RNAs of Influenza A, B, and C Viruses

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1 JOURNAL OF VIROLOGY, May 1976, p Copyright 1976 American Society for Microbiology Vol. 18, No. 2 Printed in U.SA. RNAs of Influenza A, B, and C Viruses M. B. RITCHEY, P. PALESE,* AND E. D. KILBOURNE Department of Microbiology, Mount Sinai School of Medicine of CUNY, New York, New York Received for publication 17 December 1975 The nucleic acids of influenza A, B, and C viruses were compared. Susceptibility to nucleases demonstrates that influenza C virus, just as influenza A and B viruses, possesses single-stranded RNA as its genome. The base compositions of the RNAs of influenza A, B, and influenza C virus are almost identical and comparative analysis on polyacrylamide gels shows that the genome of influenza C/GL/1167/54 virus, like that of the RNAs of influenza A and B viruses, is segmented. Eight distinct RNA bands were found for influenza A/PR/8/34 virus and for influenza B/Lee/40 virus. The RNA of influenza C/GL/1167/54 virus separated into at least four segments. The total molecular weights ofthe RNA of influenza A/PR/8/34 and B/Lee/40 virus were calculated to be 5.29 x 106 and 6.43 x 106, respectively. A minimum value of 4.67 x 106 daltons was obtained for influenza C/GL/1167/54 virus RNA. The data suggest that influenza C viruses are true members of the influenza virus group. It is well established that influenza A viruses contain a segmented genome. The nucleic acid of purified influenza A virus consists of at least seven (4, 16) or more likely eight (20) singlestranded RNA pieces. Most evidence suggests that these pieces are of negative polarity, i.e., translation takes place from a strand complementary to the one isolated from the virion (14; P. R. Etkind and R. M. Krug, Int. Congr. Virol., Madrid, 1975; M. B. Ritchey and P. Palese, unpublished data). The molecular weight of the RNA of influenza A viruses is in the range of 4 x 106 to 5 x 106 (4, 16, 20, 22; M. W. Pons, Int. Congr. Virol., Madrid, 1975). This means that influenza A viruses have a protein coding capacity of at least 400,000 daltons. Much less is known about the RNAs of influenza B viruses. Genetic evidence has been presented that influenza B viruses undergo genetic reassortment similar to influenza A viruses (30) and this suggests that influenza B viruses also possess a segmented genome. An early report shows that the base composition of influenza B viruses is similar to the one found for influenza A viruses (26), but there is no direct evidence that the genome of influenza B viruses consists of several segments. Nothing is known about the nucleic acid of influenza C viruses. Classification of influenza C viruses in the genus influenza virus was based initially on its biological and epidemiological characteristics, including its host range in experimental animals and its occasional association with disease indistinguishable from mild influenza in man (8, 18, 29). More recently, electron microscopy examination of influenza C virus (2) and studies of the viral proteins (13; V. W. Wilson, Jr., D. Bucher, and E. D. Kilbourne, unpublished data) showed that influenza C viruses share morphological and chemical characteristics with influenza A viruses. The virus has been shown to possess hemagglutinating (HA) activity (10), as do influenza A and B viruses, and to destroy receptors on red blood cells, but it does not liberate sialic acid from the usual neuraminidase substrates and therefore may have no neuraminidase (13). No definitive classification of influenza C viruses is possible, however, without knowledge of the nature of the viral nucleic acid. In this report we compare the nucleic acids of influenza A, B, and C viruses and we show that influenza C virus is most likely a true member of the genus influenza virus possessing a single-stranded, segmented RNA genome. MATERIALS AND METHODS Chemicals and radiochemicals. Proteinase K was obtained from EM Merck (Germany); DNase I (electrophoretically purified) and RNase A (type II-A, bovine pancreas) were purchased from Worthington Biochemical Corp. and Sigma Chemical Co., respectively. Sterile solutions of carrier-free 32P, as orthophosphoric acid in water, were purchased from New England Nuclear Corp. Acrylamide, NN-methylenebisacrylamide, and N,NN',N'-tetramethylethylenediamine were obtained from Bio-Rad Laboratories and urea (U-15) was purchased from Fischer Scientific Co. Viruses and purification. For this study, one strain each of influenza A, B, and C viruses was used. Influenza A/PR/8134 (HON1) virus with an 738

2 VOL. 18, 1976 unknown passage history in eggs was used earlier, in a comparative analysis of the RNAs of different influenza A viruses (20). Influenza B/Lee/40 virus was in a multiple egg passage and influenza C/GL/ 1167/54 virus (obtained through the courtesy of Walter Dowdle from the Center for Disease Control, Atlanta, Ga.) had had 16 amniotic passages in chicken embryos. It was then passaged once in primary chicken embryo fibroblasts and four more times in the amniotic sac of embryonated eggs. For the experiments described in this communication, influenza A and B viruses were propagated in the allantoic cavity of embryonated 11-day-old eggs at 37 C. Influenza C virus was inoculated amniotically and amniotic fluids were harvested after 48 h at 35 C. The inoculation for influenza A and B viruses was about 104 egg infective dose (EID50) per egg; the inoculum for influenza C virus varied between 102 to 10" EID5O. To label the RNA of influenza A and B viruses, approximately 5 mci of 32p were injected into the allantoic cavity at the time of inoculation; after 48 h, the HA titer of labeled allantoic fluids of influenza A and B virus-infected eggs varied between 2048 and To obtain labeled influenza C virus RNA, 3 mci Of 2p were used per egg. In a typical experiment, 15 eggs were infected, four embryos survived, and approximately 4 ml of amniotic fluid with an HA titer of 2048 to 4056 and a titer of 109 EID50/ml were collected. All HA titrations were performed at 4 C using chicken erythrocytes (27). On the day of harvest influenza A, B, and C viruses were purified by first clarifying the allantoic or amniotic fluids by slow centrifugation (1,000 x g for 10 min). Five-milliliter aliquots of these fluids were then directly layered on top of a 30-ml, 30 to 60% sucrose gradient containing 0.01 M Tris-hydrochloride, 0.1 M NaCl, and M EDTA (ph 7.4) and spun for 3 h at 100,000 x g in a Spinco SW27 rotor. The visible virus bands were withdrawn from the side of the tube with a syringe. All purification steps were performed at 4 C. RNA extraction. The virus suspension obtained from the gradient was made 0.2% with sodium dodecyl-sulfate and incubated for 20 min at 56 C after the addition of 500,ug of proteinase K per ml. The following steps, involving one phenol-chloroform extraction and two alcohol precipitations, were done as described previously (20). The RNAs were dissolved in a small volume of Loenings buffer (0.036 M Tris, 0.3M NaH2PO4; M EDTA; ph 7.8) for use in the polyacrylamide gel system. Right after isolation, the specific activity of influenza A and B virus RNA was between 20,000 and 80,000 counts/min per,ug of RNA and the specific activity of influenza C virus RNA was approximately 1,000 counts/min per,g of virus protein. (The percentage of RNA in influenza C virus was not determined.) Nuclease sensitivity. Nuclease sensitivity was determined using 32P-labeled RNA purified as described above. Suitable amounts were incubated at 37 C for 30 min with RNase A (10,g/ml) in 0.05 M RNAs OF INFLUENZA VIRUS 739 Tris-hydrochloride, ph 7.4, containing 0.1 M NaCl and M MgCl2 or with DNase I (10 Ag/ml) in 0.05 M Tris-hydrochloride, ph 7.4, containing M MgCl2. Next, carrier yeast RNA to 300 Ag/ ml and 50% trichloroacetic acid to 5% final concentrations were added and the samples were incubated at 0 C for 30 min. The acid-precipitable material was collected on a membrane filter (Millipore Corp.), washed four times with cold 5% trichloroacetic acid, dried, and counted in a liquid scintillation counter by using a toluene-based scintillation fluid. A control to determine total acid-precipitable counts was included. Base composition. 32P-labeled RNA, purified as described above, was hydrolyzed in 0.5 N KOH at 80 C for 1 h. The hydrolysate was desalted by applying it to Whatman CM82 paper and eluting it with H20 (12). The eluate was dried by passing a stream of N2 over the surface, resuspended in water, and applied to Whatman 3MM paper. Electrophoresis was then carried out with citrate buffer, M, ph 3.5, at 10 V/cm for 16 h (31). After drying, the paper was cut up into 0.5-cm strips and counted in a liquid scintillation counter by using a toluene-based scintillation fluid. Polyacrylamide gel electrophoresis. RNAs were analyzed on a 12-cm polyacrylamide slab gel containing 2.8% acrylamide, 0.14% N'N'-methylenebisacrylamide, 0.1% N,N,N',N'-tetramethylethylenediamine, 6 M urea, 0.2% ammonium persulfate, and Leonings buffer. This procedure was originally described by Floyd et al. (8) and has been modified by us (20). All RNA samples were heated for 30 s at 80 C and applied in a volume of 1 to 20 A.l. After the end of the run the gel was dried under vacuum and a Kodak (no screen) X-ray film was exposed to the dried gel. A total of 5,000 to 8,000 counts/min of viral RNA sample is sufficient to produce a visible pattern after 18 h of exposure. RESULTS Nuclease digestion of influenza A, B, and C virus nucleic acids. To establish whether influenza C virus possesses RNA or DNA as its genome, the three strains, influenza A/PR/8, B/ Lee, and C/GL viruses, were purified and their nucleic acids were extracted after proteinase K treatment as described above. An aliquot of each of the three 32P-labeled samples was treated with bovine pancreatic RNase A and DNase. As expected, the RNA of influenza A and B viruses was degraded by RNase but not by DNase (Table 1). Similarly, the nucleic acid of influenza C virus was also digested by TABLE 1. Nuclease sensitivity of influenza A, B, and C viral RNAs Influenza virus Addi- A B C tion Counts/ Counts/ Counts/ % % mina min min None 7, , , RNase DNase 7, , , ' Acid-precipitable counts.

3 740 RITCHEY, PALESE, AND KILBOURNE RNase but not by DNase. The high salt concentration used for the RNase treatment stabilizes double-stranded RNA and then makes it resistant to pancreatic RNase A digestion. Although no attempts were made to melt or reanneal the RNA, this result strongly suggests that the genome of influenza C virus is single-stranded RNA. Base composition of the RNAs isolated from influenza A, B, and C viruses. The RNAs of the three virus strains were hydrolyzed and the nucleotide monophosphates were analyzed by paper electrophoresis. A comparison of the values in Table 2 shows that the base compositions of influenza A, B, and C virus RNAs are very similar. The data obtained from influenza A/PR/8 virus RNA are virtually identical to those published by other laboratories (1, 7, 15, 26). The base composition data of influenza B/ Lee virus RNA differ from an earlier report in that the guanosine content given is 4% higher and the uracil content is 2% lower (26). The influenza C virus RNA has a base composition which excludes the presence of large double-stranded RNA stretches, an observation consistent with the nuclease digestion experiments (Table 1). It seems unlikely that the different guanine plus cytidine/adenine plus uracil ratios of influenza A, B, and C virus of 1.23, 1.25, and 1.38, respectively, reflect significant differences in base composition. Analysis of other influenza C virus strains should give a more complete picture. So far the base composition data clearly show that influenza C/GL virus has a single-stranded RNA genome and is very similar to influenza A and B viruses. Comparison of influenza A, B, and C viral RNAs on polyacrylamide gels. Figure 1 shows the RNA analysis of the three virus strains using a 2.8% polyacrylamide gel containing 6 M urea. Clearly, eight distinct RNA bands of influenza A/PR/8 virus are seen (Fig. 1, lane 2). This pattern is identical to the one reported for PR/8 virus grown in the chicken embryo chorioallantoic membrane system (20). Next to it on lane 3 the RNA pattern of J. VIROL. influenza B/Lee virus is shown. Except possibly for band 1, all the bands show a migration pattern different from the one obtained for influenza A/PR/8 and again, eight distinct bands can be resolved. Lane 4 shows the RNA segments of influenza C/GL virus. The RNA ofthis virus can only be separated into four distinct segments. At least three out of the four RNA bands migrate differently from the RNA pieces of influenza A/PR/8 and B/Lee virus. Using the values of 1.9 x 106 (17), 0.71 x 106, (17) 1.07 x 106 (28), 0.55 x 106 (28), and 2.6 x 104 for the molecular weights of the rrnas and trnas from mammalian cells (285, 18S) and from E. coli 23S, 16S, and 4S, it was possible to establish a standard curve. The molecular weights of the different influenza virus RNA segments were determined according to these marker RNAs (Table 3). If it is assumed that infectious particles contain one copy of each RNA segment, the total molecular weight of the RNA of influenza A/ PR/8 virus adds up to 5.29 x 106 and that of influenza B/Lee virus to a slightly higher value of 6.43 x 106. In the case of influenza C virus the total molecular weight of the four bands adds up to 4.67 x 106, assuming that each RNA segment is present in equimolar concentrations in an infectious virus particle. However, as can be seen from Fig. 1, the segments of influenza C virus are not present in equimolar concentrations in this experiment, the molar ratios being 0.27, 1, 0.76, and 0.32 for bands 1, 2, 3, and 4, respectively. Molar ratios were determined by cutting out the radioactive band, measuring the amount of radioactivity in a scintillation counter, and dividing the value by the molecular weight of the particular RNA segment. To calculate an accurate total molecular weight for the RNA of influenza C virus the molar ratios of the RNA segments were determined for five separate preparations. For all preparations, essentially the same ratios were obtained regardless of whether 102 or 106 EIDso of virus were used for the inoculum. This indicates that the molar distribution of segments is reproducible a b TABLE 2. Base composition of influenza A, B, and C viral RNAs Influenza virus Base A B C Avg, expt 2 + range Avg, expt 2 + range Avg, expt 3 + SDa Cytidine 23.6b ± ± 1.1 Adenine , ± 1.8 Guanine 21.2 ± ± ± 2.5 Uracil 31.8 ± ± SD, Standard deviation. Percentage.

4 E. coii A B C I 2 3 S 16 S 1 4ql f M0JOW' FIG. 1. Polyacrylamide gel electrophoresis of influenza A, B, and C virus RNAs. RNA was isolated from purified virus and electrophoresed as described in text. Left to right: (lane 1) Escherichia coli ribosomal RNA, 16 and 23S; (lane 2) influenza AIPRI8134 virus; (lane 3) influenza B/Lee/40 virus; (lane 4) influenza CIGLI 1167/54 virus. The arrow marks the origin of the gel. Note; in an earlier paper (20) the bands of influenza A! PR/8/34 virus RNA are labeled from top to bottom as 1, 2, and 2A through 7 instead of1 through 8 as labeled here. 741

5 742 RITCHEY, PALESE, AND KILBOURNE and does not depend on the virus inoculum. The nonequimolar distribution of RNA segments of influenza C/GL virus could therefore mean that bands 2 and 3 consist of more than one species which have not been resolved by the polyacrylamide gel system. Clearly, further work is needed to solve this problem and the 4.67 x 106 molecular weight of influenza C virus RNA probably represents a minimum value. DISCUSSION The RNase sensitivity of influenza C virus RNA shows clearly that this virus carries single-stranded RNA in its genome. Five percent of 32P-labeled RNA is not digested by ribonuclease A, but this is also true of the RNAs of influenza A and B viruses, for which the singlestranded nature of the RNA is firmly established. The similar base composition of the RNAs of TABLE 3. Molecular weights of the RNAs of influenza AIPRI8, B/Lee, and CIGL viruses Band A/PR/8/34 B/Lee C/GL/1167/ X 10" 1.11lx 10 2X 10" x x x 10" x x 10" 1.07 x x 10" 0.81 x 10" 0.49 x x 10" 0.74 x 10" x x x x x 10" 0.5 x 106 J. VIROL. influenza A, B, and C viruses examined suggests that these viruses can be classified together. Furthermore, comparison of the base composition of the influenza viruses with those of other single-stranded RNA animal viruses demonstrates not only that viral genomes can be distinguished by the analysis but that influenza viruses stand apart in their high percentage of uracil (greater than 30%) (Table 4). It should be mentioned that an exception to this has recently been observed. A fish rhabdovirus has been isolated which has a high UMP content (24) and an overall composition different from VSV virus (Table 4). Of the other groups, only the parainfluenza viruses, a genus with characteristics similar to the influenza viruses, approach this percentage of uracil. Influenza C virus, on the basis of its high percentage of uracil and low percentage of guanine, is most likely a member of the influenza rather than the parainfluenza virus genus. It should be noted that in the past, base composition analysis has been a useful tool in virus classification (3, 19). Newman et al. (19) were able to distinguish six different groups of picornaviruses by using this technique. It should also be noted that the base compositions of influenza A, B, and C viruses and other viruses are easily distinguished from contaminating host cellular RNA which has a base composition of 29.0% cytidine, 20.1% adenine, 30.9% guanine, and 20.0% uracil (25). Polyacrylamide gel electrophoresis showed TABLE 4. Base composition of single-stranded RNA viruses Base Genus Virus Reference C A G U Enterovirus Polio, type Rhinovirus Human, type Calicivirus Vesicular, exan thema A Cardiovirus Encephalomyocar ditis Aphthovirus Foot and mouth disease, type 0 Equine rhinovi- Equine, NM rus Alphavirus (to- Sindbis gavirus) Rhabdovirus VSV Leukovirus Rous sarcoma, Bryan Parainfluenza NDV, L-Kansas virus Influenza virus Influenza A/PR/8/ Present data 34 (HON1) a Abbreviations: C, Cytidine; A, adenine; G, guanine; U, uracil; VSV, vesicular stomatitis virus; NDV, Newcastle disease virus.

6 VOL. 18, 1976 that influenza A/PR/8 and B/Lee viruses each possess a segmented genome of at least eight pieces. Similar analysis of the influenza C/GL virus RNA revealed at least four different segments. It is not clear if bands 2 and 3 of influenza C/GL virus consist of more than one species or if there are more than four segments. It is highly unlikely that the bands which have been resolved so far represent breakdown products and that the influenza C virus genome is nonsegmented. Several preparations of influenza C/GL virus and one preparation of the closely related strain influenza C/1233 showed the same RNA pattern on polyacrylamide gels as shown in Fig. 1. It is unlikely that different preparations would always give identical breakdown products. Another possibility is that the smaller pieces represent incomplete genomes derived from defective particles. Even though in the amniotic system the ratio of EIDso to HA units is high, this possibility cannot be excluded. Experiments are in progress to examine the RNA of influenza C virus grown in another cell system. Definitive evidence for the segmented nature of the influenza C virus genome, however, can only be obtained by oligonucleotide sequence analysis of the different pieces (11). The calculation of the total molecular weights of 5.29 x 106, 6.43 x 106, and 4.67 x 106 for the RNAs of influenza A/PR/8, B/Lee, and C/GL viruses is based on the assumptions that all RNA segments have been separated, that the infectious virus particles contain one and only one copy of each segment, and the molecular weight determinations on polyacrylamide gels are accurate. Until these assumptions are proven true the values of the total molecular weights must remain rough estimations and further work is needed to arrive at accurate values. Influenza C virus is capable of enzymatically destroying the receptors on erythrocytes, but the viral receptor-destroying enzyme, which was first studied by Hirst (10), is most likely not a neuraminidase (13). Although the presence of a neuraminidase has always been thought of as characteristic for the group of influenza viruses, the data presented here suggest that influenza C virus, despite its lack of neuraminidase, belongs to the genus of influenza viruses. To the evidence for similar morphology, biology, host range, and disease production, we now add evidence for chemically similar genomes (on the basis of viral RNA base composition and segmentation of the RNA). These data support the present classification of influenza C virus in the influenza virus genus. Experiments are now underway to determine RNAs OF INFLUENZA VIRUS 743 whether the RNA of influenza C virus (and also of influenza B virus) is of negative (nonmessage) polarity similar to the RNA of influenza A viruses. 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