P P P P P

A A A A A

1

F

2

E

N N

P P

P P

P E

P P

P P

E E

P P F

F F N

P P F

P P P

F F P

P P P

P P P

F F F

412/A 1 2 3

4 5 412/E

412/F 1 2 3

'P, HavIN1(FPV); A, H2N2 (Singapore) ; F, H1N1 (FMI) ; N, Hav2Neq 1(Virus N) ; E, Heg2Neg2 (equi 2) .

least the RNA segment carrying the is lesion of the mutant strain (Scholtissek et al ., 1976 ; Almond et al ., 1977) . We have applied this technique to is 412 using chick embryo cells and various prototype strains as listed in Table 2 . The assignments of various RNA segments to one or the other parent strain were done by molecular hybridization using 32P-labeled vRNA segments of either FPV, virus N, or the Singapore strain, and nonlabeled cRNA of the parent and recombinant strains . As can be seen in Table 2, all recombinants obtained without exception have exchanged segment 8 against the corresponding segment of the rescuing virus, but never segment 7 . The recombinants obtained with equi 2 have exchanged in addition segments 1 or segments 1 and 5 . Two recombinants with FM1 have also exchanged segments 2 and 5, and one has exchanged segments 1 and 3 . All recombinants derived from the Singapore strain have also exchanged segments 1, 2, 3, and 5 . From the combined data of Tables 1 and 2 it can be concluded that is 412 carries

Viral RNA Synthesis in Cells Infected with is 412 Next the is mutant 412 was characterized in biological terms . First, viral RNA synthesis in is 412-infected chick embryo cells was examined at various temperatures. As can be seen in Table 3, both types of viral RNA, vRNA as well as eRNA, are synthesized in is 412-infected cells at the permissive as well as the nonpermissive temperature . The only difference to cells infected with the wild type FPV is that in cells infected with is 412 at 40°, relatively more cRNA is being synthesized. Induction of Viral RNA Potymerase by is 412 As can be seen in Table 4, after infection of chick embryo cells with is 412 and incubation at 32° similar activity of the viral RNA polymerase was found in cytoplasmic extracts when compared with 90/N3-infected cells . Under nonpermissive conditions the RNA polymerase activity is not reduced in the inn vitro test of is 412-infected cells .

TABLE 3 SYNTHESIS OF VIRUS-SPECIFIC RNA IN VIVO AT 33' AND 40' AFTER INFECTION WITH is MUTANT 412 AND WILD TYPE VIRUS' Percentage of total RNA in vivo incubation Type of virus RNA

33` (0-6)

Wild type

Virion Complementary

11 .5 6 .8

ND ND

13.1 4.3

Ta 412

Venue

10.0 7 .1

6 .4 5 .4

12.8 15.0

Strain

Complementary

33' (0-4)

40' (4-6)

40° (0-6)

° The infected cells were kept either at 33° or 40°, or were shifted 4 hr after infection from 33° to 40°, A pulse with PHln^line was started 4.5 hr after infection. Total RNA was isolated 6 hr after infection. The percentage of labeled vRNA and 0RNA was determined by specific hybridization (total RNA = 40,00) cpm) . (Scholtiesek and Rott, 1970) . ND, not done.



20

KOENNECKE, BOSCHEK, AND SCHOLTISSEK TABLE 4

INDUCTION OF VIRAL RNA POLYMERASE RECOMBINANT 90/ N3 AND IS MUTANT 412 33° AND 40°°

BY AT

The Protein Patterns of Radioactively Labeled Cells Infected with is 412

Radioactivity in RNA (cpm)

Incubation in vitro (32°) (min)

33°

40°

90/N3

10 20

2300 5400

3000 6900

Ts 412

10 20

3000 7000

2900 6400

Strain

tion activity was found in is 412-infected cells at 40° (Table 5) .

° Infected chick embryo fibroblasts were kept either at 33° or at 40° for 5 hr . Thereafter the polymerase activity was determined by incubating cytoplasmic fractions at 32° with ['H]GTP plus cofactors according to Scholtissek and Rott (1969) . The acid-precipitable radioactivity of mock-infected cultures was subtracted . Synthesis of Hemagglutinin, Neuraminidase, and Infectious Particles after Infection with is 412 The yield of hemagglutinin in chick embryo cells infected with is 412 at 33° is the same as that in 90/N3-infected cells . After raising the temperature to 40° the yield decreases to about 12% of the value obtained at 33° (Table 5) . The production of neuraminidase is not impaired at 40' (Table 5) . At the nonpermissive temperature no infectious particles were found when compared with the yield obtained at 33' (Table 5) . Also in the quantitative hemadsorption test a reduction in the hemadsorp-

The protein patterns obtained by polyacrylamide gel electrophoresis of radioactively labeled cell extracts revealed that after infection with is 412 and incubation at 40° all viral proteins were synthesized 6 hr after infection. However, the amount of labeled hemagglutinin and especially of M protein is highly reduced in is 412-infected cells at 40° (Fig. 1) . The double band in the region of HA in Fig . Ii is a constant finding. The meaning is not yet understood . A time course of the synthesis of the various proteins is shown in Fig . 2 . The synthesis of the M protein in is 412-infected cells is always highly reduced at 40° ; however, later in the infection its production ceases almost completely . In Fig. 3 using a different gel it is shown that in is 412-infected cells at 40° normal amounts of NS2 are produced . Shift-Up and Shift-Down Experiments In Table 6 results on the yield of infectious virus are presented, which were obtained after shift-up from 33° to 40° at different times after infection . The incubation at 40° was continued always for 8 hr before the plaque test was performed on extracts of cells plus medium . In order to reduce the background infectivity cells were washed immediately after infection at room tem-

TABLE 5 MULTIPLICATION OF RECOMBINANT 90/N3 is MUTANT 412 AT Two DIFFERENT TEMPERATURES IN A SINGLE-CYCLE EXPERIMENT IN TISSUE CULTURES" AND

HAU/ml

Neuraminidase (units/m])

Hemadsorption activity (0D at 575 nm)

1 .8 x 10' 2 .5 x 10'

128 256

146 480

0 .900 1 .060

1 .5 x 10' <10

128 16

146 292

0 .620 0 .330

Strain

Temperature (°C)

PFU/ml

Recombinant 90/N3

33 40

Ts mutant 412

33 40

" The titers were determined in the supernatant of cells harvested at 8 hr after infection, which were broken by three repeated cycles of freezing and thawing, followed by centrifugation . The quantitative hemadsorption activity was determined 6 hr after infection .

INFLUENZA A VIRUS RECOMBINANT MUTANT WITH GENE LESION a

b c

d e f g h i

FIG . 1 . Polyacrylamide gel electrophoresis of u S labeled cells infected with FPV, virus N, 901N3, and is 412 . For comparison, in vivo ['°S]methioninelabeled virus particles were also used . Infected cells were labeled with 10 µC [nSlmethionine for 10 min starting at 6 hr after infection . Polyacrylamide gel, 25% . (a) Uninfected control cells ; (b) FPV, 40°; (c) virus N, 40' ; (d) FPV particles; (e) virus N particles; (f) 901N3, 33°; (g) 90/N3, 40° ; (h) is 412, 33°; (i) is 412, 40° .

perature once with 0 .9% NaCl solution at pH 2 (N . J. Dimmock, personal communication) . After two further washings with phosphate-buffered saline, pH 7 .2, the cells were covered with 2 ml medium (=time 0) . It can be seen that shift-up after 2 hr of incubation at 33° does not yield any infectious virus. Even after 4 hr preincubation at 33° the virus yield at 40° is still quite low. In a corresponding shift-down experiment, virus yield is not diminished when is 412-infected cells were preincubated for 2 hr at 40° . Longer preincubation at 40° reduces the virus yield at 33° to a certain extent (Table 6) . These experiments demonstrate that the is defect in is 412 is a late one .

21

within the cytoplasmic matrix as shown in Fig . 4d . These inclusions, which appear in close association with free ribosomes were neither observed in 901N3-infected nor in uninfected cells . The same observation was

a

a

b e d e

b c d e

g h i

1 g h i

Electron Microscopic Examination of Infected Cells Chick embryo cells which were infected with is 412 or 90/N3 and incubated at 33° or 40° for 10 hr were fixed, embedded, and sectioned for examination in the electron microscope . At 33° both strains produced virus particles (Figs . 4a and c). At 40°, cells infected with 901N3 also produced virus particles as seen in Fig . 4b . No virus particles were visible on the surface of the cells infected with is 412 at 40° . Instead, numerous dense inclusions were observed

FIG . 2 . Time course of synthesis of the various viral proteins at 33° or 40°, respectively . Experimental conditions as in Fig . 1 . The isotope was added at the times after infection as indicated . (A) Isotope was added at 1 hr (a-e) or 2 hr after infection (f-i), respectively. (a) Mock-infected cells ; (b) and (c) infected with 90/N3 (b at 33°, c at 40°); (d) and (e) infected with is 412 (d at 33°, e at 40°) ; (f-i) corresponding 2-hr values . (B) Isotope was added at 3 or 4 hr after infection, correspondingly . (c) Isotope was added at 8 or 10 hr after infection, correspondingly .



22

KOENNECKE, BOSCHEK, AND SCHOLTISSEK

made when the cells were fixed 19 hr after infection . Further identification of these inclusions has not yet been attempted, however, their appearance is similar to structures which were identified as accumulations of NS protein by Shaw and Compans (1978) after infection with another influenza A strain . DISCUSSION

Since the genome of influenza viruses consists of only eight genes it may be assumed that at least some of the gene products might exhibit more than one function. By isolating and studying as many independently isolated is mutants as possible, not only the exact number of recombination groups, but also the potential multifunctional properties of a certain gene product should be recognizable . It is therefore still worthwhile to isolate new is mutants, although many such isolations have already been described in the literature . Difficulties have been encountered in isolating is mutants of the two smallest genes which code for the M and NS proteins, presumably because of the relatively small target size but also because they belong to the highly a b e d e f g h i

FIG . 3 . Polyacrylamide gel electrophoresis of °& labeled cells infected with 901N3 (b-e) and is 412 (f-i) . Infected cells were labeled as in Fig. 1 for 10 min (b,d,f,h), or were labeled for 10 min and chased for another 60 min (c,e,g,i) at 33° (b,c,f,g) or 40° (d,e,h,i) . (a) Uninfected cells . Polyacrylamide gel, 15% .

TABLE 6 YIELD OF INFECTIOUS VIRUS AFTER SHIFT-UP OR SHIFT-DOWN OF to 412-INFECTED CEI Ls° Incubation

Incubation

at

at

33° (hr)

40° (hr)

PFU/ml

1 2 3 4

8 8 8 8 8 8 0

0 0 2 .0x10° 2 .5x10' 2 .0x10' 1 .2x106 5.0 x 10°

5 6 14

40° (hr)

33° (hr)

PFU/ml

1 2 3

8 8 8 8 8 8 0

1 .0 x 10' 4 .0x10° 2 .0x10° 1 .5x10 6 2 .0xI0' 2 .0x10' 1 .2 x 10-

6 14

° Cells were infected with is 412 . Immediately after infection the cells were washed with 0 .9% NaCl, pH 2, in order to remove background infectivity . Thereafter the cells were incubated for different lengths of time either at 33° or 40° . The shift-down or shift-up, respectively, occurred at the times indicated . Further incubation for 8 hr was either at 40° or 33°, respectively.

conserved genes (Scholtissek et at ., 1976) . Thus, only two is mutants have been assigned to the NS proteins (Almond et al ., 1977, 1979) . During our efforts to mutagenize a recombinant between FPV and virus N we have discovered one is mutant (ts 412) the defect of which was assigned to the RNA segment coding for the NS proteins (Tables 1 and 2) . The biological properties of this mutant have been studied in considerable detail . In spite of the fact that the NS proteins are early proteins (Skehel and Hay, 1978 ; Inglis and Mahy, 1979) it has turned out that the is 412 mutant has a late defect : at the nonpermissive temperature all virus-specific products are being synthesized in almost normal yields except for hemaglutinin, M protein, and infectious particles . Normal RNA polymerase activities and high neuraminidase titers were found . There is relatively more cRNA synthesized that vRNA when compared to infection with wild type virus, however, the meaning of this observation is not clear . The protein patterns on polyacrylamide gels also revealed that all viral proteins were labeled normally with the exception of hemagglutinin and M protein . The latter are

INFLUENZA A VIRUS RECOMBINANT MUTANT WITH GENE LESION

23

FIG. 4. Electron micrographs of chick embryo cells infected with 90/N3 or is 412 . (a) 901N3 at 33° ; (b) is 412 at 33° ; (c) 901N3 at 40° ; (d) is 412 at 40° whereby budding virus particles were not observed . Arrowheads point to dense inclusions within the cytoplasmic matrix . Bar represents 500 nm .

synthesized in reduced amounts, although ment with a late defect in is 412 . In the the bit hemagglutinin synthesized is cleaved electron microscope deposits of dense mainto HAl and HA2 . The shift-up experi- terial can be seen after infection with is ments described in Table 6 also are in agree- 412 at 40° . This dense material was neither

24

KOENNECKE, BOSCHEK, AND SCHOLTISSEK

seen in 90/N3-infected cells nor after incubation at 33°. It is not yet clear whether this material is composed of NS protein . No virus particles were visible at 40° in is 412-infected cells. Thus, the only biological defects observed in is 412-infected cells under restriction conditions are reduced production and/or stability of the hemagglutinin and M protein and the complete absence of budding and of infectious particles . It is not clear how the NS protein might be involved in these activities . The two is mutants with defects in RNA segment 8 as described by Wolstenholme et al . (1980) also synthesize little M protein at the nonpermissive temperature . However, in contrast to is 412 very little vRNA and no NS2 are produced at 40° . ACKNOWLEDGMENTS We thank V . von Hoyningen-Huene for providing us with "P-labeled vRNA segments, and Dr . F . Bosch for helping us with the polyacrylamide gel electrophoresis of labeled proteins. The skillful technical assistance of Miss C . Hillmann and Miss R . Back is gratefully acknowledged . The work was supported by the Sonderforschungsbereich 47 . It is in partial fulfillment of the requirements for the degree Dr . rer. nat . of I . Koennecke, Fachbereich Biologic, University of Giessen .

HARMS, E ., ROHDE, W ., BOSCH, F ., and SCHOLTISSEK, C . (1978) . Biochemical studies on influenza viruses. II . Assignment of gene functions to RNA segment 5, 7, and 8 of fowl plague virus and virus N . Virology 86,413-422 . HIRST, G . K. (1973). Mechanism of influenza recombination. I . Factors influencing recombination rates between temperature-sensitive mutants of strain WSN and the classification of mutants into complementation-recombination groups . Virology 55, 81-93 . INGLIS, S . C ., and MAHY, B. W. J . (1979) . Polypeptides specified by the influenza virus genome . 3 . Control of synthesis in infected cells . Virology 95, 154-164 . INGLIS, S . C ., BARRET, T ., BROWN, C . M ., and ALMOND, J . W. (1979) . The smallest genome RNA segment of influenza virus contains two genes, which may overlap . Proc . Nat . Acad . Sci . USA 76, 3790-3794 . LAMB, R . A., and CHOFPIN, P. W . (1979) . Segment 8 of the influenza virus genome is unique in coding for two polypeptides . Proc . Nat . Acad. Sci. USA 76, 4908-4912 . NAKAMURA, K ., and HOMMA, M. (1974) . Cell fusion by HeLa cells persistently infected with hemabsorption type 2 virus . J. Gen . Viral . 25,117-124. PALESE, P. (1977) . The genes of influenza virus . Cell 10, 1-10 . RITCHEY, M . B ., and PALESE, P . (1977) . Identification of the defective genes in three mutant groups of influenza virus . J. Virl . 21, 1196-1204. SCHOLTISSEK, C . (1969) . Synthesis in vitro of RNA complementary to parental viral RNA by RNA polymerase induced by influenza virus. Biochim . Biophys . Acta 179, 389-397 .

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ALMOND, J . W ., MCGEOCH, D ., and BARRY, R . D . (1977) . Method for assigning temperature-sensitive mutants of influenza viruses to individual segments of the genome . Virology 81, 62-73 . ALMOND, J . W ., McGEOCH, D ., and BARRY, R . D .

SCHOLTISSEK, C ., and RoTT, R . (1969). Ribonucleic acid nucleotidyl transferase induced in chick fibroblasts after infection with an influenza virus . J. Gen .

(1979), Temperature-sensitive mutants of fowl plague virus : Isolation and genetic characterization . Virology 92, 416-427 . BOSCH, F . X ., HAY, A . J ., and SKEHEL, J . J . (1978) . RNA and protein synthesis in a permissive

SCHOLTISSEK, C ., and Rorr, R . (1970) . Synthesis in vivo of influenza virus plus and minus strand RNA and its preferential inhibition by antibiotics . Virology 40, 989-996 .

and an abortive influenza virus infection . In Negative Strand Viruses and the Host Cell (B . W. J . Matey and R . D . Barry, eds .), pp . 465-474 . Academic Press, New York . DAVENPORT, F . M ., RGTT, R ., and SCHAFER, W . (1960) . Physical and biological properties of influenza virus components obtained after ether treatment . J. Exp . Med . 112, 765-782 . DRZENiEK, R ., SETO, J. T ., and RGTT, R. (1966) . Characterization of neuraminidase from myxoviruses. Biochim . Biophys . Arta 128, 547-558 .

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INFLUENZA A VIRUS RECOMBINANT MUTANT WITH GENE LESION SCHOLTISSEK, C ., ROHDE, W ., VON HOYNINGEN, V .,

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