Transcription-Inhibition and RNA-Binding Domains of Influenza A Virus Matrix Protein Mapped with Anti-Idiotypic Antibodies and Synthetic Peptides

Transcription

1 JOURNAL OF VIROl OGY, Sept. 1989, p X/89/ $2./ Copyright 1989, American Society for Microbiology Vol. 63, No. 9 Transcription-Inhibition and RNA-Binding Domains of Influenza A Virus Matrix Protein Mapped with Anti-Idiotypic Antibodies and Synthetic Peptides ZHIPING YE, NORMAN W. BAYLOR, AND ROBERT R. WAGNER* Depar-tmen7t of Microbiology and Cancer- Ceniter, Uni i'ersity of Virginia Medical School, Charlottesv,ille, Vir-ginia 2298 Received 16 March 1989/Accepted 11 May 1989 We have undertaken by biochemical and immunological experiments to locate the region of the matrix (M,) protein responsible for down-regulating endogenous transcription of A/WSNI33 influenza virus. A more refined map of the antigenic determinants of the M, protein was obtained by binding of epitope-specific monoclonal antibodies (MAbs) to chemically cleaved fragments. Epitope 2-specific MAb 289/4 and MAb 7E5 reverse transcription inhibition by M, protein and react with a 4-kilodalton cyanogen bromide fragment extending from amino acid Gly-129 to Gln-164. Anti-idiotype serum immunoglobulin G prepared in rabbits immunized with MAb 289/4 or MAb 7E5 mimicked the action of M, protein by inhibiting transcription in vitro of influenza virus ribonucleoprotein cores. This transcription-inhibition activity of anti-mab 7E5 immunoglobulin G and anti-mab 289/4 immunoglobulin G could be reversed by MAb 7E5 and MAb 289/4 or could be removed by MAb 7E5-Sepharose affinity chromatography. Transcription of influenza virus ribonucleoprotein was inhibited by one of three synthetic oligopeptides, a nonodecapeptide SP3 with an amino acid sequence corresponding to Pro-9 through Thr-18 of the Ml protein. Of all the structural proteins of influenza virus, only NP and Ml showed strong affinity for binding viral RNA or other extraneous RNAs. The 4-kilodalton cyanogen bromide peptide (Gly-129 to Gln-164), exhibited marked affinity for viral RNA, the binding of which was blocked by epitope 2-specific MAb 7E5 but not by MAbs directed to three other epitopes. Viral RNA also bound strongly to the nonodecapeptide SP3 and rather less well to anti-idiotype anti-mab 7E5; these latter viral RNA-binding reactions were only slightly blocked by preincubation of anti-mab 7E5 or SP3 with MAb 7E5. These experiments suggest the presence of at least two RNA-binding sites, which also serve as transcription-inhibition sites, centered around amino acid sequences 8 through 19 (epitope 4?) and 129 through 164 (epitope 2) of the 252 amino acid M1 protein of A/WSN/33 influenza virus. A hydropathy plot of the Ml protein calculated by free-energy transfer suggests that the two hydrophilic transcription-inhibition RNA-binding domains are brought into close proximity by an a-helix-forming intervening hydrophobic domain. Virions of influenza virus consist of helical, segmented ribonucleoprotein (RNP) cores surrounded by a membrane with protruding hemagglutinin and neuraminidase glycoproteins and a matrix (M1) protein that lines the inner layer of the membrane (14). The M1 protein, encoded by RNP segment 7 (1, 13), also binds to RNP cores and apparently plays a key role in assembly and budding of progeny virions (7, 12). Interaction of the M1 protein with RNP cores is also implicit in the finding that M1 protein inhibits RNA synthesis by the RNP-associated transcriptase of influenza virus (25). The antigenic determinants of the M1 protein of A/WSN/ 33 influenza virus (HON1) were partially mapped recently by reactivity with monoclonal antibodies (MAbs) of proteolytically cleaved and partially sequenced M1 protein fragments (22). These studies were performed with MAbs directed to three epitopes that were provided by K. L. van Wyke (21); these studies have been confirmed by using MAbs generated in our laboratory, which also resulted in identification of a fourth epitope in the M1 protein (Ye and Wagner, unpublished data). Epitope 1 (MAb M2-1C6) is located between amino acids 8 and 89, whereas epitope 2 (MAb 289/4 and MAb 7E5) and epitope 3 (MAb 94/6) are located between amino acids 89 and 141 or somewhat more carboxy distal. The N-terminal 9-kilodalton (kda) fragment generated by * Corresponding author. t Present address: National Center, Frederick, MD Cancer Institute Frederick Cancer formic acid binds to phosphatidylcholine vesicles, whereas the C-terminal 15-kDa formic acid fragment inhibits influenza virus RNP transcription, an effect which is reversed by epitope 2-specific MAb 289/4 (22). These earlier findings indicated that epitope 1 and the membrane-binding site of M1 protein map N proximal to amino acid 89, whereas epitope 2 and the transcription-inhibition site map carboxy distal to amino acid 89 of the M1 protein (22). In the current studies, we undertook experiments to map more precisely the site or sites on the M1 protein responsible for in vitro down-regulation of influenza virus RNP transcription as well as more circumscribed mapping of the antigenic determinant, epitope 2, as a putative site for transcription inhibition by M1 protein. In addition to more refined chemical cleavage, these studies involve the transcription-inhibition activity of anti-idiotypic immunoglobulins and synthetic oligopeptides corresponding to M1 protein amino acid sequences as well as the affinity of Ml protein and its peptides for binding influenza virus RNA. MATERIALS AND METHODS Virus and cells. All experiments were performed as previously described (22) with influenza virus A/WSN/33 (HON1). Unlabeled virus was produced by infecting the allantoic sac of 1-day-old chicken embryos with cloned stock virus and harvesting allantoic fluids 48 h later. Viral RNA was biologically labeled with 32P (15 p.ci/ml) in phosphate-free Eagle Downloaded from on September 19, 218 by guest 3586

2 VOL INFLUENZA VIRUS Ml PROTEIN RNP-BINDING SITES 3587 minimum essential medium on confluent monolayers of MDCK cells or chicken embryo fibroblasts. Preparation of RNP cores and purified Ml protein. As previously described in detail (22), enzymatically active RNP cores of influenza virus were obtained by disrupting virions in 1% Nonidet P-4 in a ph 8. tricine buffer containing.25 M NaCl. After viral membrane protein was removed by glycerol gradient centrifugation, purified RNP cores had a full complement of RNA polymerase activity and only traces of M1 protein as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Ml protein was isolated from virions and purified by the acidic chloroform-methanol extraction procedure of Gregoriades (11) as previously described in detail by us (22). The M1 protein recovered in the organic phase, evaporated under nitrogen, and lyophilized was >9% pure as determined by SDS-polyacrylamide gel electrophoresis. MAbs to Ml protein of A/WSN/33 influenza virus. Three of the hybridoma cell lines producing MAbs to the A/WSN/33 influenza virus M1 protein were a generous gift of Kathleen van Wyke of the National Institute of Allergy and Infectious Diseases Laboratory of Infectious Diseases (21). MAb M2-1C6 has been assigned to epitope 1, MAb 289/4 has been assigned to epitope 2, and MAb 94/6 has been assigned to epitope 3 (22). Other hybridomas secreting MAbs to the Ml protein of A/WSN/33 influenza virus have been produced in the Lymphocyte/Hybridoma Center of the University of Virginia School of Medicine. One MAb used in these studies (MAb 7E5) has been assigned to epitope 2 based on enzymelinked immunosorbent assay (ELISA) competitive binding studies, and another (MAb 5C9) cross-reacts with none of the other MAbs and has been assigned to a new epitope 4 (data not published). Large amounts of each MAb were obtained by producing ascites in Pristane-primed BALB/c mice injected intraperitoneally with i7 hybridoma cells from each clone. Immunoglobulins of class immunoglobulin GI (IgGl) or IgG2a specific for each epitope were purified by chromatography on staphylococcal protein A-Sepharose columns. The specificity of each MAb IgG was determined by Western blot analysis and competitive binding of paired MAbs. Preparation of anti-idiotypic polyclonal antibody. Antiidiotypic polyclonal antibody (1) was generated in New Zealand White rabbits injected intradermally at multiple sites with 1 mg of MAb 7E5 or MAb 289/4 IgG (subclass IgGI) emulsified in complete Freund adjuvant. Control serum was obtained from each of the four rabbits, two before immunization with MAb 7E5 and two before immunization with MAb 289/4. Booster intradermal injections with.5 mg of each purified MAb IgG emulsified in incomplete Freund adjuvant were administered at 4, 7, 1. and 13 weeks after primary immunization. Serum collected from clotted blood of each rabbit exsanguinated 5 and 13 weeks after the course of immunization was clarified by centrifugation at 8, x g for 2 min at 4 C. Anti-mouse normal immunoglobulins were removed from each serum by affinity chromatography through a column of Sepharose-linked normal mouse immunoglobulin; the anti-idiotypic IgG passes through the column. The binding affinity of MAb IgG to anti-idiotypic IgG was determined by an ELISA, in which polyclonal IgG placed in wells of microdilution plates (5 pg per well) was incubated at room temperature for 16 h in IgG coating buffer consisting of.15 M Na,CO3,.35 M NaHCO, (ph 9.6), and.1cc sodium ethylmercurithiosalicylate. After 1% gamma-globulin-free horse serum was added for 1 h at room temperature, normal mouse IgG (1 pg) was added to each well and incubated for 3 min. Serial dilutions of biotin-labeled mouse MAb were then added to the wells, and finally 1 RlI of Streptoviridin peroxidase (.25 pg/ml in phosphate-buffered saline) was added to each well. The ELISA reaction for each substrate was read at a wavelength of 414 nm. Influenza virus transcription in vitro. RNP cores or whole virions were suspended in 1 LI of transcription buffer containing 1 mm Tris hydrochloride (ph 7.8), 1.25 mm MnCl,, 1 mm NaCl, 1 mm dithiothreitol, 7.5 mm MgCl.,.2% Nonidet P-4 (for whole virion transcription),.4 mm ApG, 1 mm each of ATP. GTP, CTP, and.1 mm UTP containing 5 pci of kx-32p]utp (41 Ci/mmol; Amersham Corp.. Arlington Heights, Ill.). Transcription mixtures were incubated for 2 h at 31 C, and the reactions were terminated by the addition of.6 ml of sodium pyrophosphate (67 p.m). Carrier yeast trna (5 p.g) was then added to the reaction mixture, and the total RNA was precipitated with an equal volume of 1% trichloroacetic acid. The mixture was kept on ice for 2 min, and the acid-insoluble RNA was collected by filtration through glass fiber filters (GF/A; Whatman, Inc., Clifton, N.J.). The radioactive content in the filter paper was measured by scintillation spectrometry. Viral RNA-protein interaction assay. Influenza virion proteins, purified Ml protein, and proteolytically cleaved M1 protein were tested for their capacity to bind viral RNA or rrna. Viral RNA was biologically labeled with 32P by growing influenza virus or vesicular stomatitis virus (VSV) in confluent cultures in the presence of phosphate-free Eagle minimal essential medium containing I3P,. 32P-labeled viral RNA was extracted from released virions by the modified method of Both and Air (4), in which virions are incubated in 5 ml of.1 M Tris hydrochloride (ph 7.4)-.1 M KCI-.15 M MgCl,-.2E% SDS-1 mg of proteinase K for 2 min at 56 C. After the virus suspensions were adjusted to.15 M NaCl, they were mixed with equal volumes of water-saturated phenol at 56 C for 5 min and then equal volumes of chloroform for 2 min at room temperature during nitrogen gas bubbling. Viral RNA in the aqueous phase after lowspeed centrifugation was adjusted to.3 M sodium acetate and precipitated with 2 volumes of ethanol. Viral proteins or cleaved M1 protein fragments were separated by electrophoresis on 17.5% polyacrylamide-sds gels. electroblotted onto nitrocellulose sheets, and tested for their capacity to bind 32P-labeled RNA by the method of Bowen et al. (5), with minor modifications. In some cases, M1 proteins or peptides were directly spotted onto nitrocellulose sheets, which were then washed with a probing buffer consisting of 1 mm Tris hydrochloride (ph 7.5), 5 mm NaCl, 1 mm EDTA..2% bovine serum albumin (BSA),.2% Ficoll, and.2% polyvinylpyrrolidone for 1 h at room temperature. Duplicate nitrocellulose sheets were then incubated either with 3% BSA or with MAbs. The nitrocellulose sheets were then incubated for 1 h at room temperature with the same probing buffer containing 32P-labeled RNA in a 1:4, ratio with carrier yeast trna. The sheets were then washed five times in probing buffer, dried, and subjected to autoradiography. RESULTS Further mapping the antigenic determinants of the M, protein. In previous studies (22) we partially mapped three epitopes of the Ml protein by reactivity of proteolytically cleaved fragments with MAbs prepared by van Wyke et al. (21). Of two peptides formed by cleavage with formic acid, a Downloaded from on September 19, 218 by guest

3 3588 YE ET AL. J. VIROL. N NCS FA NCS CNBr CNBr v I VV V I1 I - Ep1V[Ep3& 4 1 Ep2 I II s s s s S _ V I IC FA NCS CNBr L Met- 1 MAb M2-1C6 Asp-89 Leu-46 MAb M2-1C6,94/6,5C9 Tyr-1 19 MAb 7E5 Gly-129 GIn-164 FIG. 1. Linear map of the epitopes (Ep) on the M1 protein of A/WSN/33 influenza virus as determined by binding of four epitope-specific MAbs to peptide fragments chemically cleaved with formic acid (FA), NCS, or CNBr. CNBr-cleaved M1 peptides were separated by electrophoresis on a 1 to 2% gradient polyacrylamide gel containing 7 M urea, whereas the formic acid-cleaved and NCS-cleaved M1 peptides were separated by electrophoresis on 15% polyacrylamide-7 M urea gels. The separated peptides were electroblotted onto nitrocellulose sheets and exposed to MAb M2-1C6 (epitope 1), MAb 7E5 (epitope 2), MAb 94/6 (epitope 3), or MAb 5C9 (epitope 4). Bound MAbs were measured by reaction with 125I-labeled staphylococcal protein A, autoradiography, and laser densitometry. The M1 map location of each peptide generated and shown above was determined by N-terminal amino acid sequencing after purification by high-performance liquid chromatography or after electroblotting onto polyvinylidene difluoride and aligning the peptides with the deduced amino acid sequence of the 252 amino acid M1 protein (3). The stick model of the M1 protein portrays the deduced boundary limits of the four epitopes and the chemical cleavage sites. 9-kDa fragment at the amino-terminal third of the M1 protein was recognized by epitope 1-specific MAb M2-1C6, and a 15-kDa fragment at the carboxy-terminal two-thirds was recognized by epitope 2-specific MAb 289/4. Partial cleavage of M1 protein by staphylococcal V8 protease gave rise to a 16-kDa peptide, mapping from amino acid 8, which was recognized by MAbs to all three epitopes. These studies suggested that epitope 1 of the M1 protein lies between amino acids 8 and 89, whereas epitopes 2 and 3 are apparently located between amino acids 89 and 141 or somewhat more carboxy distal. Both the intact M1 protein and its C-terminal 15-kDa fragment markedly inhibited endogenous transcription of influenza virus RNP cores, and this transcription inhibition was reversed by epitope 2-specific MAb 289/4. To obtain a more circumscribed map of the A/WSN influenza virus M1 protein, we performed further chemical cleavage experiments and also tested some synthetic oligopeptides, corresponding to M1 amino acid sequences, for their capacity to bind MAbs. Moreover, we also prepared our own MAbs and compared them with those we obtained from van Wyke et al. (21). By competitive binding studies, we were able to identify a fourth M1 protein epitope. In the current epitope-recognition studies, we used the following four MAbs: MAb M2-1C6 (epitope 1), our own MAb 7E5 (epitope 2), MAb 94/6 (epitope 3), and our own MAb 5C9 (epitope 4). Formic acid cleavage and peptide purification were performed as previously described (22), as were cleavage and purification of M1 protein fragments cleaved by N-chlorosuccinimide (NCS) (17). Cyanogen bromide cleavage of M1 protein (2 jig) was accomplished with 125 p.g of CNBr in Tris hydrochloride buffer (ph 6.8) in the presence of 75% formic acid. Peptide fragments separated by polyacrylamide gel electrophoresis were electroeluted onto nitrocellulose sheets and identified by Western blot analysis with MAbs and 125I-labeled staphylococcal protein A. Figure 1 summarizes the linear map of the M1 epitopes of influenza A/WSN/33 virus as determined by MAb recognition of chemically cleaved protein fragments. Exposure of M1 protein to a 5-fold excess of CNBr in 75% formic acid for 16 h at room temperature resulted in production of two major peptides that migrated on a 1 to 2% gradient polyacrylamide-7 M urea gel to positions estimated as 8 and 4 kda. When blotted onto nitrocellulose, the 8-kDa peptide was recognized on Western blotting by MAb M2-1C6 (directed to epitope 1), MAb 7E5 (directed to epitope 2), and MAb 94/6 (directed to epitope 3), whereas the 4-kDa CNBr fragment was recognized only by MAb 7E5 (directed to epitope 2). The partial amino acid sequence, compared with that of the M1 protein, revealed that the 4-kDa CNBr fragment originated from Gly-129 and probably extended to Gln-164 (data not shown). The findings reveal that epitope 2 is located on M1 protein at a position somewhere between amino acids 129 and 164. Exposure of M1 protein to NCS, which cleaves at tryptophan (and secondarily at tyrosine or histidine) residues, resulted in partial cleavage and production of two major peptides that migrated on 1 to 2% gradient polyacrylamide-sds gels at molecular masses of about 1 and 8 kda. On Western blot analysis, the 1-kDa fragment reacted with three MAbs, but the 8-kDa fragment was recognized only by MAb M2-1C6 (directed to epitope 1) and by MAb 94/6 (directed to epitope 3) but not by MAb 7E5 (directed to epitope 2) (data not shown). The partial amino acid sequence revealed that the 8-kDa NCS peptide originated from N- terminal Leu-46 and, based on estimated molecular weight, probably extended to Tyr-119 at its C terminus. These data suggested that epitopes 1 and 3 are located at regions along the M1 protein extending from residues 46 to 119 (Fig. 1). Exposure of the M1 protein to formic acid resulted in a 9-kDa fragment, extending from amino acid residue 1 to residue 89, which reacted only with MAb M2-1C6 directed to epitope 1 (22) (Fig. 1). Since the partially overlapping 8-kDa NCS peptide (extending from M1 protein residues 46 to 119) also reacted with MAb M2-1C6, the assumption can be drawn that epitope 1 is located somewhere between amino acid residues 46 and 89. Conversely, the region of the M1 protein comprising epitope 3 is spanned by residues 89 and Downloaded from on September 19, 218 by guest

4 VOL. 63, 1989 INFLUENZA VIRUS Ml PROTEIN RNP-BINDING SITES These postulates are summarized in Fig. 1. No reliable evidence can be presented for location of epitope 4, because MAb 5C9 bound only weakly to the 8-kDa NCS peptide and with the 16-kDa V8 protease peptide (residues 8 through 141) and not with any of the other peptides generated by chemical or protease cleavage of M1 protein (data not shown). All of these assignments of epitope map locations must be considered tentative, owing to likely strong influences of threedimensional structure on the antigenic determinants of the A/WSN influenza virus M1 protein. Transcription inhibition by anti-idiotypic antibody directed to epitope 2-specific MAbs. Previous studies had shown that MAb 289/4 (directed to epitope 2) readily reverses transcription inhibition by the M1 protein (22), as does our own epitope 2-specific MAb 7E5 (data not reported). It seemed likely, therefore, that at least one transcription-inhibition site on the M1 protein is located somewhere between Gly- 129 and Gln-164, the amino acid sequence of the 4-kDa CNBr fragment that recognizes these two MAbs (Fig. 1). It has been difficult to obtain sufficient quantities of the purified 4-kDa CNBr fragment to test directly its capacity to inhibit influenza virus RNP transcription. As an alternative strategy, we generated polyclonal antiidiotypic serum in rabbits inoculated with IgG obtained from MAb 289/4 and MAb 7E5 as described in Materials and Methods. Anti-MAb 7E5-1, anti-mab 7E5-2 (each raised in two separate rabbits), anti-mab 289/4-1 and anti-mab 289/ 4-2, M1 protein, and, as a control, anti-igg (raised in a rabbit immunized with nonspecific mouse IgG) were tested in increasing concentrations for their capacity to inhibit transcription by A/WSN influenza virus RNP cores essentially devoid of endogenous M1 protein. As described in Materials and Methods, RNA synthesis by RNP cores was measured by incorporation of [32P]UMP into acid-precipitable RNA in a complete in vitro transcription mixture containing increasing concentrations of IgG or M1 protein. Increasing concentrations of anti-mab 7E5, anti-mab 289/4, and M1 protein exhibited equivalent capacity to inhibit influenza virus RNP transcription to levels 37 to 5% that of naked RNP cores (Fig. 2). Rabbit anti-mouse IgG had no effect on RNP transcription. Clearly, the anti-idiotypic IgG directed to epitope 2-specific MAbs inhibited RNP transcription in a manner quite similar to that of influenza virus Ml protein. To confirm that this transcription-inhibition effect of the rabbit anti-mab serum IgG is due to anti-idiotypic antibody, MAbs directed to epitope 2 and, as a control, MAbs directed to epitope 1 were tested for their capacity to reverse the transcription-inhibition effect of the rabbit anti-mab sera. In these experiments, constant concentrations (44,ug/ml) of anti-mab 7E5 IgG, anti-mab 289/4 IgG, or M1 protein were preincubated for 1 h in the presence or absence of increasing concentrations of epitope 2-specific MAb 7E5, MAb 289/4, or, as a control, MAb M2-1C6 directed to epitope 1. These polyclonal rabbit anti-idiotypic sera or M1 protein, in the presence or absence of MAbs, was then added to RNP cores in a complete transcription mixture, and [32P]UMP incorporation into newly synthesized RNA was measured for a 2-h period. Figure 3 compares the level of transcription in vitro by RNP alone, reduction of RNA synthesis by anti-idiotype serum or M1 protein, and the reversal of transcription inhibition by increasing concentrations of MAbs directed to epitope 2 or 1. Anti-MAb 7E5 or anti-mab 289/4 reduced influenza virus RNA synthesis about 4% (Fig. 3A), and Ml protein reduced viral RNA synthesis by 5% (Fig. 3B). 9 2 C' 8 Z 7 <6. z 4 a. I [PROTEIN] pg/mi FIG. 2. In vitro transcription by influenza virus RNP cores inhibited by anti-idiotypic antibodies anti-mab 7E5-1 and anti-mab 7E5-2 (, ), anti-mab 289/4-1 and anti-mab 289/4-2 (A, A), normal rabbit anti-mouse IgG (x), and Ml protein (O). As described in Materials and Methods, RNP cores were prepared relatively free of Ml protein, and each anti-idiotypic IgG was prepared by affinity chromatography on protein A-Sepharose and normal rabbit IgG- Sepharose columns. Transcription of RNA cores (2,ug) was measured in a complete transcription mixture by incorporation for 2 h at 31C of [a-32p]utp into acid-precipitable RNA in the absence or presence of increasing concentrations of anti-idiotypic IgG, rabbit anti-mouse IgG, or Ml protein. Increasing concentrations of epitope 2-specific MAb 7E5 or MAb 289/4 restored most of the transcription-inhibition activity of anti-mab 7E5 and anti-mab 289/4 (Fig. 3A) and almost all the transcription-inhibition activity of M1 protein (Fig. 3B). MAb M2-1C6 had no capacity to reverse the transcription-inhibition activity of anti-mab 289/4 at any concentration. These results reinforce the evidence that anti-idiotypic polyclonal antibody directed to epitope 2- specific MAbs inhibits influenza virus RNP transcription by a mechanism similar to that of M1 protein inhibition of transcription. Evidence has been presented that anti-idiotypic antibodies may have more than one antigenic determinant closely related to epitopes on the original antigen (16). To rule out the possibility that the transcription-inhibition activity of the anti-idiotypic IgG of rabbit anti-mab 7E5 serum might be due to recognition of an anti-framework-associated idiotype (Ab2cX) or to anti-antigen-combining site-related idiotope (Ab2-y), rather than to the Ab2,I internal image idiotope of the original antigenic epitope (1), anti-mab 7E5 antigen (the protein A-Sepharose-purified anti-idiotypic anti-mab 7E5 IgG) was passed through an MAb 7E5-Sepharose affinity column. Controls consisted of anti-mab 7E5 passed through columns of epitope 4-specific MAb 5C9-Sepharose and BSA- Sepharose. The anti-mab 7E5 IgG effluents from these affinity columns were tested at concentrations of, 1, 3, and 6,ug/ml for their capacity to inhibit [32P]UMP incorporation into RNA synthesized in vitro for 2 h by influenza Downloaded from on September 19, 218 by guest

5 359 YE ET AL..-T x z cc a. I cc z ~ CYJ CO, r+m [MAb IgG],ug/ml FIG. 3. Reversal by epitope 2-specific MAbs of the viral RNP transcription-inhibition activity of anti-idiotypic antibodies (A) and M1 protein (B). Incorporation of [32P]UMP into newly synthesized RNA was measured in duplicate in complete transcription mixtures (see Materials and Methods) by influenza virus RNP cores alone (2,ug) or after preincubation of RNP cores with anti-mab 289/4 IgG (15 p.g), anti-mab 7E5 IgG (15 p.g), or Ml protein (1,ug). RNP cores preincubated with anti-idiotypic antibodies or Ml protein were also incubated with increasing concentrations of MAb 7E5 (directed to epitope 2), MAb 289/4 (directed to epitope 2), or MAb M2-1C6 (directed to epitope 1). All transcription reactions were terminated in 2 h, and trichloroacetic acid-insoluble radioactivity was determined by scintillation spectrometry. J. VIROL. virus RNP cores in a complete transcription mixture. These experiments revealed that anti-mab 7E5 eluted from an MAb 7E-Sepharose column was completely devoid of any capacity to inhibit viral RNP transcription as was a control rabbit anti-mouse IgG (data not shown). In sharp contrast, influenza virus RNP transcription was inhibited 5 and 67%, respectively, by 3- and 6-mg/ml concentrations of anti- MAb 7E5 IgG passed through MAb 5C9-Sepharose or BSA- Sepharose columns (data not shown). These results reinforce the evidence that inhibition of influenza virus RNP transcription by anti-idiotypic polyclonal IgG raised by epitope 2-specific MAb 7E5 is caused by internal idiotypic IgG sequences complementary to those of epitope 2 of influenza virus M1 protein. Transcription-inhibition activity of synthetic oligopeptides corresponding to M, protein amino acid sequences. We originally set out to map the epitope locations and transcriptioninhibition domains of the influenza virus M1 protein with synthetic oligopeptides corresponding to regions identified by hydrophilicity and 3-turn plots. Based on these calculations, we prepared three synthetic peptides designated SP1 (16 amino acids, M1 residues 25 through 4), SP2 (15 amino acids, M1 residues 67 through 81), and SP3 (19 amino acids, M1 residues 9 through 18). None of these three synthetic oligopeptides directly recognized any of our panel of MAbs by Western dot blot or ELISA analyses (data not shown). As previously described (2), monospecific polyclonal antibodies were prepared in rabbits injected with SP1, SP2, and SP3 coupled by gluteraldehyde to BSA; anti-bsa IgG was removed by filtration through a BSA-Sepharose affinity column. These polyclonal anti-peptide IgGs were tested by ELISA for their capacity to compete for binding sites on influenza virus M1 protein for all four epitope-specific MAbs. Anti-SP1 competed for none of the four M1 protein epitopes. Anti-SP2 IgG at concentrations up to 48 pg/ml inhibited M1 protein binding of epitope 3-specific MAb 94/6 but not binding of MAb M2-1C6 (epitope 1), MAb 289/4 (epitope 2), or MAb 5C9 (epitope 4). Anti-SP3 IgG at similar concentrations inhibited M1 protein binding of epitope 4-specific MAb 5C9 but did not inhibit the other MAbs for epitopes 1, 2, and 3 (data not shown). This latter result suggests that epitope 4 is located in a region of the M1 protein adjacent to amino acids 9 through 18, consistent with the linear model shown in Fig. 1. The likelihood of a complex three-dimensional rather than a linear structure of the M1 protein, as well as the bulkiness of IgG molecules, casts some doubt on the reliability of these studies with polyclonal antibodies versus MAbs for mapping antigenic determinants. Each of the three synthetic oligopeptides corresponding to M1 amino acid sequences was compared with M1 protein for its ability to inhibit in vitro transcription by influenza virus. Increasing equimolar concentrations of SPI, SP2, SP3, and intact M1 protein were incubated with RNP cores virtually free of M1 protein, and the incorporation of [32P]UMP into acid-precipitated RNA was measured in a complete transcription reaction. SPI and SP2 had little or no effect on viral RNP transcription (Fig. 4). Quite unexpectedly, SP3 at concentrations increasing from to.6 FM had a dramatic inhibitory effect on viral RNP transcription, equivalent to the inhibitory effect of equimolar concentrations of M1 protein. These data suggest that the region of the M1 protein corresponding to amino acids 9 through 18 greatly influences the transcription-inhibition activity of influenza virus M1 protein. We also constructed a fourth synthetic oligopeptide (SP4) that corresponds to M1 protein amino acids 135 through 165, resembling the 4-kDa CNBr peptide extending from amino acids 129 to 164. SP4 inhibited transcription by influenza virus RNP cores in a manner similar to that of SP3 and native M1 protein (data not shown). Moreover, SP4 was also capable of binding viral RNA in a manner described in the following experiments. Binding affinity of influenza virus RNA to Ml protein and chemically cleaved fragments. A question that arises is whether M1 protein down-regulates influenza virus transcription by binding to viral RNA as well as to nucleocapsid RNP. Evidence for interaction of influenza virus RNA and its M1 protein has been noted by L. Wakefield and G. G. Brownlee (Virus Res. supplement 2, p. 66, 1988). Before studying viral RNA-M1 protein interaction in greater detail, it was necessary to address the question whether the RNA in influenza virus nucleocapsids is accessible to small or medium-sized proteins such as RNases, unlike the RNA in VSV nucleocapsids, which is not degraded by RNases (8). To this end, purified RNP cores (2 mg) extracted from A/WSN influenza virus and VSV (New Jersey serotype) virions were Downloaded from on September 19, 218 by guest

6 VOL. 63, 1989 INFLUENZA VIRUS Ml PROTEIN RNP-BINDING SITES 3591 C.) C,, 15 MAt M2-1C6tED1) MAb7E5 (Ep2) MAb9O4/;6(Eij3) = NP.- ^- 59E4)- - > Ev=m 2 -,:Ē E E rs > > > z a_g 'X ~ C., a.) [PEPTIDE/PROTEIN] PM FIG. 4. Comparative ability of synthetic peptides (SPI. SP'2. and SP3) and influenza virus Ml protein to inhibit transcription in vitro by influenza virus RNP cores. As described in Materials and Methods, the synthetic peptides had amino acid sequences corresponding to those of Ml protein as follows: SP1, Ala-25 to Glu-4; SP2, Thr-67 to Gln-81; SP3, Pro-9 to Thr-18. Transcription by Ml-free RNP cores alone (2,ug) or in the presence of increasing concentrations of SPI, SP2, SP3, or M1 protein was measured in duplicate for 2 h at 31 C in complete transcription mixtures. The reaction was quenched by the addition of.6 ml of sodium pyrophosphate (67,uM). [32P]UMP radioactivity in trichloroacetic acidprecipitated, newly synthesized RNA was measured by scintillation spectrometry. exposed to 3. or 3 tg of pancreatic RNase A in 1. ml of Tris hydrochloride buffer (ph 7.9) containing 4 mm NaCI and 5 mm MgCl2 at 37 C for 3 min. After the reaction was stopped with 1 mm EDTA-.5% SDS-2 mg of protease K per ml, viral RNA in RNase-treated and control samples was extracted three times with phenol and twice with phenolchloroform and ethanol precipitated, heated in formaldehyde at 55 C for 1 min, and analyzed by electrophoresis on 3% polyacrylamide gels containing 7 M urea. Bands in ethidiumbromide-stained gels visualized by UV light revealed that RNA in all eight influenza virus nucleocapsid segments was completely digested by RNase A, whereas the RNA in VSV nucleocapsids was intact after RNase exposure (data not shown). This sensitivity to RNase A of RNA in influenza virus RNP cores suggests looser RNA packaging by NP protein that would make nucleocapsid RNA accessible to Ml protein. The capacity of Ml protein to bind influenza virus RNA and the region of the Ml protein responsible for this binding were tested with virion proteins, purified M1 protein, and formic acid- and CNBr-cleaved Ml protein fragments separated by electrophoresis on 12.5% polyacrylamide-sds gels. These proteins or Ml fragments were transferred by electroblotting onto nitrocellulose sheets, which were then exposed to MAbs. As described in Materials and Methods. go V i - A B C D FIG. 5. Binding of influenza virus 32P-labeled RNA to virion proteins, purified M1 protein, and chemically cleaved M1 peptides previously reacted with MAbs directed to each of four M1 epitopes. Whole virion proteins (VIRION), purified Ml protein (M), formic acid-cleaved M1 protein fragments (M/FA), CNBr-cleaved Ml protein fragments (M/CNBr). and BSA were subjected to electrophoresis along with marker proteins on 17.5% polyacrylamide-sds slab gels and then transferred to nitrocellulose sheets by electroblotting. As described in Materials and Methods, the proteins on the nitrocellulose sheets were exposed to 3% BSA and then flooded with epitope 1-specific MAb M2-1C6 (Epl). epitope 2-specific MAb 7E5 (Ep2). epitope 3-specific MAb 94/6 (Ep3), or epitope 4-specific MAb 5C9 (Ep4). The sheets were then exposed to biologically 32P-labeled influenza virus RNA in probing buffer with yeast carrier trna at room temperature for 1 h. The nitrocellulose sheets were then washed five times in probing buffer, dried, and autoradiographed. RNA extracted from influenza virions biologically labeled with [32P]UMP and suspended in probing buffer was flooded onto the nitrocellulose sheet to measure 32P-labeled RNA binding to proteins or peptides as determined by autoradiography (4). 3-P-labeled influenza virus RNA bound only to virion NP and M1 proteins as well as to the 15-kDa formic acid M1 fragment and the 4- and 8-kDa CNBr M1 fragments (Fig. 5). As shown previously (22), the carboxy-distal 15-kDa formic acid fragment inhibits transcription in a reconstituted system and recognizes epitope 2-specific MAb 289/4; the 4-kDa CNBr fragment corresponds to a segment of the 15-kDa peptide and has similar properties (Fig. 1). The 8-kDa CNBr fragment is a partial cleavage precursor of the 4-kDa CNBr fragment and contains epitope 2. It was therefore not unexpected that the epitope 2-specific MAb 7E5 greatly reduced binding of viral RNA to the 15-kDa formic acid fragment and completely abolished binding of viral RNA to the 4- and 8-kDa CNBr fragments (Fig. SB). The other three MAbs exhibited no capacity to block binding of viral 32P-labeled RNA to the 15-, 8-, and 4-kDa fragments. However, it is also clear that other (N-terminal) regions of the M1 protein can also react with viral RNA as noted by clear, albeit reduced, binding of 32 P-labeled RNA to intact Ml protein preincubated with MAb 7E5. No explanation comes to mind for reduced binding of viral 3-P-labeled RNA to NP protein preincubated with MAb 7E5. Nonetheless, these data demonstrate the potential of viral RNA to bind M1 protein, particularly in the region reactive with epitope 2-specific MAb 7E5 and with anti-idiotypic anti-mab 7E5. It must be pointed out clearly that this binding capacity of Ml protein (and NP protein) is not specific for influenza virus RNA; 32P-labeled VSV RNA and 28S and 18S rrna also bind to M1 and NP proteins of influenza virus (data not shown). Of greater significance, however, is the fact that only the M1 Downloaded from on September 19, 218 by guest

7 3592 YE ET AL. TABLE 1. Comparative binding of influenza virus genomic 32P-labeled RNA to Ml protein, to anti-mab anti-idiotypic IgG, or to synthetic peptides pretreated or not pretreated with MAbs Protein or peptide" MAb' RN ofvirab M1 protein None 1 Ml protein 5C9 (epitope 4) 93 Ml protein 7E5 (epitope 2) 45 Anti-MAb 7E5 None 65 Anti-MAb 7E5 5C9 (epitope 4) 55 Anti-MAb 7E5 7E5 (epitope 2) 42 SP2 (Thr-67 to Gln-81) None 29 SP3 (Pro-9 to Thr-18) None 15 SP3 (Pro-9 to Thr-18) 5C9 (epitope 4) 1 SP3 (Pro-9 to Thr-18) 7E5 (epitope 2) 82 " Samples (4.5 nmol) of purified M, protein, anti-idiotypic IgG to MAb 7E5 (anti-mab 7E5), and synthetic peptides SP2 and SP3 were directly blotted on nitrocellulose paper and then incubated with 3% BSA in 5 mm Tris hydrochloride (ph 7.4)-1 mm EDTA-.25% gelatin-.5% Nonidet P-4. The sheets were then incubated with MAbs or buffer alone. Each sheet was then exposed to excess 32P-labeled influenza virus genomic RNA (4 x 17 cpm/,ug; 1 ng/15 ml of probing buffer). After autoradiography, the relative amount of 32p in each spot was measured by laser densitometry. " Nitrocellulose sheets were exposed to MAb 5C9, MAb 7E5 or buffer alone. ` Relative concentration of bound influenza virls 32P-labeled RNA was determined by laser densitometry with Ml binding in the absence of MAb considered arbitrarily as 1%. and NP proteins of influenza virus exhibit any affinity for binding RNA in a nonselective manner. Binding affinity of viral RNA to anti-idiotype antibody and synthetic oligopeptides. Two approaches for determining the regions of influenza virus M1 protein that serve as sites for binding viral RNA are the use of anti-idiotype antibody and synthetic oligopeptides, each with amino acid sequences corresponding to those in regions of the M1 protein. The capacity of the anti-idiotype anti-mab 7E5 and the synthetic oligopeptide SP3 (nonodecapeptide Pro-9 to Thr-18) to mimic M1 protein in its capacity to inhibit influenza virus RNP transcription (Fig. 3 and 4) suggested them as candidates for binding affinity for viral RNA. To explore this possibility, we compared the viral RNA binding affinity of the anti-idiotype anti-mab 7E5 and the SP3 oligopeptide with that of the M1 protein itself. In addition, we tested the capacity of epitope 2-specific MAb 7E5 and epitope 4- specific MAb 5C9 to block the viral RNA-binding capacity of anti-mab 7E5 and SP3. In these experiments equimolar concentrations of Ml protein, anti-mab 7E5 IgG, SP2, and SP3 were spotted on nitrocellulose sheets and then exposed to MAb 7E5, MAb 5C9, or no MAb. The nitrocellulose sheets were then exposed to excess 32P-labeled viral RNA and autoradiographed, and the amount of 32P-labeled viral RNA bound was determined by laser densitometry integration. Table 1 compares the binding affinity of viral RNA to M1 protein, to anti-mab 7E5 antibody, and to synthetic oligopeptides; the affinity for RNA binding of Ml protein unexposed to MAb was taken as the baseline level of 1% binding. By comparison, equal concentrations of anti-mab 7E5 IgG bound 65% of viral RNA, SP3 bound 15% of viral RNA, and SP2 only 29% of RNA, each in the absence of MAb. Preincubation of M, protein with epitope 2-specific MAb 7E5 reduced its RNA binding to 45%, whereas preincubation with epitope 4-MAb 5C9 had only a negligible effect on viral RNA binding to M1 protein (93%). In contrast to its effect on M1 protein, preincubation with MAb 7E5 had lesser effects on viral RNA binding to anti- J. VIROL. idiotype anti-mab 7E5 IgG (65% down to 42%) or binding of viral RNA to SP3 (15% down to 82%), presumably due to variations in three-dimensional structure and conformation of the M1 protein. However, in each case epitope 2-specific MAb 7E5 reduced viral RNA binding to anti-mab 7E5 more significantly than did epitope 4-specific MAb 5C9 (Table 1). Not unexpectedly, viral RNA binding to SP3 was not significantly inhibited by MAbs directed to other regions of the M1 protein. Although not conclusive, these experiments suggest that M1 protein amino acid sequences Gly-129 to Gln-164 (epitope 2) and Pro-9 to Thr-18 (SP3) are potential recognition sites of influenza virus RNA that may influence down-regulation of viral RNP transcription. DISCUSSION The most abundant protein of the influenza virion is the Ml protein encoded by RNA gene segment 7, which also codes for another protein, of low abundance, designated M2, which was originally thought to be nonstructural (1, 15). The M2 protein has now been found in virions as well as in infected cells, and it may well play a role in influenza virus growth (23, 24). However, it is the Ml protein that plays the critical role in virion assembly by apparently joining the RNP core segments to a plasma membrane region in which HA and NA glycoproteins have previously been inserted (7). The M1 protein has at least one site for binding to membrane lipid bilayers (6, 11, 12) as well as a unique site that promotes binding to RNP cores (22). Interaction of Ml protein with RNP cores results in down-regulation of endogenous influenza virus transcription (25). Clear evidence for the role of Ml protein in assembling RNP cores was provided recently by Patterson et al. (18), who used anti-np and anti-m1 antibodies differentially labeled with ferritin and gold for locating M1 and RNP by electron microscopy in various compartments of influenza virus-infected cells. By such techniques, these authors were able to identify NP protein of RNP cores in close proximity to M1 protein in the nucleus of infected cells. The NP-M1 complexes were also apparent on the cytoplasmic surface of the plasma membrane in the proximity of newly formed and budding virions bearing surface glycoprotein spikes. The critical role of the M1 protein in RNP-membrane assembly and budding to form progeny virions is becoming more apparent. The M protein of VSV has a similar size and similar functions but, unlike the influenza virus M, protein, the VSV M protein is basic, does not have membrane-intercalating hydrophobic sequences of amino acids, and has an N-terminal RNP-binding site that down-regulates transcription and a membranebinding site positioned toward the C terminus (17, 2). We had previously used MAbs supplied by van Wyke et al. (21) as probes for locating the antigenic and functional domains on the Ml protein of influenza virus A/WSN/33 (22). These earlier studies used proteolytic cleavage to map the membrane-binding site and epitope 1 to a region between amino acids 8 and 89 of the Ml protein, whereas the transcription-inhibition site and epitope 2 mapped carboxy distal to amino acid 89. MAb M2-1C6 (directed to epitope 1) partially blocked binding of M1 protein to liposomal membranes, and MAb 289/4 (directed to epitope 2) reversed the transcription-inhibition activity of the M1 protein (22). The current studies permit more circumscribed mapping of epitope I to a region between amino acids 46 and 89, epitope 2 to between amino acids 129 and 164, and epitopes 3 and 4 to a region extending from amino acids 45 to 119. The present studies also attempt by three separate techniques to define more precisely the transcription-inhibition Downloaded from on September 19, 218 by guest

8 VOL. 63, 1989 INFLUENZA VIRUS M1 PROTEIN RNP-BINDING SITES 3593 domain of the M1 protein. By preparing anti-idiotypic antibody against the IgG of epitope 2-specific MAb 289/4 and MAb 7E5, both of which reverse transcription inhibition, we were able to confirm the location of the transcriptioninhibition site of M1 protein at or near its epitope 2 (Gly-129 to Gln-164). Both anti-idiotypes anti-mab 289/4 IgG and anti-mab 7E5 IgG inhibited transcription by influenza virus RNP cores as efficiently as did M1 protein itself. Moreover, anti-idiotype inhibition of transcription was reversed by epitope 2-specific MAbs. However, transcription inhibition and RNA binding are not synonymous, since anti-mab 7E5 anti-idiotypic antibody binds viral RNA in an amount only 65% of that of M1 protein. The second technique used to locate the M1 protein transcription-inhibition site(s) was by preparing synthetic oligopeptides designed to correspond to hydrophilic amino acid sequences of the M1 protein. None of the three synthetic oligopeptides competed with M1 protein for MAbbinding sites or reacted with anti-m1 MAbs, and only one (SP3) gave rise to polyclonal antibodies that competed for an M1 protein-binding site with MAb (epitope 4-specific MAb 5C9). No anti-sp antibodies reversed M1 protein transcription inhibition. However, SP3, corresponding to M1 sequence Pro-9 to Thr-18, inhibited transcription by influenza virus RNP cores as efficiently as did M1 protein itself. This finding implicates a second site on the M1 protein, other than the epitope 2 sequence Gly-129 to Gln-164, as an RNP-binding site concerned with transcription inhibition by M1 protein. The third technique used here to investigate transcription inhibition by the M1 protein was to assay its capacity to bind naked viral RNA, which ostensibly could be exposed in RNP cores because we found them permeable to RNase A. Only viral NP and M1 proteins bound viral genomic RNA, as did the synthetic peptide SP3 corresponding to M1 sequence Pro-9 to Thr-18. Although this finding suggested that epitope 4 of M1 protein is the RNA-binding site, this RNA binding to M1 was not blocked by epitope 4-specific MAb 5C9, which also showed no capacity to reverse transcription inhibition by M1 protein. The anti-idiotype anti-mab 7E5 also exhibited some capacity to bind viral RNA, but less efficiently than M1 protein or SP3, suggesting a second region of the M1 protein as another possible viral RNAbinding site in the vicinity of epitope 2. Of potential significance is the existence of a zinc finger motif (2, 19) extending from amino acids 145 through 165 of the M1 protein (Wakefield and Brownlee, Virus Res.; Ye and Wagner, unpublished data). It must be emphasized that the potential RNAbinding affinity of at least two sites on the M1 protein is not specific for influenza virus RNA; VSV genomic RNA and rrna bind equally readily to the M1 protein. To visualize more clearly the structural properties of those regions of the influenza virus M1 protein responsible for transcription inhibition and RNA binding, we analyzed the hydropathicity of the M1 protein as an indication of its three-dimensional structure. We used the technique of Engelman et al. (9), which is based on calculating the transfer of free energy for amino acid side chains in a-helical polypeptides. Figure 6 depicts the hydropathy index of the M1 protein as measured by the free energy required for transfer of each 2-amino-acid helix from the membrane to the water phase plotted as the first amino acid in the window. The hydropathy plot reveals three hydrophobic domains of the M1 protein extending from amino acids 1 to 2, 45 to 7, and 17 to 15, based on positive free energy transfer from membrane to water. This model is consistent with previous w czx I z cc r -o w w < atl: 1- w z I 1- Ah I FIRST AMINO ACID RESIDUE IN WINDOW FIG. 6. Hydropathy plot of AIWSN/33 influenza virus M1 protein by the method of Engelman et al. (9). The computations are based on the free energy required for transferring a helix of 2 amino acid residues from a membrane to the water phase. The data are plotted as a function of the first M1 protein amino acid in the window of 2 amino acids. Peaks below indicate hydrophilicity, and peaks above zero (in black) indicate hydrophobicity. Peaks that exceed +2 kcal (ca kj)/mol are indicative of potential transmembrane helices. data indicating the existence of a membrane-binding site and epitope 1 in a region of the M1 protein between amino acids 4 and 8 (22). Another, but somewhat less likely, transmembrane domain of the M1 protein was postulated to be present between amino acids 13 and 17 (12). Figure 6 also provides evidence for four hydrophilic domains exhibiting negative free energy transfer to water corresponding to amino acid sequences 2 to 4, 6 to 11, 13 to 17, and 22 to 24 presenting at the surface of the M1 protein. The third hydrophilic domain (amino acids 13 to 17) encompasses the putative site of epitope 2 (amino acids 129 to 164; Fig. 1), which also represents one of the two regions of the M1 protein responsible for down-regulation of influenza virus transcription. Hydrophilic domain 2 (amino acids 6 to 11) encompasses the second putative transcription-inhibition and viral RNA-binding site represented by the synthetic peptide SP3 (corresponding to M1 amino acids 9 to 18). Connecting hydrophilic domains 2 and 3 is a hydrophobic domain, extending from amino acids 17 to 15, which could serve to bring hydrophilic domains 2 and 3 into close proximity in a helical three-dimensional structure in this region of the M1 protein. This hypothesis that the M1 protein can assume a conformation that brings into proximity its two putative transcription-inhibition and RNA-binding domains requires confirmation by further study. ACKNOWLEDGMENTS This research was supported by Public Health Service grant R37 Al from the National Institute of Allergy and Infectious Diseases and by grant MV-9 from the American Cancer Society. LITERATURE CITED 1. Allen, H., J. McCauley, M. Waterfield, and M. J. Gething Influenza virus RNA segment 7 has the coding for two polypeptides. Virology 17: Barbosa, M. S., D. R. Lowy, and J. T. Schiller Papillomavirus polypeptides E6 and E7 are zinc-binding proteins. J. Virol. 63: Baylor, N. W., Y. Li, Z. Ye, and R. R. Wagner Transient expression and sequence of the matrix (M1) gene of WSN Downloaded from on September 19, 218 by guest

9 3594 YE ET AL. J. VIROL. influenza virus in a vaccinia vector. Virology 163: Both, G. W., and G. M. Air Nucleotide sequence coding for the N-terminal region of the matrix protein of influenza virus. Eur. J. Biochem. 96: Bowen, B., J. Steinberg, U. K. Laemmli, and H. Weintraub The detection of DNA-binding proteins by protein blotting. Nucleic Acids Res. 8: Bucher, D. J., I. G. Kharitonenkov, J. A. Zakomirdin, V. B. Grigoriev, S. L. Klimenko, and J. F. Davis Incorporation of influenza M protein into liposomas. J. Virol. 36: Compans, R. W., and H.-D. Klenk Viral membranes, p In H. Fraenkel-Conrat and R. R. Wagner (ed.), Comprehensive virology, vol. 13. Plenum Publishing Corp., New York. 8. Emerson, S. U Transcription of vesicular stomatitis virus, p , In R. R. Wagner (ed.), The rhabdoviruses. Plenum Publishing Corp., New York. 9. Engelman, D. M., T. A. Steitz, and A. C. Goldman Identifying nonpolar transbilayer helices in amino acid sequences of membrane proteins. Annu. Rev. Biophys. Biophys. Chem. 15: Gaulton, G. N., and M. I. Greene Idiotypic mimicry of biogical receptors. Annu. Rev. Immunol. 4: Gregoriades, A Interaction of influenza M protein with viral lipid and phosphatidylcholine vesicles. J. Virol. 36: Gregoriades, A., and B. Frangione Insertion of influenza M protein into the viral lipid bilayer and localization of site of insertion. J. Virol. 4: Lamb, R. A The influenza virus RNA segments and their encoded proteins, p In P. Palese and D. W. Kingsbury (ed.), Genetics of influenza virus. Springer-Verlag K.G., Vienna. 14. Lamb, R. A., and P. W. Choppin The structure and replication of influenza virus. Annu. Rev. Biochem. 52: Lamb, R. A., and S. L. Zebedee Influenza M, protein is an integral membrane protein expressed on the infected cell surface. Cell 4: Nisonoff, A., and E. Lamoyi Implications of the presence of an internal image of the antigen in anti-idiotypic antibodies: possible application to vaccine production. Clin. Immunol. Immunopathol. 21: Ogden, J. R., R. Pal, and R. R. Wagner Mapping regions of the matrix protein of vesicular stomatitis virus which bind ribonucleocapsids. liposomes, and monoclonal antibodies. J. Virol. 58: Patterson, S., G. Gross, and J. S. Oxford The intracellular distribution of influenza virus matrix protein on nucleoprotein in infected cells and their relationship to haemagglutinin in the plasma membrane. J. Gen. Virol. 69: Sehnke, P. C., A. M. Mason, S. J. Hood, R. M. Lister, and J. E. Johnson A "zinc-finger"-type binding domain in tobacco streak virus coat protein. Virology 168: Shipley, J. B., R. Pal, and R. R. Wagner Antigenicity, function, and conformation of synthetic oligopeptides corresponding to amino-terminal sequences of wild-type and mutant matrix proteins of vesicular stomatitis virus. J. Virol. 62: van Wyke, K. L., J. W. Yewdell, J. M. Reck, and B. R. Murphy Antigenic characterization of influenza A virus matrix proteins with monoclonal antibodies. J. Virol. 49: Ye, Z., R. Pal, J. W. Fox, and R. R. Wagner Functional and antigenic domains of the matrix (M,) protein of influenza A virus. J. Virol. 61: Zebedee, S. L., and R. A. Lamb Influenza A virus M, protein: monoclonal antibody restriction of virus growth and detection of M, in virions. J. Virol. 62: Zebedee, S. L., and R. A. Lamb Growth restriction of influenza A virus by M, protein antibody is genetically linked to the M, protein. Proc. Natl. Acad. Sci. USA 86: Zvonarjev, A. Y., and Y. Z. Ghendon Influence of membrane (M) protein on influenza A virus virion transcriptase activity in vitro and its susceptibility to rimantadine. J. Virol. 33: Downloaded from on September 19, 218 by guest