ESCAPE OF A HIGHLY DEFECTIVE INFLUENZA A VIRUS MUTANT FROM ITS TEMPERATURE SENSITIVE PHENOTYPE BY...

ESCAPE OF A HIGHLY DEFECTIVE INFLUENZA A VIRUS MUTANT FROM ITS TEMPERATURE SENSITIVE PHENOTYPE BY EXTRAGENIC

SUPPRESSION AND OTHER TYPES OF MUTATION

Brian R. Murphy,* Mark D. Tolpin, Judith G. Massicot, Hyun Y. Kim, Robert H. Parrott, and Robert M. Chanock

Laboratory of Znfectious Diseases National Institute of Allergy and Infectious Diseases

National Institutes of Health Berhesda, Maryland 20205

and

Children's Hospital National Medical Center 111 Michigan Avenue, N.W.

Washington, D.C. 20010

INTRODUCTION

Genetic instability represents one of the major obstacles impeding current efforts to develop safe, effective mutants for use in live virus vaccines. Although certain temperature-sensitive (1s) and cold-adapted ( c a ) mutants, with defined mutations responsible for attenuation, have been shown to be safe and immunogenic in man, on occasion infected individuals have yielded virus with altered ts or ca This type of genetic instability has been observed most frequently during unrestricted viral replication in a completely susceptible

For example, the influenza A/Hong Kong/68-ts-l [El (H3N2) recombinant, which exhibited ii 38" C shutoff temperature for plaque formation, retained its ts phenotype after replication in adult volunteers who had detectable immunity to the viral neuraminidase surface-glycoprotein but not to the viral hemagglutinin surface-gly~oprotein.~. In contrast, when the ts-1 [El recombinant was given to young vaccinees who had not been previously infected with an influenza A virus, 25% of these individuals shed virus that had lost its temperature sensitivity ( t ~ ) .1

In an attempt to produce a ts virus more stable than the influenza A/Hong Kong/68 ts-1 m] donor, two single-lesion ts mutants, which exhibited an unusual degree of genetic stability. were mated and their rs genes were transferred into a recombinant.? The resulting influenza A/ Udorn/ 72-ts-1A2 recombinant possessed ts mutations on the P3 and P1 polymerase genes and exhibited a 37" C shutoff temperature for plaque f~ rma t ion .~ , * The two mutant genes were then transferred by genetic reassortment to the influenza A/Victoria/ 75 (H3N2) wild-type virus.9 The A/Victoria/75-ts-l A2 recombinant proved to be more attenuated and more stable genetically in hamsters, adults, and children than a series of ts-1 [El recombinants studied previously. (Wright, et al. Un-

* Mailing address: National Institutes of Health, NIAID, LID, Building 7, Room 301, Bethesda, Maryland 20205.

172 0077-8923/80/354-172 $1.75/0 @ 1980, NYAS

Murphy et al.: Escape of Influenza A Virus 173

published observations.) 9-15 The ts P1 and P3 genes from the A/Udorn/72 ts-1A2 donor virus were then transferred to the influenza A/Alaska/77 (H3N2) wild-type virus to determine if they would confer the same satisfactory level of attenuation and genetic stability on another recombinant that possessed contemporary surface antigens.lG The A/ Alaska/77-ts-l A2 recombinant, like the A/Udorn/72-ts-lA2 donor virus and its A/Victoria/75-ts-l A2 derivative, was markedly restricted in its replication in the hamster's lung and did not exhibit evidence of instability of its ts phenotype.lG Subsequently, this recombinant was administered to adult volunteers and was found to be satis- factorily attenuated, immunogenic, and genetically ~tab1e.l~ However, when the A/ Alaska/ 77-ts-1 A2 recombinant was administered to a child who lacked antibody to both the hemagglutinin and neuraminidase glycoproteins, virus with the ts+ phenotype was shed. In this paper we describe the manner in which the highly defective A/ Alaska/ 77-ts-1A2 recombinant escaped its ts phenotype.

METHODS AND MATERIALS

Viruses

The isolation and cloning of the influenza A/Alaska/6/77 (H3N2) wild- type virus has been described.16 The production and characterization of the A/Alaska/6/77-ts-l A2 recombinant has also been described." Virus recovered on the ninth day from a seronegative child infected with the A/Alaska/77-ts- 1A2 recombinant is referred to as isolate FV 1319. Two suspensions of FV 1319 virus were examined: (1) an uncloned suspension of virus isolated in primary calf kidney (BK) monolayer culture (passage history BK-1) and (2) a cloned suspension of virus that underwent plaque (34" C) to plaque (39" C) passage in BK monolayer culture. The latter virus was passaged once more in BK culture and grown in the allantoic cavity of avian leucosis virus-free, specific pathogen-free eggs (Spafus, Inc., Storrs, Conn.) . The cloned suspension of FV 1319 virus was passaged a total of 4 times in BK cells and once in eggs.

Administration of the Znfluenza A/ Alaska/ 77-ts-I A2 Recombinant to Children

The Alaska/77-ts-IA2 virus was administered intranasally at a dose of 106.s TCID,, to a seropositive and a seronegative child as previously described.* Virus was recovered from nasal wash specimens as previously described.2 The studies were conducted at a time when influenza A viruses were not isolated from the community.

Efficiency of Plaque Formation (EOP) and Genetic Analysis

Influenza A virus was titered on Madin Darby canine kidney (MDCK) monolayer cultures grown on 12-well Linbros plastic plates (Flow Labora- tories, McLean, Va.). 1 pg/ml of TPCK trypsin (Worthington Biochemicals,

174 Annals New York Academy of Sciences

Freehold, N.J.) was used in the Liebowitz Medium 15 and agarose (0.8%) overlay. Plates were incubated at the restrictive temperature of 36", 37", 38", 39", 39.5". or 40°C in a constant temperature water bath and the clear plaques that developed were scored after 2 days.

To determine if two viruses shared ts lesions on the same gene, the plate complementation-recombination assay was employed 18 using MDCK monolayer cultures on 12-well plates. Temperature-sensitive parental and recombinant viruses were tested in pairwise matings with ts viruses known to contain a ts mutation on the P1, P3, or NP gene.

Recombination

MDCK cells in 24-well Co-star plastic plates were mixedly infected with the FV 1319 virus and A/Alaska/77 wild-type virus at a multiplicity of infection of 1 for each virus. The virus mixture was adsorbed for 1 hr after which the cultures were washed four times with 1 ml of medium consisting of equal parts of medium 199 and Eagles No. 2 supplemented with 66 pglrnl of gentamicin, 10 pg/ml of amphotericin, and 1 pgIml of trypsin. The cultures were then maintained with this medium at 34" C. The individual cultures were harvested 24 hours later. Virus in 9 such suspensions was derived from a mating of the uncloned FV 1319 virus and the A/Alaska/77 wild-type virus, and six other suspensions represented the progeny of a mating of the same wild-type virus and the cloned FV 1319 virus. Since the properties of recombinants derived from the cloned and uncloned FV 1319 were in all respects comparable, the data for the two sets of recombinants have been combined for clarity of presentation.

Initially, 10 to IS plaques were harvested from plates inoculated with each of the fifteen harvests. A total of 179 plaques were characterized for their ts phenotype. The 179 plaque populations were screened for their temperature sensitivity of replication by inoculating each plaque suspension in duplicate onto MDCK cells maintained under a fluid overlay and incubating one plate at 34" C and the other at 39" C. The cultures were scored for cytopathic effects (CPE) after 48 hours. The FV 1319 ts+ and A/Alaska/77 wild-type parent viruses, which were passaged and tested in parallel, produced confluent CPE at both 34" C and 39" C. Virus with the ts phenotype induced extensive CPE at 34" C but failed to produce appreciable CPE at 39" C. Putative ts virus was harvested from the culture incubated at 34" C and its ts phenotype was con- firmed by plaque titration at 34" C and 39" C. Those harvests that exhibited a 100-fold or greater reduction of plaquing efficiency at 39" C were considered ts. One ts recombinant derived from each of the 15 separate mixed infections was then cloned by plaque-to-plaque passage in MDCK culture.

Polyacrylamide Gel Electrophoresis (PAGE)

The methods used to purify virus, extract viral RNA, and perform PAGE have been described.Rr18-21

Murphy et 01.: Escape of Influenza A Virus 175

RESULTS

Administration of Influenza A / Alaska/ 77 ts-I A2 Recombinant to Children

The A/ Alaska/ 77-ts-1 A2 recombinant was administered intranasally to a seropositive and a seronegative child. Both children remained clinically well. Virus recovered on the second day from both children exhibited the same level of temperature sensitivity as the A/Alaska/77-ts-lA2 recombinant administered (TABLE l ) , i.e., a 37°C shutoff temperature for plaque formation. Subse- quently, virus with decreased temperature sensitivity was recovered from the

TABLE 1

EFFICIENCY OF PLAQUE (EOP) FORMATION OF INFLUENU A VIRUS PRESENT IN NASAL WASH SPECIMENS FROM CHILDREN WHO RECEIVED THE

A/ALAsm/77-t~- 1 A2 VIRUS

Shutoff temperature Titer (log,, pfu/ml * ) of virus Day after in nasal wash specimen ( O C) for

Child istration 34" 37" 38" 39" 40" formation virus admin- PlaSUe

Seropositive 2 5.5 <1.7 <1.7 <1.7 <1.7 37 Seronegative 2 4.9 <1.7 <1.7 <1.7 <0.7 37

3 4.4 2.6 <1.7 <1.7 <0.7 38 7 4.7 4.1 3.5 3.2 <0.7 40 8 5.2 5.2 4.9 4.2 <0.7 40 9 t 4.5 4.5 4.5 3.7 <0.7 40

A/Alaska/77-ts-l A2

A/Alaska/77 wild-type 6.5 6.5 6.3 6.9 6.5 >40 vaccine virus 6.9 3.3 <2.7 <2.7 <2.7 37

* Specimens titrated on MDCK monolayer culture. t Isolate referred to as FV 1319. Note: Quantity of virus recovered on days 1, 4, 5, 6, and 10 was insufficient for

determination of EOP. However, the isolates from days 4, 5, 6, and 10 had a 40" C shutoff temperature.

seronegative vaccinee, while the seropositive vaccinee stopped shedding virus. Virus isolated on the third day from the seronegative child had a 38" C shutoff temperature for plaque formation, while on the seventh, eighth, and ninth days virus with a 40" C shutoff temperature was recovered. Since the shutoff temperature of wild-type virus was greater than 40" C, the phenotypically altered virus shed by the vaccinee nonetheless retained some temperature sensitivity for viral replication. It was, however, considerably less temperature sensitive than the virus which was given to the seronegative child. Virus recovered on the ninth day was chosen for further genetic characterization and will be referred to as isolate FV 1319; this virus was rsc when tested at the restrictive temperature of 39" C.


Evidence for an Extragenic Suppressor Mutation in the F V 1319 tst Virus

Ramig and Fields have shown for reovirus that the most common event leading to loss of the ts phenotype was not reversion of the ts gene to wild-type, but rather the development of a suppressor mutation on another gene that corrected the ts phenotype.zz For this reason, evidence for an extragenic suppressor mutation in the FV 1319 ts+ virus was sought by mating this ts+ virus with the A/Alaska/77 wild-type virus (TABLE 2). If extragenic suppression were responsible for the tst phenotype of the FV 1319 virus, it should be possible to detect ts progeny in which a ts gene had been segregated from the gene bearing the putative suppressor mutation. Twenty-five percent of the progeny should be ts if the Is+ "revertant" contained one ts gene and one suppressor gene. The observed percentage of ts progeny was 24%, which closely approximated the expected frequency of 25 % . Each of the 43 ts recombinants was restricted 100 to 1000 fold in plaquing efficiency at 39" C compared to 34" C. In contrast, ts virus was not generated by parallel passage of either of the two parental viruses. These findings suggested that an extragenic suppressor mutation was responsible for the ts+ phenotype of the FV 1319 tx+ virus.

EOP of ts Segregarits at Several Restrictive Temperatures

The EOP of 15 independent ts recombinants derived from the FV 1319 tp virus was evaluated more fully at 36", 37", 38", 39", 39.5", and 40" C. These recombinants were also compared with their tst parents and the A/Alaska/77- ts-1A2 recombinant originally administered to the vaccinee. Representative data for the EOP of two such recombinants is shown in TABLE 3. Each of the 15 ts recombinants was significantly restricted at 39" C in contrast to their FV 1319 tst parent which showed significant restriction of plaque formation at 40" C and above. However, segregation of the ts gene from the suppressor gene did not restore the original level of restriction exhibited by the A/Alaska/ 77-ts- 1A2 recombinant, i.e., a 37" C shutoff temperature for plaque formation.

TABLE 2

EVIDENCE FOR EXTRAGENIC SUPPRESSOR MUTATION: GENERATION OF ts VIRUS BY MATING A/ALAsKA/~~-~s- I A2 ts+ "REVERTANT"

VIRUS WITH A/ALAsKA/I~ WILD-TYPE VIRUS

Number of % of Number of plaques yielding plaques

A/Alaska/77 Virus plaques tested t s virus rs

wild-type 33 0 0 ts+ (FV 1319) 31 0 0 wild-type X FV 13 19 progeny 179 44 24

Note: The A/Alaska/77-ts-lA2 virus was parallel passaged and each of 27 plaques picked were ts.


TABLE 3

EFFICIENCY OF PLAQUE FOWTION AT PERMISSIVE AND RESTRICTIVE TEMPERATURES

AND WILD-TYPE VIRUS OF THE ts VIRUS PRODUCED BY MATING A/ALASU/77 1319 ts' VIRUS

Efficiency of plaque formation on MDCK cultures (loglo pfu/ml) at indicated permissive or restrictive

Shutoff temperature ( O C)

VUUS 34 37 38 39 39.5 40 (" C) * A/Alaska/77 temperature

~~

wild-type 6.1 5.9 5.9 5.2 5.7 5.0 >40 FV 1319 ts+ 6.9 6.7 6.1 5.5 6.2 2.9 40 rs-1A2 6.3 2.0 <1.7 <1.7 <1.7 <1.7 37 FV 1319 X wild type

ts recombinant clone 65 6.6 6.4 5.2 <1.7 <1.7 <1.7 39 clone 84 6.9 6.7 5.5 <1.7 2.2 <1.7 39

* Shutoff temperature was defined as the lowest temperature at which a 10Gfold or greater reduction of virus titer (pfu/ml) was observed.

Genetic Analysis of the ts Segregants

Since the A/Alaska/77-ts-lA2 recombinant possessed two ts genes, it was possible that the ts segregants contained one or both of these ts mutations. The location of ts lesions in fifteen independently derived ts segregants derived from FV 1319 was determined by mating each of these IS segregants with single lesion ts viruses known to contain a ts mutation on the P3, P1, or NP gene (TABLE 4). The matings were carried out at 39.5" C, a temperature at which the ts segregants were markedly restricted in plaque formation. The parental A/ Alaska/ 77-ts-1A2 recombinant administered to the vaccinee possessed one or more ts mutations on the P1 and P3 genes as indicated by its failure to complement a single lesion ts P1 or ts P3 mutant. In contrast, each of the 15 ts recombinant viruses derived from the FV 1319 ts+ virus possessed only the ts P3 lesion. Ts segregants possessing a ts P1 gene were not identified. A t s P1 lesion was not identified in the FV 1319 tst virus even when this virus was tested for its ability to complement at 40" C.

Previously, it had been shown that the original ts P3 lesion specified a 37" C shutoff temperature, whereas the P3 gene in the ts segregants was clearly less defective since it specified a 39" C shutoff temperature.lB This indicated that the P3 gene of the FV 1319 virus had also undergone genetic modification.

Polyacrylamide Gel Electrophoresis of FV 1319 Virion RNA

To confirm that the FV 1319 ts+ virus was indeed derived from the A/ Alaska/ 77-ts-1A2 recombinant, virion RNA from the A/Udorn/ 72 and A/ Alaska/77-ts-lA2 recombinants, the FV 1319 tst virus, and the A/Alaska/77 wild-type virus was analyzed by PAGE (FIGURE 1). RNA 1, i.e., the P3 gene, from the FV 1319 tst virus migrated more slowly than its counterpart in the


TABLE 4

MATING THE FV 1319 t.s+ "PSEUDOREVERTANT" AND THE A/&~sw/77 WILD-TYPE VIRUS

PRESENCE OF t$ P3 LESION M THE tS RECOMBINANT VIRUSES DERIVED BY

Ratio *: of the number of plaques observed at 39.5" C to the expected number of cells dually

infected with the indicated pairs of virus

Clone 291 Clone 315 Clone 368 on indicated ts lesion

AlAlaska/77 virus (P3) t ( P l ) (NP) gene

tslA2 recombinant 0.001 0.0003 88 P3, PI FV 1319 X wild type

ts recombinant clone 98 0.0008 1.7 13 P3 clone 120 0.0006 1.4 4.5 P3

* The number of dually infected cells was estimated on the assumption that the distribution of the virus to the cell fits the Poisson distribution using the formula (l-e-mA)(l-e-"B)(number of cells) where mA and mB were the multiplicities of infection of the two infecting viruses determined at the permissive temperature of 34" C. A value of approximately 1 was arbitrarily taken to indicate that complementation-recombination had occurred and values <O. 1 indicated the absence of complementation-recombination.

t Aiaska/77-rs-lA2 recombinant clone 291 has the ts P3 gene; l* A/HK/68-ts-315 virus has a ts PI gene;' and A/Udorn/72-ts-368 has a ts NP gene (Markoff et al., unpublished observations). Matings between these viruses at 39.5" C gave ratios of 21. Note: Fifteen independently derived ts recombinants possessed only the ts P3 lesion.

A/Alaska/77-ts-lA2 recombinant from which it was derived. At all other loci (including RNAs 4-8 not shown in this figure) FV 1319 virion RNA co- migrated with virion RNA of the A/ Alaska/ 17-ts-1A2 recombinant. This suggested that the FV 1319 t.r+ virus was derived from the A/Alaska/77-ts-lA2 virus and that it acquired a mutation in its P3 gene that affected the electrophoretic mobility of this gene.

Mapping of the Suppressor Mutation

A preliminary attempt was made to map the locus of the extragenic suppressor mutation in the FV 1319 rsc isolate. This was done by examining a series of independently derived ts recombinants and by identifying the gene in each of the ts segregants that was always derived from the wild-type parent. The parental origin of the P1, P2, P3, and M genes in ts segregants could be determined because at these loci differences in migration during PAGE were discernable between the corresponding RNA segments of FV 1319 and the A/Alaska/l l wild-type virus. At the HA, NA, NP, and NS loci the genes in FV 1319 were ultimately derived from the A/Alaska/77 wild-type virus and therefore, could not be distinguished by migration in PAGE. The P3 gene in each of eight cloned, independently derived ts segregants came from


the FV 1319 parent. This was not unexpected since the complementation- recombination analysis identified a ts P3 lesion in each ts segregant. The P1 and M genes were excluded as the site of the suppressor mutation since these genes were not always inherited from the A/Alaska/77 wild-type parent. Significantly, each of the 8 ts segregants from the FV 1319 virus inherited a P2 gene from wild-type virus. Thus, the P2 gene could be the site of the suppressor mutation that affects the ts P3 gene. Additional ts segregants are being generated to further explore this possibility.

DISCUSSION

Alteration of the A/Alaska/77 ts-1A2 recombinant to the ts+ phenotype occurred during infection of a completely susceptible child. This alteration of phenotype was associated with a microcosm of genetic changes that included: (1) an extragenic suppressor mutation affecting the ts P3 gene; (2) a mutation on the ts P3 gene affecting the level of temperature sensitivity it specified; (3) a mutation on the rs P3 gene that retarded its mobility in PAGE; and (4) a mutation affecting the temperature sensitivity of the P1 gene. The two changes that were identitied on the P3 gene could reflect the same mutation, but this remains to be determined. In any case the new mutations located on the P1 and P3 genes, as well as the suppressor mutation that developed on another gene, apparently permitted the highly defective A/Alaska/77-ts-lA2 recom-

P3-

P1-

P2- *P1 - P 2

FIGURE 1. PAGE of virion RNAs 1, 2, and 3 from A/Uclorn/72 ts-lA2 recombinant, A/Alaska/77-ts-lA2 (clone 190) recombinant, A/Alaska/77 FV 13 19 ts+ isolate, and A/Aiaska/77 wild-type virus. The 16 cm gel contained 2.6% acrylamide and 6 M urea. PAGE was carried out for 21 hr at 27°C with 70 volts.


binant to escape its ts phenotype. As a result, the AlAlaskal77-ts-lA2 recombinant (37” C shutoff temperature) was replaced by a ts+ virus during infection of the young seronegative vaccinee. This ts+ virus replicated to a high titer in the upper respiratory tract (IO5.O TCID,,/ml of nasal wash) suggesting that transmission of the altered virus to susceptible contacts might occur under the appropriate conditions of exposure. Poliovirus vaccine strains commonly undergo a change from ts to ts+ phenotype after replication in the human intestinal tract, but virulence for man is rarely For this reason, the clinical significance of the ts to tp change should be assessed for the A/Alaska/ 77-1s- 1A2 recombinant.

If ts mutations are to be used for the construction of live influenza A virus vaccines, it is essential that such ts mutations regularly confer the properties of attenuation, genetic stability, and immunogenicity upon any new epidemic or pandemic virus into which they are transferred by genetic reassortment. Emergence of ts+ virus during infection of a young vaccinee with the A/Alaska/ 77-ts-1A2 recombinant indicates that the goal of genetic stability in the completely permissive human host has not been met. Of interest, other ts-1A2 recombinants bearing the surface antigens of A/Victoria/75 (H3N2) or A/ Hong Kong/77 ( H I N l ) did not undergo a change in ts phenotype similar to that seen with the A/Alaska/77-rs-lA2 recombinant. (Wright et al, Unpub- lished observation. Murphy et al. Unpublished observations.) The A/Hong Kong/ 77-ts-1 A2 recombinant was stable in doubly-seronegative adults. (Murphy et al. Unpublished observations.) The A/Victoria/ 75-rs-1 A2 recombinant exhibited a slight decrease in temperature sensitivity during infection of 10 completely susceptible young children; however, virus was not recovered at 39” C although the vaccinees shed the recombinant for an average of 7.7 days. (Wright et al. Unpublished observations.) It should also be noted that these 3 ts-1A2 recombinants, as well as the parent A/Udorn/72-ts-lA2 virus, were completely stable genetically when tested in over 200 experimentally infected hamsters.*. 9 * l6 (Murphy et al. Unpublished observations.) The basis for difference in genetic stability amongst the three rs-1A2 recombinants in man is unclear.

The mechanism by which ts mutants of reovirus type 3 revert to the ts+ phenotype has been studied by Ramig and Fields.** They examined 28 independent spontaneous t ~ + “revertants,” generated from a diverse set of ts mutants, for the presence of intragenic or extragenic mutation. Twenty-five of 28 fs+ “revertants” contained the original ts mutation demonstrable by backcross to the wild-type parent indicating that in this situation extragenic suppression was the most common mechanism for loss of the ts phenotype. Jarvik and Botstein also found that extragenic suppression of ts mutants of phage P2 could produce a shift to the tst phenotype.** They also observed that the suppressor mutations were located on genes whose products physically interacted with the product of the ts gene. These observations may be relevant if our preliminary assignment of the M 1319 suppressor mutation to the P2 locus is correct since P2 and P3 proteins are known to be associated in the influenza A virus polymerase com- plex.Z5 Since the P3 and P1 proteins are both involved in complementary RNA synthesis, the P1 gene was considered a strong candidate for the suppressor locus, but preliminary mapping eliminated the PI gene as the site.26 This indicates that the mutation responsible for the loss of temperature sensitivity of the P1 gene was not responsible for suppression of the ts P3 gene. Ramig and Fields suggested that the product of the suppressor mutation in reovirus


can itself be k2* In this situation, two ts gene products may interact to yield the tp phenotype. However, this does not appear to be the case with the suppressor mutation of the ts-1A2 ts P3 gene. If the suppressor gene itself specified a ts gene product, one would expect to identify a ts mutant in the backcross progeny that contained a ts mutation at a locus other than the P1 and P3 genes. However, such a mutant was not identified among 43 backcross ts recombinants.

Evidence for intragenic mutation was obtained for the t s P3 gene of the A/Alaska/77-ts-lA2 recombinant; electrophoretic mobility of this gene was altered and the level of temperature sensitivity it specified changed from a 37” C shutoff temperature for plaque formation to a 39” C shutoff temperature.l@ These changes could represent intragenic suppression or true reversion of one of several ts mutations, as has been observed for group A tp revertants of r e o v i r u ~ . ~ ~ Altered mobility of virion RNA associated with is mutation was observed previously with influenza A/ WSN/33 virus.2E Ts mutation on the P3 or P1 gene was accompanied by an accelerated migration of virion RNA segment 1 or 2 during PAGE.26 The RNA segments that did not bear a ts mutation exhibited a normal migration pattern during PAGE. In the present study, partial loss of the ts phenotype specified by the ts P3 gene was associated with retardation of migration of the P3 gene during PAGE. In contrast, there was no alteration in the migration of the other seven virion RNAs. These observations suggest that the two alterations in the ts P3 gene could be a manifestation of a single mutation on this gene, but this remains to be demon- strated. Presumably intragenic changes were also responsible for the loss of the temperature sensitivity of the ts P1 gene product since an extragenic suppressor mutation affecting this gene was not identified. Such intragenic mutation was not reflected in a change in the migration of the P1 virion RNA segment.

The extraordinary capacity of a highly defective influenza A virus to undergo extragenic suppression and intragenic mutation and thus escape from the constraints imposed by its ts mutations suggests that genetic stability may be an unrealistic goal for such mutants. This may also apply to the ca recombinants, which exhibit both the cu and ts phenotypes, although “revertants” of cu recombinants remain to be analyzed in the manner described here. Demonstration of suppression and/or intragenic mutation in ca recombinants should prove considerably more difficult since the ca and ts phenotypes appear to involve more genes than is the case for the A/Alaska/ 77-rs-lA2 recombinant.z8 One could perhaps speculate that genetic stability of highly defective, attenuated influenza viruses may require the construction of viable deletion mutations that cannot be corrected by suppression, intra- or extragenic. Recent advances in cloning DNA copies of influenza viral RNA segments make this a real possibility whose success is dependent on the development of techniques for converting viral DNA copies back into virion RNA.28

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