JOURNAL OF VIROLOGY, Jan. 1979, p. 361-373 Vol. 29, No. 1 0022-538X/79/01-0361/13$02.00/0 Influenza B Virus Genome: Assignment of Viral Polypeptides to RNA Segments VINCENT R. RACANIELLO AND PETER PALESE* Department ofMicrobiology, Mount Sinai School ofMedicine, City University of New York, New York, New York 10029 Received for publication 1 August 1978 It was shown that all eight RNA segments of influenza B viruses are most likely monocistronic and code for eight virus-specific polypeptides. A genetic map of the influenza B virus genome was established, and six polypeptides (P1 protein, nucleoprotein, hemagglutinin, neuraminidase, M protein, and nonstructural pro- tein) were unambiguously assigned to specific RNA segments. Molecular weight estimates ofthe eight individual genes were obtained by using the glyoxal method. These results suggest that each influenza B virus RNA segment has a greater molecular weight than the influenza A virus RNA segment which codes for the analogous gene product. Influenza B viruses were first isolated in 1940 cluding a previously unrecognized viral non- from patients afflicted with respiratory illness structural (NS) protein (30). (10, 22). Since then influenza B viruses have We now report our complete results on the been recognized as important human pathogens. establishment of a genetic map of the influenza Although influenza due to the type A virus is B viruses. Our analysis shows that there are more frequent than influenza B, the clinical at- most likely eight virus-specific polypeptides tack rate of influenza B can be very high (24), coded for by the eight influenza B virus RNA and influenza B virus infections occur in every segments. In addition to the surface proteins HA month of the year (16). Furthermore, influenza and NA, three P polypeptides, a nucleoprotein B viruses have been associated with Reye's syn- (NP), the M protein, and one NS protein can be drome, an acute encephalopathy in children (9). distinguished. (The nomenclature for these pro- Extensive serological studies on different in- teins is the same as that for the influenza A fluenza B virus isolates have demonstrated a viruses.) The genes coding for P1 protein, NP, lesser degree of antigenic variation than among NA, M protein, and NS protein are assigned to the influenza A viruses (5, 6, 14, 34). Other specific RNA segments. Finally, molecular biochemical studies on influenza B viruses have weight estimates of influenza B virus RNA seg- been scarce. Four to six influenza B virus poly- ments and influenza B virus-specific polypep- peptides have been identified previously (4, 12, tides are presented, and the gene structure of 13, 15, 20, 26, 37), and very little is known about influenza B viruses is compared with that of the genome of influenza B viruses (31). influenza A viruses. As part of a study to characterize the genome (This work was carried out by V.R.R. in par- of influenza B viruses and to compare it with tial fulfillment of the requirements for a Ph.D. that of influenza A viruses, we recently began degree in biomedical sciences at the Mount Sinai work on the establishment of a genetic map for School of Medicine of the City University of influenza B viruses. Our earlier observation (31) New York.) that the RNA of influenza B virus consists of eight segments was confirmed (30), and in a MATERIALS AND METHODS preliminary report we showed that RNA 5 of Cells and medium. Madin-Darby canine kidney influenza B viruses codes for the hemagglutinin cells (MDCK; 17) were propagated in Earle-based (HA) polypeptide (30). Our finding that RNA 5 minimal essential medium (Grand Island Biological of influenza B viruses codes for HA was con- Co.) supplemented with 10% fetal bovine serum. Viruses. Seed stocks of influenza B/Lee/40 firmed by Ueda et al. (38). Furthermore, these (B/Lee) and B/Maryland/59 (B/Md) viruses were authors succeeded in identifying RNA 6 of influ- prepared by inoculation of the allantoic cavity of 11- enza B viruses as the gene coding for the neur- day-old embryonated hen eggs as described previously aminidase (NA) polypeptide. We also presented (31). evidence for seven virus-coded polypeptides, in- Isolation ofrecombinant viruses. Recombinants 361 362 RACANIELLO AND PALESE J. VIROL. between B/Lee and B/Md viruses were isolated by by 23 cm by 1.5 mm and consisted of a resolving gel using techniques employed for the isolation of influ- with a 5 to 13% gradient of polyacrylamide and a 0 to enza A virus recombinants (27). Briefly, MDCK mono- 20% sucrose gradient. The spacer gel was 5% polyacryl- layers on 25-cm2 plastic dishes were mixedly infected amide. The gels also contained 6 M urea, 0.37% with B/Lee and B/Md viruses. It has been noted (35) (vol/vol) Triton X-100, and 5% acetic acid. The run- that, in such a mixed infection with influenza A viruses, ning buffer was 5% acetic acid. Pulse-labeled infected the multiplicity of infection (MOI) can influence the cell pellets were dissolved in a mixture containing 6 M gene composition of the resulting recombinants. urea, 5% acetic acid, 4% /3-mercaptoethanol, 0.02% Therefore, depending on which parental RNAs we pyronine Y, and 0.37% (vol/vol) Triton X-100. Elec- wished to predominate in the recombinant, one parent trophoresis was performed at room temperature at 100 was present at a MOI of 10 PFU/cell and the other V until the dye front left the resolving gel (length, 20 parent was present at 1 PFU/cell. After a 45-min cm). After electrophoresis, gels were dried and exposed adsorption at 37°C, monolayers was washed, and to X-ray film. maintenance medium was added. Cells were washed One-dimensional peptide maps. Individual viral again after 3 h and incubated at 37°C for an additional polypeptides from pulse-labeled infected cells or from 14 h until most cells were detached. Culture fluids continuously labeled purified virus preparations were were centrifuged briefly to remove cell debris and then subjected to one-dimensional peptide mapping by the plaqued in MDCK cells in the presence of hyperim- procedure of Cleveland et al. (7) with minor modifi- mune rabbit antisera directed against the surface pro- cations. After labeled viral polypeptides were sub- teins of B/Lee or B/Md virus. The resulting plaques jected to electrophoresis on a polyacrylamide gel as were picked and grown up in MDCK cells, and seeds described above, the gel was stained, and two lanes were made in 11-day-old embryonated hen eggs. which contained samples of infected and uninfected Serological analysis. The derivation of the sur- cells were cut out, dried, and autoradiographed. The face proteins of recombinant viruses was identified by labeled viral polypeptides were located on the film, HA inhibition and NA inhibition assays (2, 40). To which was used to identify viral polypeptides in the avoid steric interference of the viral HA in NA inhi- remaining lanes of the wet, stained gel. (The HA, NP, bition assays, purified virus was used which was and NS and M proteins could often be identified by treated with Pronase to destroy the viral HA (6). staining alone.) Bands were then cut out from the wet Labeling and extraction of viral RNA. The gel and subjected to partial digestion in the stack of a RNA of recombinant viruses and their parents was second polyacrylamide gel (the resolving gel contained labeled with 32p in MDCK cells and extracted from a 10 to 15% gradient of acrylamide). The enzymes used purified virus as described previously for influenza A were Staphylococcus aureus V8 protease (0.5 ytg/slot) viruses (28, 29). and chymotrypsin (20 tig/slot). All enzyme solutions Polyacrylamide gel electrophoresis of viral used to overlay the gel slices contained 2% /8-mercap- RNA. 32P-labeled RNAs of parent and recombinant toethanol (19). Gels were subjected to electrophoresis viruses were separated on 2.6% polyacrylamide gels at 80 V until the dye front reached the resolving gel containing 6 M urea under established conditions (32). and then run at 100 V until the dye marker ran off the Labeling of viral polypeptides. Pulse-labeling of resolving gel (length, 15 cm). Gels were then fluoro- influenza B virus-infected cell polypeptides with L- graphed (3), dried onto Whatman 3MM filter paper, [35S]methionine was performed as described previ- and exposed at -70°C to Kodak X-Omat RC film. ously (33). Unless indicated otherwise, at 6 h postin- Estimation of molecular weights of viral RNA fection cells were first incubated for 15 min in Earle segments and viral polypeptides. The method of salt solution containing an additional 150 mM NaCl. Weber and Osborn (39) was used to determine molec- The use of hypertonic medium to selectively suppress ular weights of viral polypeptides. Pulse-labeled poly- host protein synthesis in RNA virus-infected cells has peptides of B/Lee virus-infected cells were analyzed been reported previously (25). For pulse-labeling ex- on a SDS-polyacrylamide gel with a 3% stacking gel periments L-[35S]methionine was added directly to the and a gradient-resolving gel of 5 to 13% polyacryl- medium after a 15-min preincubation in hypertonic amide. The discontinuous buffer system used was that medium. Virus continuously labeled with L-[u5S]me- of Laemmli (18). Marker proteins used and their mo- thionine was purified as described previously for influ- lecular weights were: phosphorylase a, 94,000; bovine enza A viruses (33). serum albumin, 67,000; ovalbumin, 45,000; lactic de- Polyacrylamide gel electrophoresis of viral hydrogenase, 36,000; chymotrypsinogen, 25,000; and polypeptides.