JOURNAL OF VIROLOGY, Sept. 1994, p. 5748-57540022-538X/94/$04.00+0Copyright © 1994, American Society for Microbiology

Mutations at Palmitylation Sites of the Influenza VirusHemagglutinin Affect Virus Formation

Vol. 68, No. 9

THOMAS ZURCHER,t GUANGXIANG LUO,t AND PETER PALESE*

Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029

Received 17 March 1994/Accepted 20 May 1994

The carboxy terminus of the hemagglutinin (HA) of influenza A viruses contains three cysteine residueswhich are highly conserved among HA subtypes. It has previously been shown for the H2, H3, and H7 subtypesof HA that these cysteine residues are modified by the covalent attachment of palmitic acid. In order to studythe role of the acylated cysteines in the formation of infectious influenza viruses, we introduced mutations intothe HA of influenza A/WSN/33 virus (Hi subtype) by reverse-genetics techniques. We found that the cysteineat position 563 of the cytoplasmic tail is required for infectious-particle formation. The cysteine at position 560can be changed to alanine or tyrosine to yield virus strains that are attenuated in cell cultures. The change fromcysteine at position 553 to serine or alanine does not significantly alter the phenotype of the virus. Therequirement for a cysteine at position 563 suggests a functional role for palmitylation of the cytoplasmic tail.This interpretation is further supported by experiments in which two or more of the cysteine residues weremutated, eliminating potential palmitylation sites. None of these double or triple mutations resulted ininfectious virus. Selection of revertants of the attenuated cysteine-to-tyrosine mutant (mutation at position560) always resulted in reversion to cysteine rather than to other amino acids. Although our data indicate abiological role for the conserved cysteine residues in the cytoplasmic tail of the HA of influenza viruses, wecannot exclude the possibility that structural constraints in the cytoplasmic tail of the HA-rather than alteredpalmitylation-are the determining factors for infectious-particle formation.

The hemagglutinin (HA) of influenza virus is the majorsurface antigen and is one of the best-characterized membraneglycoproteins. It has receptor-binding and fusion activities,which are both necessary for the initiation of viral infection.The protein contains a large ectodomain that carries receptorand fusion activities, a stretch of hydrophobic amino acidswhich constitutes the transmembrane domain, and a shortcytoplasmic tail. This cytoplasmic tail contains 9 to 11 aminoacids, depending on the subtype of the HA (30) (Fig. 1).Comparison of the HA sequences in this region reveals thatfive amino acids are highly conserved and that they areidentical in 11 of the 14 known subtypes (2, 8, 17, 25). Two ofthese conserved residues are cysteines, and a third highlyconserved cysteine is located in the membrane-anchoringdomain (Fig. 1). It has previously been shown that the con-served cysteines of subtype H2, H3, and H7 HAs are posttrans-lationally modified by covalent addition of palmitic acids (14,15, 23, 27, 29). The levels of palmitylation have been quantifiedfor the A/Japan/305/57 HA (H2), whose 17 carboxy-terminalamino acids are identical to those of the A/WSN/33 HA (Hi).The cysteines at positions 560 and 563 (C-560 and C-563,respectively) (Fig. 1) appear to be highly modified, with C-563incorporating at least half of the fatty acid label, whereas C-553incorporates only about 10% of the total palmitate (15).The extent and significance of palmitylation of viral proteins

are not yet fully understood (for a review, see reference 13).The fact that HeLa cells are refractory to influenza virus

* Corresponding author. Mailing address: Department of Microbi-ology, Box 1124, Mount Sinai School of Medicine, Annenberg Build-ing, Rm. 16-20, 1 Gustave L. Levy P1., New York, NY 10029. Phone:(212) 241-7318. Fax: (212) 722-3634.

t Present address: Centro Nacional de Biotecnologia (CSIC), Uni-versidad Autonoma, 28049 Madrid, Spain.

t Present address: Department of Virology, Bristol-Myers SquibbPharmaceutical Research Institute, Princeton, NJ 08543.

growth has been ascribed to a defect in palmitylation of thiscell line, but other mechanisms responsible for the abortiveinfection in HeLa cells cannot be ruled out (19). Experimentsusing hydroxylamine to remove lipid from viral proteins havesuggested that the fatty acid moiety is important for membranefusion (23). Another study used the antibiotic cerulenin toinhibit acylation and has implicated the lipid in viral release(22). However, these interpretations are not definitive sincehydroxylamine might affect the protein structure and ceruleninis known to exert a general toxic effect. For vesicular stomatitisvirus it has been reported that the elimination of the palmity-lation site in the G protein has no effect on membrane fusionor glycoprotein incorporation into virions (31). Studies withalphaviruses have shown that the elimination of either of thetwo palmitylation sites in the carboxy terminus of the glyco-protein E2 decreases the efficiency of virus budding (7) andthat a mutant with changes in both palmitate addition sites wasnot viable (5).There is one report which suggests that the AlJapan/305/57

HA (H2) requires palmitate for membrane fusion (14). How-ever, this finding has not been supported by other workersusing either H2, H3, or H7 subtype HAs (15, 25, 27, 29). It hasbeen shown that the substitution of the conserved cysteines atposition 553, 560, or 563 by serine (H2 and H3) or alanine(H7) did not significantly affect HA biosynthesis, intracellulartransport, or receptor-binding activity when the HA is ex-pressed from recombinant plasmid DNA or simian virus 40vectors (2, 9, 14, 15, 25, 27, 29). In fact, HA proteins withmutations in two (25) or all three (15) palmitate addition sitescomplemented an influenza virus with a temperature-sensitivemutation in the HA. However, data presented here obtainedwith a different assay system suggest a role for these cysteineresidues in the formation of infectious influenza virus.

Recently, Naim and Roth (16) have extended their studies ofthe role of the cytoplasmic tail by expressing HA variants fromrecombinant simian virus 40 strains in influenza virus-infected

5748

PALMITYLATION SITES OF INFLUENZA VIRUS HEMAGGLUTININ 5749

Ectodomain CytoplasmicTM Domain

NH2- COOH

Hi

H2

H3

H4

H5

H6

H7

H8

H9

H10Hll

H12

H13

H14

M C c

M C c

A C C

A C Q

ILCJG M

A M S

CL

A

C SG

CQ

A

Cc

MICIs

5605-NGSLQ

-NGSLQK-GNIR-NGNIR-NGSLQ-NGSMQKNGNMR

-NGSCR

-NGSCRKNGNMR

-NGSCR

-NGNYR

--GNCR-NGSLQ

ccccccccccccF

C

RI

RI

NI

QIRIRI

TI

MF

NI

TI

TI

TF

NYRI

C

c

ccccC

C

C

C

C

C

C

FIG. 1. Comparison of the carboxy-terminal amino acid sequencesof the HAs of 14 different influenza A virus subtypes (2, 8, 17, 25).Cysteine residues are boxed, and the vertical bar indicates the divisionbetween the transmembrane (TM) and the cytoplasmic tail (for a

review, see reference 30). The numbering of the conserved cysteines isfor the Hi subtype HA (6).

cells. Although chimeric HAs with foreign cytoplasmic se-quences were efficiently excluded from viral envelopes, HAswhich lacked a cytoplasmic tail were incorporated into virions.Similarly, Simpson and Lamb (25) showed that HA mutantslacking a cytoplasmic tail were incorporated into viral particles,but the particles were found to be noninfectious. This wouldindicate that the conserved cytoplasmic sequences are notrequired for the incorporation of HA into virions but that thecytoplasmic tails of the HAs are necessary for optimal infec-tivity of the virus.By reverse-genetics techniques, it is now possible to con-

struct transfectant influenza viruses which have changes intheir HA genes (3). The advantage of this procedure is that theeffects of mutations can be analyzed in the context of theinfectious virus. In the present study we isolated transfectantinfluenza viruses containing mutations at the conserved palmi-tate addition sites of the HA, in order to study the effects of thechanges on the phenotypes of the viruses.

MATERIALS AND METHODS

Virus and cells. Influenza A HK-WSN (H3N1) virus wasused as a helper virus for transfection experiments as describedpreviously (3). HK-WSN virus is a reassortant virus whichcontains seven genes from A/WSN/33 (H1N1) virus and theHA gene from A/Hong Kong/8/68 (H3N2) virus. HK-WSNvirus was propagated in Madin-Darby bovine kidney (MDBK)cells and titrated by plaquing on MDBK cells. MDBK cellswere used for ribonucleoprotein (RNP) transfection and forthe selection of the transfectant viruses as well as for thepreparation of virus stocks (12). Multiple passaging of virusesin MDBK cells or in embryonated eggs was done with inoculaderived from the sample of the previous passage having thehighest titer. Dilutions between 1/10 and 1/1,000 were used forinoculation.Growth curves in MDBK cells. Confluent monolayers of

MDBK cells in 60-mm-diameter dishes were infected with

virus at a multiplicity of infection of 0.001 for 1 h at roomtemperature and incubated at 37°C and 5% CO2 after additionof the culture medium. The virus titer at 12-h intervalspostinfection was determined by plaque assay or 50% tissueculture infective dose (TCID50) assay. The latter assay wasused for titrating mutant viruses which did not form discreteplaques.

Oligonucleotide-directed mutagenesis of the palmitate ad-dition sites of HA. Plasmid pT3/WSN-HA containing thefull-length cDNA of the wild-type HA gene of influenzaA/WSN/33 virus (3) was used for the construction of all themutant constructs. In order to facilitate the introduction ofmutations into the 3' end of the cDNA, pT3/WSN-HA wasmodified in the following way. A HindlIl restriction enzymesite was introduced at nucleotide position 1679 (16 amino acidsaway from the carboxy terminus), and the Hindlll site betweenthe T3 promoter and the pUC18 vector of the originalpT3/WSN-HA clone was replaced by a PstI site. For thispurpose pT3/WSN-HA was first linearized with HindlIl, andthe ends were filled in with Klenow enzyme (Bethesda Re-search Laboratories). Then the linearized vector was digestedwith BstXI and purified by gel electrophoresis. A PCR frag-ment was generated with pT3/WSN-HA DNA as a templateand two synthetic oligonucleotides as primers. The upstreamprimer (5'-CTGTCGCCAGTTCACTGGTGCIT1l TGGTCTCCCTGGGGGCAATAAGCTTCTGGATGTGT-3') covers thesequences from nucleotide positions 1636 to 1695 of the HAcDNA and contains a BstXI site and a Hindlll site (under-lined) generated by introducing silent mutations at nucleotidepositions 1679 and 1682 (boldface). The downstream prim-er HAO1 (5'-CCGGCCJXCAGAATTAACCCTCACAAA-3') contains a T3 promoter and a PstI site (underlined). ThePCR fragment was digested with BstI and ligated into thepreviously described vector. The modified plasmid was desig-nated pT3/WSN-HA/HindIII.The mutant plasmids shown in Fig. 2 were constructed by

oligonucleotide-directed mutagenesis. To create pC553S, forinstance, a DNA fragment was amplified by PCR with pT3/WSN-HA/HindIII as the template and with the primers HAO1and the oligomucleotide (5'-CCGGAAGXJ2TCTGGATGTCTTCTAATGGGTC-3') containing the HindIll site (under-lined) and the appropriate mutation (boldface) in the openreading frame of the HA. The PCR fragment was digested withHindIll and PstI and inserted into pT3/WSN-HA/HindIIIdigested with HindlIl and PstI. All mutant plasmids wereconstructed in this manner with the appropriate primers andtemplates. The inserts of all the mutant plasmids were con-firmed by sequencing (21).

In order to express wild-type and mutant HA proteins, theXbaI-PstI fragments of pT3/WSN-HA/HindIII and of themutant constructs containing the entire coding and noncodingregions of the HA were subcloned into the expression vectorpSVK3 (Pharmacia), downstream of the T7 promoter.

Purification of influenza A virus RNA polymerase complexand RNP transfection of MDBK cells. The RNA polymerasecomplex was purified from influenza A/PR/8/34 virus or X-31as described previously (18) and was then used for RNPtransfection of MDBK cells. The transfection procedure fol-lowed the protocol provided earlier (3), except that the trans-fection yield was passaged twice in liquid on MDBK cells in thepresence of 0.5% rabbit anti-HK antiserum (24). Viruses wereplaque purified in the presence of 0.5% rabbit anti-HK anti-serum.

Virus purification and RNA extraction. The wild-type andmutant transfectant viruses were propagated in MDBK cells

VOL. 68, 1994

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5750 ZURCHER ET AL.

Ectodomain

NH2-

TM Cyto

-

TCT TCT AAT GGG TCT TTG CAG TGC AGA ATA TGC ATCname of Cys Ser Asn Gly Ser Leu Glu Cys Arg Ile Cys Ileconstruct 53560 563

pT3WSNwt Cys

pC553SpC553A

pC560SpC560ApC560YpC56OF

pC563SpC563A

pC553A/C560A AlapC56OA/C563ApC553S/C560S SerpC553S/C560S Ser/C563SpS558C/C560SpC56OS/WTpC563S/WT

SerAla

SerAlaTyrPhe

AlaAlaSerSer

Cys SerCys

Cys Cys (+)

(+) C553S(+) C553A

(- )

(+) C560A(+) C560Y(+) C560F

Ser (-)

Ala (-)

Ala (-)

(-)Ser (-)

(-)(+)

Cys (+)

FIG. 2. Amino acid sequence of the carboxy terminus of wild-type influenza A/WSN/33 virus and mutant HA proteins. The left column liststhe names of plasmids used in this study. For example, in mutant C553S the cysteine at position 553 is replaced with serine. In the right column,rescued mutants are indicated by a plus sign in parentheses. TM, transmembrane region; Cyto, cytoplasmic tail; pT3WSNwt, pT3WSN-HA/HindIII(wild type).

and then purified by 30 to 60% sucrose gradient centrifugation.Virion RNA was then extracted as described previously (11).RNA and DNA sequencing. The sequences of the HA genes

of the transfectant viruses C553S, C553A, and C560A wereverified by direct RNA sequencing with the primer 5'-GGTGTATCAGATTCTGGCGATC-3' (corresponding to positions1607 to 1628 of the A/WSN/33 HA gene) and avian myeloblas-tosis virus reverse transcriptase. The sequences of the HAsegments of all the other mutant viruses were obtained as

follows. RNA from purified virus was transcribed into a cDNAwith avian myeloblastosis virus reverse transcriptase and theprimer 5'-ACGTGGATCCGAAAGTGTAAGAAATGGG-3', which is complementary to positions 1509 to 1526 of theA/WSN/33 HA virion RNA and contains a BamHI site (un-derlined). This oligonucleotide and the primer 5'13/HindIII(ATGCTCTAGAAGCTI'AGTAGAAACAAGG) were usedfor subsequent PCR amplification of the cDNA. The sequenceof oligonucleotide 5'13/HindIII corresponds to the 13 con-served nucleotides at the 5' end of the virion RNA andcontains a HindlIl site. HindlIl fragments of the PCR productswere subcloned into pUC19 and sequenced by standard meth-ods (20, 21).

Expression of HA in transfected COS-7 cells. For transientexpression of HA cDNA cloned into pSVK3, subconfluentmonolayers of COS-7 cells were infected with a recombinantvaccinia virus expressing the T7 RNA polymerase (vTF7.3) (4)for 1 h at a multiplicity of infection of 5. Ten micrograms ofplasmid DNA was transfected into a 60-mm-diameter dish bythe calcium phosphate precipitation method (32). At 16 hposttransfection the cells were fixed with 3% paraformalde-hyde and the surface expression was determined by indirectimmunofluorescence (26). The Hi HA-specific monoclonalantibody 2G9 (10) was used as the first antibody, and a

rhodamine-coupled goat anti-mouse antibody (BoehringerMannheim) was used as the second antibody.

RESULTS

Design and construction of carboxy-terminal mutants ofinfluenza A/WSN/33 virus HA. Oligonucleotide-directed mu-

tagenesis was performed on a cDNA copy of the A/WSN/33HA to introduce mutations into the region encoding thecarboxy terminus. C-553, C-560, and C-563 have been associ-ated with palmitylation of the HA in other HA subtypes (14,15, 27, 29). Cysteine at these positions was first changed toserine or alanine, and the biological effects of these mutationswere analyzed (Fig. 2). We also constructed mutant S558C/C560S (containing an S-to-C change at position 558 and a

C-to-S change at position 560), since the HA of subtype H13contains a cysteine at position 558 but not at position 560 (Fig.1). This mutant thus possessed a potential palmitylation site atposition 558 instead of position 560. In addition, C-560 was

changed to phenylalanine or tyrosine. The C-560-to-phenylal-anine change was done because this sequence is present in theH13 subtype of the HA (Fig. 1). The single substitution ofC-560 with tyrosine was previously shown to result in a proteinwith a phenotype quite different from that of the wild-typeprotein (1, 9). Whereas the wild-type HA was excluded fromcoated pits, the mutant HA protein was efficiently internalizedand was in addition transported to the basolateral rather thanthe apical membrane.

Rescue of mutant viruses. We were first interested in findingout whether viruses with these mutant HA proteins could forminfectious virus particles. In order to rescue the mutated HAgenes we performed RNP transfections with HK-WSN (H3N1)as a helper virus (3). As shown in Fig. 2, viruses were isolatedafter transfection of RNAs encoding single changes of C-553 toserine or to alanine, suggesting that the cysteine at this positionin the HA is not crucial for the formation of viable virus. Thecysteine at position 560 could be changed to alanine, tyrosine,or phenylalanine, and viable viruses could still be rescued.

transfectantvirus rescued

J. VIROL.

I- -LAJUHIrrnu

PALMITYLATION SITES OF INFLUENZA VIRUS HEMAGGLUTININ 5751

A

A

C

E

B

D

FI

109

108

10 7

106

105

104

103

FIG. 3. Surface expression of wild-type and mutant HA proteins intransiently transfected, vaccinia virus-infected (vTF7.3) COS-7 cells.Transfected plasmids included pSVK3/HA (wild type) (A), mock-transfected plasmid (B), pSVK3/C56OA/C563A (C), pSVK3/C563A(D), pSVK3/C560S (E), and pSVK3/C553S/C560S/C563S (F) (fordetails, see Materials and Methods).

However, a change to serine at this position did not result in aviable transfectant virus. In order to rule out the possibility thatan unaccounted-for mutation in the main portion of the HAabrogated the rescue of mutant C560S virus, we replaced theregion encoding the cytoplasmic portion of pC560S with thecorresponding one of the wild type. Transfection of thisplasmid (pC560S/wt) resulted in the rescue of a transfectantinfluenza virus. Mutants with changes of C-563 to alanine orserine were also not rescued. Replacement of the regionencoding the cytoplasmic portion of pC563S with the corre-sponding wild-type sequence again resulted in the rescue of atransfectant virus, suggesting that the cysteine at position 563is critical. Furthermore, mutants in which two or three of theconserved cysteine residues were substituted by alanine or byserine were not rescued. Mutant S558C/C560S was also notrescued, suggesting a structural incompatibility of serine withposition 560. Alternatively, the cysteine introduced at position558 may not be able to serve as a palmitate addition site.

In all instances when a mutant virus was not rescued, RNPtransfections were repeated two or more times under well-controlled conditions. In these experiments, the rescue ofapproximately 103 wild-type transfectants per ml of virussupernatant was routinely achieved. Furthermore, the mostattenuated mutant (C560A) was consistently rescued (seebelow). In addition, all mutant plasmids were shown to yieldRNA levels in vitro similar to those obtained with pT3/WSN-HA/Hindlll (wild type). We thus suggest that mutations whichdo not result in the rescue of virus under our conditionsabrogate or seriously impair the function of the HA.

Expression of mutated HAs in transfected cells. In order toconfirm that the mutant constructs encode HA proteins whichare expressed on the cell surface, the HA cDNAs weresubcloned into the expression vector pSVK3 and tested for cellsurface expression by indirect immunofluorescence (see Mate-rials and Methods). All the mutant HA genes that were notrescued into live viruses encoded proteins whose expression at

B

9g

8 -

7.-

E0In)

I~-

6-

5 -

4-

3

0--N-

-U

12 24 36

time (h)

WSN wtC553AC553SC560A

48 60

-0-- C560Y-*- C560F0-- WSNwt

,rI l*0 1 2 24 36 48 60

time (h)FIG. 4. Growth characteristics of wild-type and transfectant viruses

on MDBK cells determined by plaque titration (A) and TCID50 assayon MDBK cells (B). It should be noted that mutant C560Y does notform discrete plaques, and thus the TCID50 assay was used for titrationof virus yields (for details, see Materials and Methods).

the cell surface was indistinguishable from that of the wild-typeHA. Immunofluorescence photographs of the wild-type HAand of the mutants C560S, C563A, C560A/C563A, and C553S/C560S/C563S are shown in Fig. 3. We conclude that theintroduced mutations do not significantly affect the biosynthe-sis or the transport of the mutant HA proteins, confirmingpreviously published results (14, 15, 25, 27, 29).Growth properties of transfectant viruses. The growth fea-

tures of the transfectants in MDBK cells were characterized(Fig. 4). The mutation of the conserved cysteine in themembrane-anchoring region barely affected viral growth. Mu-tant C553A behaves like the wild-type control, whereas themutant virus C553S showed a reproducible delay in virus

VOL. 68, 1994

5752 ZURCHER ET AL.

release and grew only to a fivefold-lower titer than thewild-type virus (Fig. 4A). However, mutant C560A grew to50-fold-lower titers than the wild-type virus (Fig. 4A). Inter-esting results were obtained when C-560 was replaced bydifferent amino acids. Whereas mutant C560Y was as attenu-ated as C560A virus, mutant C56OF reached-with delays-final titers comparable to those of wild-type virus (Fig. 4B).

Generation of revertants by passaging of attenuated mu-tants. We examined the importance of the conserved cysteineat position 560 of the cytoplasmic tail of HA by attempting toselect for revertant viruses of the attenuated mutants C560Yand C560A. Whereas only one nucleotide change is sufficientto change the codon for tyrosine (TAC) to that for cysteine(TGC), two nucleotide changes are necessary to change analanine codon (GCC) to a cysteine codon (TGC). Bothmutants could alter position 560 by single point mutations,resulting in a variety of amino acids other than cysteine.A preparation of mutant virus C560Y was grown by inocu-

lating a 75-cm2 tissue culture flask of MDBK cells with virusfrom a single plaque. The resulting preparation, with a titer of4 x 106 TCID50s per ml, contained about 104 particles whichformed plaques with wild-type characteristics. Viruses fromtwo large plaques of two independent virus preparations werefurther plaque purified, and viral RNA was prepared forsequencing. In all four cases the tyrosine had reverted to acysteine. Since there is a mutation in the 5' noncoding region(at position 1739; a T-to-C change) of the original plasmidconstruct (pC560Y), these viruses can be identified as truerevertants. Furthermore, viruses obtained from individualsmall diffuse C560Y plaques were passaged in MDBK cells(seven passages) or in eggs (eight passages). Again, reversionto cysteine occurred as confirmed by sequencing.

Similar experiments were performed with the mutantC560A, and neither reversion of the attenuated phenotype norchanges in the mutated sequences were observed. This result ismost likely due to the fact that a reversion from alanine tocysteine would require the simultaneous change of two nucle-otides. As expected, seven passages in MDBK cells of themarginally attenuated mutant C560F did not result in asequence change at this position (as confirmed by sequenceanalysis).

DISCUSSION

The carboxy-terminal sequences of the influenza virus HAsof 14 subtypes show remarkable conservation of three cysteineresidues. However, it has been reported (15, 25, 27, 29) thatsubstitution of the conserved cysteines does not affect the rateof intracellular transport or the receptor-binding or fusionactivities of the mutant HA proteins and that the cytoplasmictail cysteine residues are not required for the virus to beinfectious. We found these results surprising, since influenzavirus genome sequences are not strongly conserved in theabsence of a functional requirement.We therefore engineered single or multiple mutations of the

conserved cysteines in the cytoplasmic tail of the HA and triedto introduce these changes into the genomes of influenza virustransfectants. It was found that several changes of the cysteinesdid not lead to rescued viruses and that other cysteine changesresulted in viruses with attenuated phenotypes in cell cultures.These findings support the notion that the conserved cysteinesplay a biological role in maintaining the structure of thecytoplasmic tail and/or in offering palmitate attachment atthese sites.

Is the attachment of palmitate to the conserved cysteines inthe cytoplasmic tail of the HA required for the formation of

infectious influenza viruses? Mutants which lack a cysteine atposition 553 could be rescued, and in fact, mutant C553Agrows like the wild type. It is thus unlikely that palmitylation atthis site would be of importance for virus formation. Inaddition, Naim et al. (15) have shown that C-553 incorporatedonly about 10% of the total [3H]palmitate covalently attachedto the HA. Although Naim et al. (15) used an H2 subtype HA(A/Japan/305/57) for the quantification of the palmitylationlevels, we assume similar modification levels for the A/WSN/33virus HA (Hi), since this virus' 17 carboxy-terminal aminoacids are identical to those of the A/Japan/305/57 virus. Thecysteine residue at position 560 was shown to contain about40% of the incorporated palmitate (15). When C-560 wasreplaced by alanine, the mutant (C560A) was found to behighly attenuated. This finding is compatible with the idea thatpalmitylation at position 560 is not absolutely required for theformation of infectious virus but that it has an accessory role.Finally, viruses with mutations at position 563 of the HA couldnot be rescued. C-563 is conserved in the HAs of all 14subtypes, and according to Naim et al. (15), it has the highestpalmitylation level. Also, all mutant viruses with changes in twoor three of the palmitylation sites were not viable. Takentogether, these data suggest that palmitylation is significantand that the level of palmitylation of the three conservedcysteine residues (with C-563 possessing the highest level andC-553 possessing the lowest) correlates with the ability to forminfectious influenza virus particles.

Is a conserved structure of the cytoplasmic tail of the HArather than palmitylation required for the formation of infec-tious influenza virus? The fact that substitutions of cysteines bydifferent amino acids had different effects on the phenotypes ofviruses indicates a role for the structure in this region. Muta-tions to serine are more disruptive to viral growth than those toalanine: mutant C553S shows a delay in particle formation andgrows to about a fivefold-lower titer than the wild type,whereas mutant C553A grows normally (Fig. 4A). Further-more, mutant C560S was not viable, but C560A was rescued. Amutant HA protein with a serine at position 560 incorporatedonly about 50% of the fatty acid, in contrast to mutant proteinscontaining either tryptophan or tyrosine (15). Thus, a serine atposition 560 can affect the efficiency of palmitylation at otherpositions, presumably by changing the peptide structure in theHA tail. This indirect effect of serine at position 560 on thepalmitylation at position 563 might be the reason that themutant C560S was not rescued. Alternatively, the structuralchanges in the cytoplasmic tail may in themselves be sufficientto abrogate the viability of this mutant.

Surprisingly, mutant C56OF was only marginally attenuated.Since a phenylalanine is present at position 560 in the subtypeH13 HA, this amino acid might provide the optimal alternativestructure to cysteine. This idea is supported by the finding ofSugrue and colleagues (28), who showed that the conservedand palmitylated cysteine in M2 (at position 50) is changed toa phenylalanine in some equine influenza virus strains. Sincephenylalanine is a large hydrophobic amino acid, it mightfunctionally replace a modified cysteine.The mutant C560Y grows to peak titers about 2 log units less

than the wild type. Several independent revertants have beencharacterized, and the tyrosine was always changed back tocysteine by a single A-to-G nucleotide change. Single nucle-otide changes of the tyrosine codon can result in a codon thatencodes other amino acids, such as histidine (transition) orasparagine, aspartic acid, phenylalanine, and serine (transver-sion). However, none of these amino acids were observed inour revertants. This suggests that a cysteine is indeed thepreferred amino acid at position 560. However, since we

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PALMITYLATION SITES OF INFLUENZA VIRUS HEMAGGLUTININ 5753

sequenced only four revertants and since we did not test everypossible amino acid replacement at position 560, we cannotexclude the possibility that other amino acids are also welltolerated. An explanation for the growth advantage of thewild-type virus is that a palmitylated cysteine in this positionfavors virus assembly and/or that the wild-type sequence has apreferred structure allowing for appropriate interaction amongviral components. It was reported previously that a mutant HAprotein with a tyrosine at position 560 is no longer transportedto the apical membrane but instead is directed to the basolat-eral membrane (1). This change in transport rather than thealtered palmitylation site could represent a disadvantage forthe virus and provide an additional pressure for reversion ofthe virus at this site. Further experiments are necessary tocharacterize the C560Y mutant in polarized cells and toexamine the intracellular transport of the mutant HA protein.Using the reverse-genetics approach, we mutated the con-

served cysteines in the HA segment of transfectant influenza Aviruses. Our data suggest a biological significance for theconserved cysteines in the cytoplasmic tail of the HA ofinfluenza viruses and thus add a new dimension to the dataobtained by others (15, 25). In those earlier studies, HAmutants (subtypes H2 and H3) with substituted cysteine resi-dues were shown to complement the temperature-sensitive,transport-defective influenza virus mutant ts61S. The tran-siently expressed mutant HA proteins were incorporated intoinfectious viral particles (15, 25). However, this type of comple-mentation experiment cannot address the question of whetherthe cytoplasmic sequences of the HA are required for virusbudding or other biologically important steps, because atnonpermissive temperatures palmitylated fragments of thets61S HA may have been synthesized and they would havebeen capable of initiating virion assembly, as recently discussedby Naim and Roth (16). On the other hand, we cannot rule outthe possibility that the cysteines in the H2 or H3 subtype HAsused for the earlier studies play a less crucial role than in theinfluenza A/WSN/33 virus HA, which belongs to the Hisubtype. In summary, we were able to demonstrate a biologicalfunction for the conserved carboxy-terminal cysteines in an HiHA subtype for the formation of infectious virus particles.However, the determining factor responsible for this phenom-enon remains unclear: a change of one or more of the cysteineresidues may alter the overall level of palmitylation of the HAand thus affect the assembly process. Alternatively, the palmi-tylation of a distinct cysteine (C-563) in the HA may be crucialfor infectivity. Another possibility is that a change in theoverall structure of the cytoplasmic tail of the HA, by affectingthe protein-protein interactions required for virus formation, isthe determining factor in infectious-virus formation.

ACKNOWLEDGMENTS

We thank Wendy Barclay and Richard Peluso for critically readingthe manuscript, Adolfo Garcia-Sastre and Michael Bergmann forhelpful discussions, and JoEllen Barnett for editorial help. We alsothank Sandra Masur for help in making the immunofluorescencepictures. We thank Bernard Moss for kindly providing the recombi-nant vaccinia virus vTF7.3.

This work was supported by a grant from the Swiss NationalFoundation and the Swiss Foundation for Medical and BiologicalResearch (Schweizerische Stiftung fur Medizinisch-Biologische Sti-pendien [T.Z.]) and by grants from the NIH (P.P.).

REFERENCES1. Brewer, C. B., and M. G. Roth. 1991. A single amino acid change

in the cytoplasmic domain alters the polarized delivery of influ-enza virus hemagglutinin. J. Cell Biol. 114:413-421.

2. Doyle, C., M. G. Roth, J. Sambrook, and M.-J. Gething. 1985.

Mutations in the cytoplasmic domain of the influenza virushemagglutinin affect different stages of intracellular transport. J.Cell Biol. 100:704-714.

3. Enami, M., and P. Palese. 1991. High-efficiency formation ofinfluenza virus transfectants. J. Virol. 65:2711-2713.

4. Fuerst, T. R., E. G. Niles, F. W. Studier, and B. Moss. 1986.Eucaryotic transient-expression system based on recombinantvaccinia virus that synthesizes bacteriophage T7 RNA polymerase.Proc. Natl. Acad. Sci. USA 83:8122-8125.

5. Gaedigk-Nitschko, K., and M. J. Schlesinger. 1991. Site-directedmutations in the Sindbis virus E2 glycoprotein's cytoplasmicdomain and the 6K protein lead to similar defects in virusassembly and budding. Virology 183:206-214.

6. Hiti, A. L., A. R. Davis, and D. P. Nayak. 1981. Complete sequenceshows that the hemagglutinin of the HO and H2 subtypes of humaninfluenza virus are closely related. Virology 111:113-124.

7. Ivanova, L., and M. J. Schlesinger. 1993. Site-directed mutationsin the Sindbis virus E2 glycoprotein identify palmitoylation sitesand affect virus budding. J. Virol. 67:2546-2551.

8. Kawaoka, Y., S. Yamnikova, T. M. Chambers, D. K. Lvov, andR. G. Webster. 1990. Molecular characterization of a new hemag-glutinin, subtype H14, of influenza virus. Virology 179:759-767.

9. Lazarovits, J., and M. Roth. 1988. A single amino acid change inthe cytoplasmic domain allows the influenza virus hemagglutininto be endocytosed through coated pits. Cell 53:743-752.

10. Li, S., V. Polonis, H. Isobe, H. Zaghouani, R. Guinea, T. Moran, C.Bona, and P. Palese. 1993. Chimeric influenza virus inducesneutralizing antibodies and cytotoxic T cells against human immu-nodeficiency virus type 1. J. Virol. 67:6659-6666.

11. Luo, G., M. Bergmann, A. Garcia-Sastre, and P. Palese. 1992.Mechanism of attenuation of a chimeric influenza A/B transfec-tant virus. J. Virol. 66:4679-4685.

12. Luytjes, W., M. Krystal, M. Enami, and P. Palese. 1989. Amplifi-cation, expression, and packaging of a foreign gene by influenzavirus. Cell 59:1107-1113.

13. Mcllhinney, R. A. J. 1990. The fats of life: the importance andfunction of protein acylation. Trends Pharmacol. Sci. 15:387-391.

14. Naeve, C. W., and D. Williams. 1990. Fatty acids on the A/Japan/305/57 influenza virus hemagglutinin have a role in membranefusion. EMBO J. 9:3857-3866.

15. Naim, H. Y., B. Amarneh, N. T. Ktistakis, and M. G. Roth. 1992.Effects of altering palmitylation sites on biosynthesis and functionof the influenza virus hemagglutinin. J. Virol. 66:7585-7588.

16. Naim, H. Y., and M. G. Roth. 1993. Basis for selective incorpora-tion of glycoproteins into the influenza virus envelope. J. Virol.67:4831-4841.

17. Nobusawa, E., T. Aoyama, H. Kato, Y. Suzuki, Y. Tateno, and K.Nakajima. 1991. Comparison of complete amino acid sequencesand receptor-binding properties among 13 serotypes of hemagglu-tinins of influenza viruses. Virology 182:475-485.

18. Parvin, J. D., P. Palese, A. Honda, A. Ishihama, and M. Krystal.1989. Promoter analysis of influenza virus RNA polymerase. J.Virol. 63:5142-5152.

19. Portincasa, P., G. Conti, and C. Chezzi. 1992. Role of acylation ofviral haemagglutinin during the influenza virus infectious cycle.Res. Virol. 143:401-406.

20. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecularcloning: a laboratory manual, 2nd ed. Cold Spring Harbor Labo-ratory Press, Cold Spring Harbor, N.Y.

21. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencingwith chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA74:5463-5467.

22. Schlesinger, M. J., and C. Malfer. 1982. Cerulenin blocks fattyacid acylation of glycoproteins and inhibits vesicular stomatitis andSindbis virus particle formation. J. Biol. Chem. 257:9887-9890.

23. Schmidt, M. F. G., and B. Lambrecht. 1985. On the structure ofthe acyl linkage and the function of fatty acyl chains in theinfluenza virus haemagglutinin and the glycoproteins of SemlikiForest virus. J. Gen. Virol. 66:2635-2647.

24. Schulman, J. L., and P. Palese. 1977. Virulence factors of influ-enza A viruses: WSN virus neuraminidase required for plaqueproduction in MDBK cells. J. Virol. 24:170-176.

25. Simpson, D. A., and R. A. Lamb. 1992. Alterations to influenza

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5754 ZURCHER ET AL.

virus hemagglutinin cytoplasmic tail modulate virus infectivity. J.Virol. 66:790-803.

26. Staeheli, P., 0. Haller, W. Boll, J. Lindenmann, and C. Weiss-mann. 1986. Mx protein: constitutive expression in 3T3 cellstransformed with cloned Mx cDNA confers selective resistance toinfluenza virus. Cell 65:147-158.

27. Steinhauer, D. A., S. A. Wharton, D. C. Wiley, and J. J. Skehel.1991. Deacylation of the hemagglutinin of influenza A/Aichi/2/68 hasno effect on the membrane fusion properties. Virology 184:445-448.

28. Sugrue, R. J., R. B. Belshe, and A. J. Hay. 1990. Palmitoylation ofthe influenza virus M2 protein. Virology 179:51-56.

29. Veit, M., E. Kretzschmar, K. Kuroda, W. Garten, M. F. G.

Schmidt, H.-D. Kienk, and R. Rott. 1991. Site-specific mutagenesisidentifies three cysteine residues in the cytoplasmic tail as acyla-tion sites of influenza virus hemagglutinin. J. Virol. 65:2491-2500.

30. Ward, C. W. 1981. Structure of the influenza virus hemagglutinin.Curr. Top. Microbiol. Immunol. 94/95:1-74.

31. Whitt, M. A., L. Chong, and J. K. Rose. 1989. Glycoproteincytoplasmic domain sequences required for rescue of a vesicularstomatitis virus glycoprotein mutant. J. Virol. 63:3569-3578.

32. Wigler, M., R. Sweet, G. K. Sim, B. Wold, A. Pellicer, E. Lancy, T.Maniatis, S. Silverstein, and R. Axel. 1979. Transformation ofmammalian cells with genes from procaryotes and eucaryotes.Proc. Natl. Acad. Sci. USA 77:3567-3570.

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