Cell,

Vol. 10, l-10,

January

1977,

Copyright

0 1977 by MIT

The Genes of Influenza

Peter Palese Mount Sinai School of Medicine New York, New York 10029

of CUNY

In 1931 influenza viruses were first isolated from pigs (Shope, 1931), and subsequently the first human influenza virus isolates were obtained (Smith, Andrewes, and Laidlaw, 1933). This marked the beginning of modern influenza virus research, and from then on many scientists have been fascinated by this virus and the disease it causes. Unlike any other known virus, influenza A virus undergoes dramatic changes with respect to its surface antigens, and whenever these changes take place (recently every lo-15 years new subtypes emerged), the virus causes severe pandemics in the human population (Table 1). The most dramatic example of such a pandemic was the 1918 influenza virus outbreak, which was estimated to be directly or indirectly responsible for the death of 20 million people worldwide. But even between pandemic periods, the virus changes constantly and thereby successfully overcomes attempts to protect people fully by vaccination. By the time vaccines are ready for distribution, the virus has changed significantly with respect to its surface antigens, and vaccines, prepared with slightly different strains, become less effective against the new variant. This phenomenon of constant antigenic change and the fact that different influenza viruses undergo extensive genetic interactions (recombination) have generated a deep interest in the peculiar genetic makeup of influenza viruses. The purpose of this paper is to review briefly the current state of knowledge of influenza virus genetics, with particular emphasis on recent findings regarding the characterization and function of individual genes and their gene products. influenza A, 6, and C Viruses influenza viruses exist as three different types-A, B, and C. Of these, the influenza A viruses cause generally the most severe disease in humans and have been analyzed biochemically to the greatest extent. Influenza A viruses share immunologically related internal proteins (nucleoprotein and membrane protein) which can be differentiated from those of influenza B and C viruses. These internal antigens provide the basis for their classification into types. Although pleomorphic and long filamentous forms have been observed, in most instances influenza viruses appear roughly spherical in shape (diameter approximately 100 nm) with characteristic spikes projecting from the surface. Influenza A and B viruses-the C types have not been as well characterized-consist of approxi-

Virus

mately 1% RNA, 7% carbohydrate, 22% lipid, and 70% protein. Figure 1 shows a schematic view of an influenza virus characterized by its spikes, the lipid membrane, an underlying protein layer, and the internal ribonucleoprotein complex. Such a model is generally accepted for influenza A and B viruses; although influenza C viruses seem to possess a similar morphology in the electron microscope (Flewett and Apostolov, 1967), not much is known about the localization of the proteins within the virion and how influenza C viruses compare to the A and B types with respect to their biochemical structure (Kendal, 1975). The RNAs of Influenza Virus Analysis of the nucleic acid of influenza A viruses demonstrated the presence of single-stranded RNA of several million daltons in the virion (Ada and Perry, 1954; Frisch-Niggemeyer and Hoyle, 1956). The observation of high rates of genetic recombination after co-infection of cells with genetically distinct influenza viruses (Burnet and Lind, 1949; Simpson and Hirst, 1961; Hirst 1962) later led to the proposal that influenza viruses possess a segmented RNA genome, and that the frequent exchange of genetic information reflects the reassortTable

1. Major

Antigenic

Subtypes

of (Human)

Year

Influenza

A Viruses

SubtVDe’

Around

1918

(H~wlNl)~

1929-1947

(Swine)

HONl

1946-1957

HlNl

1957-l

H2N2

(Asian)

H3N2

(Hong

966

1966a Serologic differences and neuraminidase (N) ruses (Chanock et al., b No virus isolates from able.

Kong)

of the surface proteins hemagglutinin determine the subtype of influenza 1971). this period, only serologic evidence

(H) A viavail-

Hemagglutlnln; Neuromlnldose

; LLlpld

membrane Matrix

protein

Nucleoproteln; P proteins RNA Figure

1. Schematic

The particles glutinin and protein layer,

Diagram

are characterized neuraminidase, and the internal

of Influenza

A Virus

by spikes consisting of hemaga lipid membrane, an underlying ribonucleoprotein complex.

Cell 2

ment of individual RNA segments (Burnet 1956; Hirst 1962). This concept was supported by the finding that the RNA of influenza virions is indeed segmented when analyzed by polyacrylamide gel electrophoresis (Duesberg, 1968; Pons and Hirst, 1968). It should be noted that in the influenza virus literature, the term recombination is used for reassortment of genes, and that recombination in the true genetic sense, which requires the breakage and reunion of nucleic acid molecules, has not been observed with influenza viruses. Recent technical developments in the analysis of RNAs have helped to define the number of individual RNA segments. Three different groups independently found that influenza A viruses possess eight RNA genes (Pons, 1976; Bean and Simpson, 1976; Ritchey, Palese, and Kilbourne, 1976a; Palese and Schulman, 1976b, 1976c). This is based on the fact that eight virus-specific RNA segments have been identified in the virion, as well as in infected cells, and that all eight genes have been mapped (see below). The finding that influenza virus consists of eight distinct RNAs was later also confirmed by McGeoch, Fellner, and Newton (1976). Earlier reports, which postulate 5-7 RNA pieces for influenza A viruses, are therefore most probably incorrect. The finding that influenza WSN virus grown under high multiplicities of infection may possess up to nine RNA segments probably reflects the appearance of defective genes, which are only seen when many defective particles are produced (Palese and Schulman, 1976a). Not much is known about the structure of individual segments. Young and Content (1971) showed that three size classes of virion RNAs have pppAp at their 5’ terminal end, and Lewandowski, Content, and Leppla (1971) found unphosphorylated uridine at the 3’ terminal as the only base. Furthermore, the data of Content and Duesberg (1971), Horst et al. (1972), and subsequently of McGeoch et al. (1976) indicate that different RNAs possess different nucleotide compositions and different oligonucleotide maps. All these results clearly support the notion that the virion RNAs are physically separate, that they are not derived from a large precursor, and that they represent unique genes. Preliminary investigation of the polarity of the influenza virus virion RNA resulted in controversy. Siegert, Bauer, and Hofschneider (1973) reported that RNA isolated from influenza A/PR/8/34 virus directed the translation of virus-specific proteins in a cell-free system, a finding which could not be verified by other investigators (Kingsbury and Webster, 1973). At present, the overwhelming evidence suggests that influenza A viruses contain virion RNA which is not mRNA. First, influenza viruses contain a virus-associated RNA polymerase

(Chow and Simpson, 1971; Simpson and Bean, 1975). Second, it has been found that purified RNA from infected cell polysomes completely protects virion RNA after hybridization, which suggests that virion RNA is complementary to mRNA isolated from polysomes (Etkind and Krug, 1975; Glass, McGeoch, and Barry, 1975). Third, RNA from polysomes directs the synthesis of virus-specific proteins in eucaryotic cell-free systems, whereas RNA from highly purified virus does not (Etkind and Krug, 1975; Ritchey and Palese, 1976; Content, 1976). In several instances, virion RNA has been shown to stimulate nonspecifically cell-free protein synthesis systems, but no viral proteins were made (Tekamp and Penhoet, 1976). Finally, only virusspecific RNA from polysomes but not virion RNA contains 7-methyl-G capped 5’ terminals (Krug, Morgan, and Shatkin, 1976) and poly(A) at the 3’ end (Etkind and Krug, 1974), which also suggests that virion RNA is not mRNA. These data together identify influenza A viruses as negativestrand viruses according to Baltimore’s classification. Much less is known about the RNAs of influenza B and C viruses. Genetic evidence was presented that influenza B viruses also undergo genetic recombination similar to influenza A viruses (Tobita and Kilbourne, 1974). In addition, the base composition of influenza B virus RNA is virtually identical to that of influenza A viruses (Schuster, 1960; Ritchey et al., 1976a), suggesting that influenza B virus RNA may be segmented as well. This was verified by analysis on polyacrylamide gels which showed that influenza B virus also contains eight RNA segments (Figure 2) (Ritchey et al., 1976a). As shown in Figure 2, the RNAs of influenza B/Lee/40 virus differ significantly, however, in their migration pattern from that of influenza A/PR/8/34 virus RNAs. Finally, influenza C viruses also contain a segmented RNA genome. A comparison with the RNA pattern of influenza A and B viruses shows that influenza C viruses possess at least four RNA segments (Figure 2) (Cox and Kendal, 1976; Ritchey et al., 1976a). It is not clear at this time whether influenza B and C viruses are also negative-strand RNA viruses. Genetic Map of Influenza Virus Correlations in size of the RNAs and proteins of influenza A viruses have been used as a basis to speculate which gene codes for a particular viral protein. Recently, however, we showed that this approach is not feasible because different influenza viruses possess different genetic maps, and the apparent size of RNA molecules does not always correlate with the size of the proteins for which they code. In the following, I describe briefly our approach

The Genes 3

of Influenza

Virus

which led to the elucidation of the complete genetic map of two different influenza viruses. Polyacrylamide gel electrophoresis of the RNAs of influenza A/PR/8/34 virus (HONl) (PR8 virus) and of A/ Hong Kong/a/68 virus (H3N2) (HK virus) revealed first, that eight RNA segments of each virus can be identified, and second, that corresponding RNA segments show a different migration rate. This is not surprising, since PR8 virus and HK virus were first isolated more than 30 years apart. To identify

E.coli

A

B

C

23 S

specific genes and their gene products, recombinant viruses were obtained after co-infection of cells with PR8 and HK virus, and analyzed with respect to the derivation of their RNAs and proteins. A schematic illustration of this strategy is shown in Figure 3. For example, by selecting a virus with antiserum against PR8 hemagglutinin and the HK neuraminidase, a recombinant was isolated which contained the HK hemagglutinin and the PR8 neuraminidase (HK-PR8 virus). After ultraviolet light irradiation of the HK parent, most viable recombinants had derived only the hemagglutinin from the HK parent (because of selection with antiserum) and all other genes from the PR8 parent not treated by ultraviolet light. Conversely, the PR&HK recombinant, containing the PR8 hemagglutinin and the HK neuraminidase, was obtained from HK virus and ultraviolet-irradiated PR8 virus after selection with antiserum against the HK hemagglutinin and the PR8 neuraminidase. Analysis of the RNAs of the recombinant viruses clearly showed that in each case only RNA 4 (counting the slowest moving segment RNA 1) was exchanged, indicating that RNA 4 codes for the hemagglutinin (Figure 4; HK- PR8

PR8

IP-

IP-

2P-

2P-

3P-

3P-

----

4P-HA ---..._---..__

4HLHA

16S

7

5P-

5P-

6P-

6P-

7P-

7P-

8P-

8P-

HK

IH-

IH-

2H-

2H-

3H,c__...--.....__. .___. -__ 4H LHA

Figure 2. Polyacrylamide and C Virus RNAs

Gel Electrophoresis

of Influenza

A, B.

Lane 1: E. coli ribosomal RNA (23s and 16s); lane 2; RNA of influenza A/PR/8/34 virus; lane 3: RNA of influenza B/Lee/40 virus; lane 4: RNA of influenza C/GL/1167/54 virus. Influenza A and B viruses each contain eight RNA segments; influenza C virus possesses at least four RNAs. From Ritchey et al. (1976a).

3. Strategy

3H _.__ -----.~ 4P-HA

5H-

5H-

6H-

6H-

7H-

7H -

BHFigure

PR8-HK

to Map the Genome

8Hof Influenza

Virus

Analysis of the RNAs and proteins of two recombinants and their parent viruses permits the identification of individual genes. The RNA segments of influenza AIPR16/34 and AIHK/6/68 viruses (PR6 and HK viruses) and their recombinants HK-PR6 (HK hemagglutinin and PR8 neuraminidase) and PRB-HK (PR8 hemagglutinin and HK neuraminidase) are schematically drawn as dark lines. The letter P or H next to the RNA segments (numbered l-8) identifies them as PR8 or HK genes, respectively. The HK-PR8 recombinant contains all genes from the PR8 virus except for RNA 4, which is derived from HK virus. This makes it very probable that gene 4 of the recombinant codes for the HK hemagglutinin. The reverse recombinant PRB-HK derives all genes from HK virus, except the fourth RNA which is derived from PR8 virus. Again, this identifies RNA 4 as the hemagglutinin gene.

Cell 4

HKPR8 PR8

PR8HK HK

Palese and Schulman, 1976b). The remaining genes of the recombinants were clearly derived from the parent which was not treated by ultraviolet light (Figure 4). Using the same technique, it was found that the fifth RNA of HK virus codes for the HK neuraminidase, but the sixth RNA of PR8 virus codes for its respective neuraminidase (Palese and Schulman, 1976b) (Figure 6). A more complicated approach had to be used to identify the remaining six genes. Analysis of many recombinant viruses derived from HK and PR8 viruses were necessary to obtain recombinants which were identical with one of the parents except for one (or few) RNA segments. Figure 5 shows, for example, the RNA pattern of three recombinants which permitted the identification of the genes coding for nucleoprotein (NP), membrane protein (M), and nonstructural protein (NS) (Ritchey, Palese, and Schulman, 1976b). Recombinant number 1 in Figure 5 is identical with HK virus except for RNA 8; analysis of the proteins of the recombinant and the two parent viruses on gradient polyacrylamide gels (results not shown) revealed that recombinant number 1 possesses only the nonstructural protein from PR8 virus, but that all other proteins were derived from HK virus (Ritchey et al., 1976b). This identifies RNA 8 as the gene coding for the nonstructural protein NS. A similar analysis of recombinants number 2 and number 3, whose RNA patterns are shown in Figure 5, and of other recombinants revealed the genes coding for membrane protein, nucleoprotein, and the three P proteins. Figure 6 shows the RNA genome of PR8 and HK virus and the successful identification of all eight genes, including the RNAs coding for the three P proteins, Pl, P2, and P3 (Palese, Ritchey, and Schulman, 1977a; Ritchey et al., 1977). It should be noted that RNA 5 of HK virus codes for its neuraminidase, but RNA 5 of PR8 virus codes for its nucleoprotein, and that the slowest moving RNA (RNA 1) of both viruses codes for the third largest protein, P3, and not for the largest protein, Pl. Recently, McGeoch et al. (1976) reported a genetic map of influenza virus based on unpublished experiments proposing that RNA 1, 2, and 3 code for Pl, P2, and P3 proteins, respectively. This Figure

4. Mapping

of the Hemagglutinin

Gene

The RNAs of influenza AIPRkTl34 and AIHKl8l60 virus (PRB and HK virus) and of two recombinants derived from them are analyzed on polyacrylamide gels. (For explanation, see Figure 3 and text.) Lane 1: HK-PRB recombinant deriving all genes from PR8 virus except RNA 4 (the hemagglutinin gene, arrow); lane 2: PR8 virus; lane 3: HK virus; lane 4: PRI-HK recombinant deriving all genes from HK virus except RNA 4 (the hemagglutinin gene, arrow). It should be noted that RNA 7 of PRB and HK viruses are not separated in this particular gel. The letter P or H next to the RNA segment identifies it as a PR8 or HK gene, respectively. From Palese and Schulman (1976b).

The 5

Genes

of Influenza

Virus

PR8 HK #I

#2

#3

PR8

is in contrast to our data concerning map of human and animal influenza

the genetic viruses.

Comparison of the RNA Patterns of Different Influenza A Viruses Careful analysis of the RNAs of PR8 and HK virus revealed that each gene has a characteristic migration rate on polyacrylamide gels. Consequently, it was possible to identify recombinant viruses derived from HK and PR8 viruses with respect to the derivation of all eight genes (Palese and Schulman, 1976a, 1976b; Palese et al., 1976; Ritchey et al., 1976b, 1977; Schulman and Palese, 1976). This technique therefore extends our ability to characterize recombinant viruses far beyond pre-

P3 PROTEIN PI PROTEIN P2 PROTEIN

H~~AGGL~T~N~~ :i” NUCLEOPROTEI N NEURAMINIDASE

Figure

6. Complete

HEMAGGLUTININ MEURAMINIRASE NUCLEOPROTEIN

tr ‘,,

Map of the Influenza

Virus

Geome

The RNAs of influenza AIPR16/34 and A/HK/8/68 viruses (PR6 and HK viruses) were separated on a polyacrylamide gel as shown in Figures 2,4, and 5. All eight RNA segments have been identified with respect to their gene products. From Ritcheyet al. (1976b). Figure 5. Polyacrylamide Gel RNA Analysis of Influenza A/PR/W 34 and AIHK/8/68 Viruses and Three Recombinant Viruses Derived from Them Lanes 1 and 6: PR6 virus; lane 2: HK virus: lane 3: recombinant virus number 1, identical with HK virus except for RNA 8; lane 4:

recombinant virus number 2 identical with HK virus except for RNAs 4 and 7; lane 5: recombinant virus number 3 identical with HK virus except for RNAs 4,6,7, and 8. The letter P or H next to an RNA segment identifies it as a PRB or HK gene, respectively. From Ritchey et al. (1976b).

Cell 6

vious methods which were restricted to the antigenie characterization of the surface proteins hemagglutinin and neuraminidase or to the identification of some individual biochemical markers (Bean and Simpson, 1975; Laver and Downie, 1976). RNA analysis of many of the 256 possible recombinants of PRB and HK viruses revealed recombinants with very interesting gene combinations. For example, two PRB-HK recombinants with identical surface proteins (PRB hemagglutinin and HK neuraminidase) were isolated, one of which contained all remaining six genes from PRB virus, and in contrast, the other derived the remaining six genes from HK virus (Schulman and Palese, 1976). Parenthetically, it is worth noting that we never observed the existence of influenza virus heterozygotes. Recombinants of other human and animal influenza viruses also have been identified with respect to the derivation of their genes (M. B. Ritchey and P. Palese, unpublished data). It therefore seems that this technique will be quite useful in identifying all kinds of influenza viruses and their recombinants, and in helping us to correlate biological properties with specific genes. An interesting application of this technique permitted the analysis of the RNA pattern of the New Jersey “swine” influenza virus. In February 1976 at Fort Dix, New Jersey, influenza viruses were isolated from recruits. The isolates were later shown to resemble influenza viruses isolated from pigs with respect to their surface antigens hemagglutinin and neuraminidase. Alarmed by the possibility that this episode might forecast the next pandemic, the U. S. government launched a massive vaccination program against “swine” influenza. RNA analysis of the human New Jersey “swine” virus and comparison of its RNAs with those of true swine viruses and of human influenza virus strains isolated at the same time revealed that the New Jersey “swine” virus is almost identical to other swine viruses with respect to all of its eight RNAs, but very much different from the current human isolates (Figure 7; Palese and Schulman, 1976c). This would indicate that the New Jersey “swine” virus does not contain genes from the current human Figure 7. Comparison of the RNA “Swine” Influenza Virus (A/NJ/ii/76) Isolate and That of a Swine Influenza

Pattern of the New Jersey with That of a Victoria-Like Virus

RNAs were electrophoresed on polyacrylamide gels. Lane 1: influenza AINJ/743/76 (H3N2) virus, a Victoria-like isolate, obtained at Fort Dix, New Jersey; lane 2: New Jersey “swine” influenza A/NJ/ 11776 (HswlNl) virus; lane 3: influenza A/Swine/Taiwan/l/75 (HswlNl) virus, an isolate obtained from pigs. Arrows identify the hemagglutinin and neuraminidase genes. There are remarkable similarities between the RNA patterns of the New Jersey “swine” virus and that of an animal strain obtained from pigs, but great differences between the RNA patterns of the Victoria-like A/NJ/ 743176 (H3N2) virus and that of the New Jersey “swine” influenza virus. From Palese and Schulman (1976c).

NJ/76 (H3N2)

NJ/76 (SW)

Tail75 (SW)

The Genes

of Influenza

Virus

strain and therefore is not a recombinant derived from swine viruses and the current Victoria (H3N2) isolates. Based on popular theories that new pandemic strains emerge through recombination of animal and human strains (Kilbourne, 1968; Laver and Webster, 1973), it seems improbable that this New Jersey “swine” virus will cause the next influenza virus pandemic. Although it is conceivable that animal strains acquire virulence for man by mutation, it should be pointed out that the viruses from pigs, which resemble the New Jersey “swine” virus, have been isolated years before 1976 and have not caused a pandemic since that time. It is therefore most probable that the occurrence of New Jersey “swine” virus in humans in Fort Dix represents an isolated event without serious consequences. Reports have also appeared that occasionally laboratory personnel, veterinarians, or abattoir workers, who were in contact with swine virus, contracted the disease (Kluska, Macku, and Mensik, 1961), transmitted the disease (Kluska et al., 1961), or showed a rise in serum antibody titers against swine influenza virus (Schnurrenberger, Woods, and Martin, 1970). To date, no epidemics have resulted from these recent occurrences of swine viruses in man. Temperature-Sensitive Mutants The study of conditional lethal mutants provides the molecular biologist with a most useful tool for elucidating biochemical steps involved in virus replication. This is the reason why many different groups attempted to isolate such mutants from influenza viruses. Although influenza virus is known to form deletion defective mutants (von Magnus particles), only temperature-sensitive mutants have been successfully used to characterize the influenza virus genome (Simpson and Hirst, 1968; Mackenzie, 1970; Ueda, 1972; Sugiura, Tobita, and Kilbourne, 1972; Hirst, 1973; Markushin and Ghendon, 1973; Scholtissek and Bowles, 1975; Spring et al., 1975; Sugiura et al., 1975). Most data suggested that there are 5-8 nonoverlapping recombinationcomplementation groups, and the working hypothesis was that mutants in each group possess a defect in one RNA segment only. Physiological characterization of the available mutants which were derived from different influenza virus strains revealed that at least four mutant groups have defects in virus-specific RNA synthesis (RNA-) (Ghendon et al., 1975; Sugiura et al., 1975; Scholtissek and Bowles, 1975). A more detailed examination of the WSN virus mutant collection isolated by A. Sugiura, K. Tobita, M. Ueda, and E. D. Kilbourne demonstrated that at least two of these four RNA- gene functions are required for complementary RNA synthesis (genes defective in groups I and Ill) (Krug, Ueda, and Palese, 1975). In addition, mutants in

group I were shown to possess a temperaturesensitive RNA polymerase activity in vitro (Mowshowitz and Ueda, 1976). Defects of mutants belonging to the remaining two RNA- groups (groups II and V) are most probably associated with virion RNA synthesis (Krug et al., 1975). Further analysis of this mutant collection and of fowl plague virus mutants revealed that one group each is defective in hemagglutinin and neuraminidase, respectively (Palese et al., 1974; Scholtissek and Bowles, 1975; Ueda and Kilbourne, 1976). All these experimental findings were compatible with the postulate that each mutant group was defective in one monocistronic RNA segment coding for an individual virusspecific protein. Definite proof for this hypothesis was recently obtained by identifying the defective genes and gene products of all seven WSN virus mutant groups originally isolated by A. Sugiura and his co-workers (Palese, Ritchey, and Schulman, 1977b; Ritchey and Palese, 1977). The experiment was performed as follows. Temperaturesensitive mutants were grown at nonpermissive temperature with a “rescuing” virus strain which was not temperature-sensitive. Recombinants which formed consist of genes derived from the temperature-sensitive mutant and one or a few genes derived from the “rescuing” virus. Because such recombinants were selected at nonpermissive temperature, the temperature-sensitive gene of the mutant was replaced by the wild-type gene of the “rescuing” virus. RNA analysis of several recombinants permitted the identification of the temperature-sensitive genes and their gene products, because all genes of the “rescuing” virus had previously been mapped by established procedures (Palese and Schulman, 1976b; Palese et al., 1977a; Ritchey et al., 197613, 1977). Identification of the defective genes of all seven complementation-recombination groups revealed that PI and P3 proteins are necessary for complementary RNA synthesis (Palese, Ritchey, and Schulman, 1977b), whereas P2 protein and NP are most probably associated with the synthesis of virion RNA (Ritchey and Palese, 1977). Finally, it was shown that mutants in group VII have a defect in the M protein (Ritchey and Palese, 1977). No mutant group has been identified as yet with a defect in the eighth viral protein (NS protein). Table 2 summarizes the physiological defects observed with the seven complementation-recombination groups of WSN virus mutants and shows the correlation of physiological defects with individual gene products. By analyzing the RNAs of wild-type recombinants derived from “mapped” influenza virus strains and other mutants, it is now feasible to compare mutant virus groups from different laboratories and to clarify which genes are defective. The identification of

Cell 0

defective genes will be particularly important for mutants which serve as candidates for attenuated live influenza virus vaccines such as those developed by Chanock and his co-workers (Murphy et al., 1976) and by Maassab (1969). Virulence Genes Clearly, a complete discussion of virulence of influenza viruses on a molecular basis must await further developments in our understanding of this virus. It is also obvious that conclusions reached for one host virus system may not be valid for another, and that much more work is required to identify the gene(s) responsible for transmissibility or viral pathogenicity as such. Nevertheless, progress has been made recently to evaluate individual gene products with respect to their contribution to virulence.lt was shown that the surface glycoprotein hemagglutinin must be cleaved into two subunits for optimal infectivity of the virus (Lazarowitz and Choppin, 1975; Klenk et al., 1975). This phenomenon may in part explain the cell tropicity of influenza viruses. Less clear results have been obtained for the neuraminidase. Although temperature-sensitive defects in neuraminidase (Palese et al., 1974a; Scholtissek and Bowles, 1975) and inhibition of neuraminidase by chemicals (Meindl et al., 1971; Palese et al., 1974b; Schulman and Palese, 1975; Palese and Compans, 1976) are both associated with inhibition of virus replication, there is no clear correlation between virulence and neuraminidase activity. Padgett and Walker (1964) described a relationship between neuraminidase ac-

Table 2. Characterization Influenza A/WSN Virus

of Temperature-Sensitive Mutants”

Mutants Defective

Defect

Protein

Groups

Physiological

I

Complementary sisb,c

II

Virion

Ill

Complementary sisb

IV

Neuraminidase activity, formation of neuraminic acid containing virus aggregates, autoaggregation*

Neuraminidased

V

Virion

Nucleoproteinh

VI

Hemagglutinin

VII

No defect in RNA synthesis’

RNA synthe-

RNA synthesis RNA

RNA synthesis

(?)” synthe-

(?)b

activitr

P2 protei+ Pl proteins

HemagglutinirF

virus-specific

a Isolated by Sugiura et al. (1972, b Krug et al., 1975. c Mowshowitz and Ueda, 1976. d Palese et al.. 1974. e Ueda and Kilbourne, 1976. r Sugiura et al.. 1975. B Palese et al., 197713. h Ritchey and Palese. 1977.

P3 protein%

1975).

M protein”

of

tivity and growth rate of influenza viruses, but Mayer, Schulman, and Kilbourne (1973) found no linkage of neurovirulence either to hemagglutinin or neuraminidase. Similarly, the rate of replication of antigenically identical viruses with 8-10 fold differences in neuraminidase depended only on the host system used and did not correlate with neuraminidase activity (Palese and Schulman, 1974). Besides the surface proteins hemagglutinin and neuraminidase, some or all of the remaining six influenza virus genes contribute to virulence. This, for example, has been clearly demonstrated with recombinant viruses which share the hemagglutinin and neuraminidase with one parent but derive genes coding for nonsurface proteins from the good growing laboratory strain A/PR/8/34 (Kilbourne, 1969). These recombinants show some of the growth and virulence characteristics of the A/ PRl8l34 virus without sharing its hemagglutinin or neuraminidase (Kilbourne et al., 1971; Beare and Hall, 1971). Recently, a recombinant which was derived from the “swine” influenza virus A/NJ/II/ 76, isolated at Fort Dix, New Jersey, and influenza A/PR/8/34 virus was shown by RNA analysis to derive six genes from the A/PRl8/34 virus and only the hemagglutinin and neuraminidase genes from the “swine” virus (Palese et al., 1976). This recombinant virus, used as a vaccine strain, grows to a 32 fold higher titer in the allantoic cavity of eggs than the “swine” virus parent. This provides direct evidence, therefore, that genes coding for nonsurface proteins contribute to virulence and growth characteristics. Similar conclusions that the surface proteins do not by themselves determine the virulence of influenza viruses in animals have been obtained by Rott, Orlich, and Scholtissek (1976). Together these results confirm the original hypothesis that influenza virus pathogenicity is of polygenie nature (Burnet, 1959). Clearly, we are now in a position to ask which gene(s) is involved during different replication steps and to identify genes contributing to virulence in a particular host system. Furthermore, attempts will be made to understand why some influenza viruses are virulent for man and what the factors are that determine the emergence of new pandemic strains. Summary and Outlook Influenza A viruses were recently shown to contain eight RNA segments which code for the three P proteins, PI, P2, P3, the hemagglutinin, the neuraminidase, the nucleoprotein, the matrix (M), and the nonstructural (NS) protein. A genetic map of the genes coding for these proteins was established, and it is now clear that all eight RNA segments found in the virion are of negative polarity. RNA analysis of wild-type recombinants derived

The Genes 9

of Influenza

Virus

from temperature-sensitive mutants and “mapped” marker viruses permitted the identification of the defective genes in seven complementation-recombination groups. It was found that Pl and P3 proteins are necessary for complementary RNA synthesis, and that P2 protein and NP are most probably associated with virion RNA synthesis. Two mutant groups have defects in the hemagglutinin and neuraminidase molecules, respectively, and one mutant group was found to possess a defective in M protein. RNA analysis was further used to compare different influenza virus strains. Thus it was shown that the New Jersey “swine” influenza virus is most probably not a recombinant virus derived from swine viruses and the current human isolates, but resembles other “true” swine influenza viruses. RNA analysis of different influenza viruses and their recombinants will open new avenues of investigation into the capricious nature of influenza virus. At this time, we know little about the mechanism of recombination of viral genes, and we do not know which enzymatic apparatus is necessary for the synthesis of virus-specific RNAs, including the obscure involvement of the host cell and its nucleus. Theories regarding the origin of influenza viruses are at best unproved at present, and we know practically nothing about the nature of virulence genes. These questions will no doubt be answered as we drift away from classical genetic approaches and move closer to a molecular understanding of influenza virus.

Work in the author’s laboratory was supported by the NIH and the NSF. The author also wishes to acknowledge the pleasant and successful collaboration with Dr. J. L. Schulman and Dr. M. 8. Ritchey on many aspects of the work described here, and to thank Dr. E. D. Kilbourne for many stimulating discussions. The author is also indebted to Dr. M. 6. Ritchey for critically reading this manuscript. I also thank Carol DeBlasio for the excellent typing of this and other manuscripts.

Cox,

Duesberg.

B. T. (1954).

W. J., Jr.. and Simpson, and Simpson,

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F. M. (1959). In The Viruses, 3, F. M. Burnet eds. (New York: Academic Press), p. 275.

123. 1101.

Burnet,

F. M., and Lind.

P. E. (1949).

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