Proc. Nat. Acad. Sci. USAVol. 80, pp. 3563-3567, June 1983Biochemistry

Defects in functional expression of an influenza virushemagglutinin lacking the signal peptide sequences

(vectorial discharge/polypeptide transport/glycosylation/hemagglutination)

KENJI SEKIKAWA AND CHING-JUH LAIMolecular Viral Biology Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20205

Communicated by Robert M. Chanock, March 7, 1983

ABSTRACT We have investigated the requirement of the sig-nal sequence for expression of influenza virus hemagglutinin (HA).For this purpose we used a recombinant prepared from a late-re-gion deletion mutant of simian virus 40 (SV40) and cloned influ-enza HA DNA; the influenza DNA was inserted into the late re-gion of SV40 previously occupied by the deleted sequences codingfor SV40 capsid proteins. A simple in-phase deletion was made inthe HA DNA, resulting in loss of 11 internal amino acids from the16 amino acid signal peptide. This deletion HA recombinant wasthen used to infect African green monkey kidney cells. Mutant HAwas not detected on the cell surface but stably accumulated in thecytoplasm at a level similar to that of wild-type HA. NaDodSO4/polyacrylamide gel analysis of lysates from infected cells showedthat mutant HA was not glycosylated. Significantly, the amountof mutant HA synthesized was not affected by tunicamycin. Incontrast, wild-type HA was decreased more than 90% by tuni-camycin. These findings suggest that mutant polypeptide is syn-thesized on free polyribosomes rather than on membrane-boundpolyribosomes. The mutant HA failed to agglutinate erythrocytes,probably due to a defect directly or indirectly associated with thelack of carbohydrate side chains.

The hemagglutinin (HA) of influenza virus constitutes the ma-jor viral envelope glycoprotein and is responsible for bindingof virus to sialic acid receptors on the cell surface (1). HA alsomediates membrane fusion that initiates uncoating of the virioninside the infected cell (2-5). During infection HA is synthe-sized in the cytoplasm, glycosylated, and transferred to the outercell membrane where the final stage of virus assembly takesplace. Functional HA contains three HA polypeptide subunitsand a specific protease cleavage of each subunit is required forviral infectivity (6, 7).

Sequence analysis of HA from many influenza virus stainsshows the presence of a hydrophobic sequence at the aminoterminus and another hydrophobic sequence near the carboxylterminus (8-11). The hydrophobic carboxyl terminus is re-sponsible for anchoring the polypeptide on the outer mem-brane because HA lacking these sequences is secreted extra-cellularly (12). On the other hand, the hydrophobic aminoterminus is cleaved during maturation and is therefore absentin the membrane-integrated HA (13). These transient hydro-phobic sequences, commonly referred to as the signal peptide,exist in all known eukaryotic transmembrane and secretory pro-teins with several notable exceptions, such as ovalbumin andthe influenza virus neuraminidase (14-16). Similar transientsignal peptides also exist in the periplasmic proteins of pro-karyotes. It has been postulated that the signal peptide seg-ments play a crucial role in mediating the transfer of polypep-tide across the membrane by a specific mechanism known as

vectorial discharge of nascent polypeptide (17, 18). Synthesisand transfer of proteins utilize a specialized machinery of mem-brane-bound polyribosomes and the Golgi apparatus in eukary-otes. Mutational analysis of the signal sequences to further elu-cidate the transfer process has not been reported in eukaryoticsystems.

Previously we constructed a recombinant consisting of a late-region deletion mutant of simian virus 40 (SV40) and clonedinfluenza DNA representing the complete sequences of the genecoding for the HA surface glycoprotein. The cloned influenzaDNA was inserted into the late region of SV40 previously oc-cupied by the deleted sequences coding for SV40 capsid pro-teins. This HA-SV40 recombinant expressed fully functionalHA in African green monkey kidney (AGMK) cells. The HAwas glycosylated and inserted into the outer membrane of theinfected cell (19). This system was used in the present study toexamine the role of the signal peptide in HA maturation andfunction. In this study, we describe the construction of a dele-tion recombinant that produced HA lacking the signal peptidesequences. Analyses of functional defects in the mutant HA thataffect glycosylation, maturation at the cell surface, and hem-agglutination activity are presented.

MATERIALS AND METHODSPreparation of DNA and Construction of HA-SV40 Dele-

tion Mutants. Previously we cloned a full-length DNA copycoding for the HA of influenza virus strain A/Udorn/72 (H3N2).To achieve functional expression the cloned HA DNA was in-serted into the late region of a SV40 vector and a recombinantof HA-SV40 that produced a functionally active HA was iso-lated. Subsequently the HA-SV40 DNA recombinant was clonedin pBR322 by using the unique BamHI cleavage site (19).

The recombinant pHA-SV40 DNA was prepared from a clonedEscherichia coli transformant as described (19). DNA frag-ments prepared by restriction enzyme digestion were sepa-rated on 3.5% polyacrylamide gels and eluted electrophoreti-cally. The Mbo II DNA fragment downstream of the cleavagesite was digested with A exonuclease (28 units/ml) in a buffercontaining 67mM glycine (pH 9.4), 3 mM MgC12, 3 mM 2-mer-captoethanol at 0°C for 30 min (20). Other enzyme digestionswere carried out according to the prescribed conditions by theirsuppliers. Constructed plasmid recombinants carrying a signalpeptide deletion were subjected to sequence analysis by themethod of Maxam and Gilbert (21). Mutant HA-SV40 DNAwas isolated after BamHI digestion, circularized, and used forcoinfection of AGMK cells with a tsA28 SV40 early-region mu-tant helper virus.

Radiolabeling and Analysis of HA Polypeptides. AGMK cellsinfected with the mutant HA-SV40 and incubated at 40°C for

Abbreviations: HA, hemagglutinin; SV40, simian virus 40; AGMK, Af-rican green monkey kidney.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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72 hr were labeled with [35S]methionine (100 ACi/ml; 1 Ci =3.7 X 1010 Bq) in a methionine-free medium for 5 hr. Labelingof cells during infection with the wild-type HA-SV40 was car-ried out similarly. Cells were directly lysed in radioimmuno-precipitation assay buffer and the supernatant fraction was im-munoprecipitated for analysis on NaDodSO4/polyacrylamidegels (19).

Indirect Immunofluorescence Assay. Three days after in-fection with either wild-type or mutant HA-SV40, unfixed cellmonolayers were stained directly in a live cell fluorescence as-say with sheep antiserum against HA and fluorescein-conju-gated rabbit anti-sheep IgG. Fixed cell fluorescence assay wasperformed on cells treated with 3.7% formaldehyde for 10 minand 0.1% Triton X-100 for 5 min as described by Wehland etal. (22).

Hemagglutination Test. Lysates of recombinant virus-in-fected cells were prepared in phosphate-buffered saline and se-rially diluted in a microtiter plate. Guinea pig erythrocytes (0.1%suspension in phosphate-buffered saline) were added to eachdilution and hemagglutination activity was examined after 30min at room temperature.

RESULTSConstruction of Mutant HA Encoding Deleted Signal Se-

quences. The wild-type HA-SV40 recombinant cloned previ-ously at the unique BamHI site of pBR322 was used for thederivation of HA deletion mutants. Fig. 1 illustrates the strat-egy used to construct HA mutants lacking the signal peptidesequences. EndoR Mbo II cleaves the HA-SV40 plasmid onceat a site 12 base pairs downstream of the initiation codon of theHA and several other sites outside the HA gene. Digestion ofpHA-SV40 with Mbo II yielded two DNA fragments containingHA-specific sequences. The DNA fragment downstream of thecleavage site was briefly treated with A exonuclease to shortenboth DNA strands progressively, whereas the other Mbo II

Bam

Mbo 11 /

Mbo 11

KpnXba

Kpn

Xba

Kpn

Bam Hi

FIG. 1. Construction of signal peptide deletion mutants of HA-SV40.

fragment was not treated. After nuclease S1 digestion, both HADNA fragments were joined. In this manner, a series of dele-tion mutations were introduced at the Mbo II cleavage site whileretaining the upstream Mbo II enzyme recognition site. Thejoined HA 'DNA from the ligase reaction mixture was cleavedwith Kpn I and Xba I to generate cohesive termini. This spe-cific DNA fragment was isolated and rebuilt into the pHA-SV40plasmid by joining it to the other fragment from the Kpn I/XbaI digest of pHA-SV40. In this manner, the signal deletion wasintroduced into the wild-type HA DNA molecule and repre-sented the only mutation in the HA-SV40 recombinant. Theconstructed DNA was used directly for cloning by transfor-mation of E. coli.

Sequence Analysis of Mutant HA Lacking the Signal Pep-tide. The constructed deletion mutant DNAs were isolated fromE. coli transformants and characterized by digestion with EndoRAva II and polyacrylamide gel electrophoresis. For compari-son, similarAva II digestion of the wild-type pHA-SV40 yieldeda 338-nucleotide fragment containing the signal sequences andthis type of analysis allowed us to estimate the size of the cor-responding fragments of mutant DNA. These estimates al-lowed us to determine the extent of deletion and served to guideus in choosing mutant recombinants for sequence analysis.Among six mutant isolates shortened by 30-100 nucleotides,we found one mutant that contained an in-phase deletion,whereas the other five showed either a + 1 or a -1 frameshiftdeletion. The latter were not analyzed further. The wild-typenucleotide sequence and the encoded signal peptide sequenceat the amino terminus of HA were compared with the se-quences of the HA-SV40 mutant (Fig. 2). -The HA signal pep-tide consists of 16 amino acids-1 charged amino acid (Lys), 11hydrophobic amino acids (Met, Ile, Ala, Leu, Phe, Val, and Gly),and 4 uncharged polar amino acids (Thr, Ser, Tyr, and Cys).The proteolytic cleavage site is located at the Gly-Gln juncturebecause Gln is the amino terminus of the membrane-incor-porated HA molecules. The mutant HA sustained a deletion of33 base pairs or 11 amino acids, all within the signal peptide.As a result, the remaining signal region contains three hydro-phobic amino acids (Met, Ile, and Gly), a polar uncharged aminoacid (Thr), and a charged amino acid (Lys). The signal peptidecleavage site of Gly-Gln and the subsequent sequences re-mained unchanged in the mutant.

Analysis of HA by Indirect Immunofluorescence Assay. Anindirect immunofluorescence assay was employed to identifythe intracellular site of accumulation of the mutant HA in theinfected cell. Initially, the deletion mutant genome of HA-SV40was packaged within SV40 virions and propagated together withthe SV40 ts helper virus at the restrictive temperature (400C).AGMK cells were infected with the SV40 ts helper and eitherthe mutant or the wild-type HA-SV40 recombinant for 3 daysprior to assay for HA by live cell fluorescence staining with HAantibody and an appropriate fluorescein conjugate. Fluores-cence was not detected on mutant infected cells, whereas cellsinfected with wild-type HA-SV40 showed positive surfacestaining (data not shown). When infected cells were fixed andstained for HA fluorescence, wild-type HA was detected in thecytoplasm and on the membrane. In contrast, the mutant HAwas found only in the cytoplasm. The mutant HA was stainedas intensively as the wild-type HA (Fig. 3). These results dem-onstrate that mutant HA lacking the signal sequences accu-mulates in the cytoplasm but is not incorporated into the outermembrane.

Analysis of HA by NaDodSO4/Polyacrylamide Gel Elec-trophoresis. We estimated the molecular size of the mutant HAproduct to determine whether the mutant polypeptide is post-translationally modified by glycosylation in the same manner as

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MboI I

wt-HA ATG MG ACT ATC ATT GCT TTG AGC TAC ATT TTC TGT CTG GTT CTC GGC CM GAC ---TVA TTC TGA TAG TM CGA MC TCG ATG TM MG ACA GAC CM GAG CCG GTT CTG ---

Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Phe Cys Leu Val Leu Gly Gin Asp ---

dl-HA ATG AAG ACT ATCTAC TTC TGA TAG (Met Lys Thr Ile

33-bp deletion

FIG. 2. Nucleotide sequences of the wild-type and the derived deletion mutant. The nucleotide sequences and the encoded amino acids at theamino terminus are shown. The wild-type HA contains a signal peptide of 16 amino acids (underlined) which is cleaved by a protease. Also indicatedis the Mbo II cleavage site where deletions are introduced to obtain this series of mutants. The mutant analyzed in this study lacks 11 of the 16amino acids. The mutant DNA retains an Mbo II cleavage site because the G-A-A-G-A Mbo II recognition sequence is still present. bp, base pair.

the wild-type HA. Tunicamycin was used as an inhibitor of gly-cosylation to determine if the mutant HA was modified by ad-dition of carbohydrate side chains. If the HA was glycosylatedthere should be a reduction in size of the HA in the presenceof the inhibitor. On the other hand, if the mutant HA was notglycosylated it would be the same size whether or not tunica-mycin was present. Immunoprecipitation of radiolabeled ex-

tracts from infected cells was performed and the precipitateswere analyzed on NaDodSO4/polyacrylamide gels. As a con-

trol, a lysate from AGMK cells infected with the wild-type HA-SV40 recombinant was used. Molecular mass of HA from thewild-type recombinant was estimated to be 70,000-75,000 dal-

wt- HA

tons, similar in size to the glycosylated HA produced duringinfluenza virus infection (Fig. 4). In the presence of tunica-mycin, an inhibitor that prevents polypeptide glycosylation (23),this value was decreased to 56,000 daltons, the predicted sizefor unglycosylated HA. It should be noted that addition of tuni-camycin also decreased the accumulation of unglycosylated wild-type HA by =90%. Similar analysis showed that the mutantrecombinant produced a HA polypeptide of 56,000 daltonsequivalent to the predicted molecular size for unglycosylatedHA. When tunicamycin was added during radiolabeling, themutant HA product that corresponded to the most prominentband was not decreased in size and remained at 56,000 daltons.Other minor bands from the deletion mutant were probablyderived from rearranged HA sequences that arose during pas-sages in AGMK cells. These results provide evidence- that themutant polypeptide synthesized during infection was not mod-ified by glycosylation posttranslationally. In contrast to the wild-type HA tunicamycin did not show an inhibitory effect on thesynthesis of mutant HA.

Hemagglutination Activity of Unglycosylated Mutant HA.Lysates from AGMK cells were prepared after infection with

M wt-HA di-HA Mock

Tunicomycin - + - + -

0m~

d - H A 70-75 - '

(HAo)

56 -

FIG. 3. Indirect immunofluorescence assay ofHA in infected cells.AGMK cells were infected with the SV40 ts helper and either the wild-type HA-SV40 virus (Upper) or the mutant HA-SV40 virus (Lower) for72 hr. Cells were fixed and stained as described in the text. Accumu-lation of the wild-type HA on the outer membrane is discernible. Themutant HA stains as intensely as wild-type HA in the cytoplasm butdoes not accumulate on the membranes.

28- _26 -

FIG. 4. NaDodSO4/polyacrylamide gel-analysis of the wild-type andmutant HAs. Monolayers ofAGMK cells were infected with the wild-type HA-SV40 (wt-HA) and mutant HA-SV40 (dl-HAY for 72 hr. In-fected cells were pretreated with tunicamycin (1 ,ug/ml) for 60 min priorto labeling with [3Slmethionine (100 ,uCi/ml) in methionine-free me-dium in the presence of 1 ug of tunicamycin per ml. Cell lysates wereimmunoprecipitated with HA antiserum and analyzed on 15% Na-DodSO4/polyacrylamide gels. M indicates the polypeptide markers(shown in daltons x 10-3) prepared from [35S]methionine-labeled in-fluenza virions.

GGC CM GAC ---

) CCG GTT CTG ---

Gly Gln Asp ---

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either the wild-type HA-SV40 or the mutant HA-SV40 and wereexamined for ability to agglutinate guinea pig erythrocytes. Ina control experiment, a hemagglutination titer of 1:8 was ob-served for the wild-type HA. The lysate containing the ungly-cosylated mutant HA at an antigen concentration comparableto the wild-type HA did not show detectable hemagglutinationactivity (data not shown).

DISCUSSIONThis study concerns the functional role of HA signal sequencesin the processing and maturation of the H3 subtype HA at thecell surface. We constructed deletion mutants containing adeletion of the signal peptide at the Mbo II cleavage site. MboII cleaves at a site eight nucleotides downstream from its rec-ognition sequence 5' G-A-A-G-A 3' such that a one-nucleotide3' protrusion is generated. The current construction schemeallowed the retention of sequences including the Mbo II rec-ognition site and those encoding the first three amino acids.After construction the deletion juncture should become a newMbo II cleavage site; however, in our mutant, two additionalbases were deleted from the upstream fragment for reasons notclear to us. This created a new Mbo II cleavage site three basesafter the juncture. Nonetheless, it should be possible to applythe scheme used in our study for the construction of deletionsof any length and thereby obtain a series of progressively ex-tended HA signal peptide mutants. Analysis of these HA mu-tants should yield information concerning the signal sequencesrequired for functional activity. The mutant constructed in thisstudy, with a deletion of 11 of the 16 amino acids in the signalsequence, was suitable for characterization of defective func-tions. This mutant retained the presumptive amino-terminalsignal-peptide protease cleavage site and therefore specificcleavage at this site did not preclude the observed abnormal-ities in HA function.Our results demonstrate that the deleted HA polypeptide

from the constructed mutant is not posttranslationally modifiedby glycosylation despite the presence of all normal Asn-X-Thrand Asn-X-Ser sequences. The signal hypothesis predicts thatwild-type HA mRNA is translated on membrane-bound poly-ribosomes of the endoplasmic reticulum and the nascent HApolypeptides are subsequently transferred to the lumen by amechanism of translocation and vectorial discharge. There isconsiderable evidence suggesting that glycosylation takes placeinitially on nascent polypeptides on endoplasmic reticulummembranes and subsequently the carbohydrate components aremodified in the Golgi apparatus (24, 25). It is interesting to notethat production of wild-type, fully glycosylated HA (in the ab-sence of tunicamycin) was 10-fold higher than that of ungly-cosylated HA (in the presence of tunicamycin). One possibleexplanation is that tunicamycin inhibition freezes these gly-cosylation steps and hence translation of HA mRNA is sloweddown, although a low level synthesis of unglycosylated wild-typeHA is eventually completed. Alternatively, tunicamycin in-creases the breakdown of unglycosylated wild-type HA becauseglycosylation confers intracellular stability and resistance toproteolytic digestion (26). On the other hand, translation of mu-tant HA mRNA probably proceeds on free polyribosomes andtherefore the nascent polypeptide lacking the signal sequencesfails to translocate. The nascent as well as the completed mutantpolypeptides do not encounter glycosyl transferases, which aremembrane-associated, and the unglycosylated HA moleculesremain in the cytoplasm. The finding that tunicamycin exertsno detectable inhibition on the accumulation of mutant HA fur-ther supports the model of synthesis on free polyribosomes.Our results show that the HA polypeptide lacking the tran-

sient signal sequences is not detectable at the cell surface. Thetransport of polypeptides from the cytoplasm to the outermembranes or for extracellular secretion is a complex process,presumably initiated by the signal sequences. Recent evidencesuggests that the signal sequences at the amino terminus ofpolypeptides specify the initial recognition by a multiple-com-ponent structure known as signal peptide recognition particles(27, 28). This recognition process did not occur with the mutantHA polypeptide in simian cells. In E. coli, similar hydrophobicsequences that provide a transient signal for polypeptide trans-port also exist in several periplasmic proteins. Deletion or al-teration of these sequences results in accumulation of theseproteins in the cytoplasm (29, 30).The HA-SV40 mutant produces unglycosylated HA that is

functionally inactive, as assayed by the hemagglutination test.This failure to agglutinate erythrocytes suggests at least twopossibilities. First, the mutant HA may be monomeric and thusfail to assemble into a trimeric structure, which is required forfunctional activity. Second, the defect of mutant HA may bedue to the lack of carbohydrate components and as a result themutant HA subunit fails to assemble properly. Because 50% ofthe carbohydrate side chains of HA can be removed withoutaffecting hemagglutination of infectivity (31), it would seem thatat least some carbohydrate components are not crucial. There-fore, it is most likely that the assembly process to form matureactive surface HA may require a defined configuration of thepolypeptide subunit that is programmed by posttranslationalmodifications such as glycosylation.

Other observations support the view that deletion within thesignal peptide of the HA polypeptide is most likely responsiblefor the functional defects described in this paper. Recently wehave found that several HAs containing substitutions of aminoacids in the signal peptide produced by point-mutation in HA-SV40 also showed similar defects of glycosylation and cell sur-face expression (unpublished data). This finding effectively rulesout the possibility that a mutation other than the signal se-quence deletion in our mutant was responsible for the observedfunctional abnormalities.

There are a number of differences evident when our resultsare compared with those of a similar recently published study(32). First, we constructed mutants that contained a simple in-ternal deletion in the HA signal sequence. The deletion HAthat was studied extensively preserved the first four amino-ter-minal amino acids and the remaining sequences.. Therefore,characterization of this mutant allowed us to correlate specificfunctional defects with this deletion. In contrast, using a dif-ferent construction procedure, other investigators derived amutant that sustained a deletion in the 5'-noncoding region ofthe HA recombinant. In addition, three different amino-ter-minal amino acids were added to replace the entire signal pep-tide (except the carboxyl-terminal Gly). Presumably as a con-sequence of either or both of these alterations, the mutant HAwas synthesized at a level 0. 16% that of wild-type HA. Hence,it is difficult to interpret the lack of glycosylation of the smallnumber of HA molecules that were produced. In contrast, mu-tant HA polypeptides directed by our mutant were made in theamount similar to that of wild-type HA molecules. Conse-quently, the absence of glycosylation of our mutant HA and itsfailure to migrate to the cell membrane can be convincingly at-tributed to deletion within the signal peptide.The authors thank Dr. Robert M. Chanock for helpful discussions

and useful suggestions about the manuscript. We also thank Ms. Jo AnnBerndt and Ms. Salome Kruger for their excellent technical assistanceand Ms. Linda Jordan for typing the manuscript.

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