Journal of General Virology (1994), 75, 3742. Printed in Great Brita#l 37 A small percentage of influenza virus M1 protein contains zinc but zinc does not influence in vitro M1-RNA interaction Christine Elster, ~ Eric Fourest, 2 Florence Baudin, ~ Kjeld Larsen, ~ Stephen Cusack 1 and Rob W. H. Ruigrok 1. 1 EMBL Grenoble Outstation, c/o ILL, BP 156, 38042 Grenoble Cedex 9 and 2 CEN-G/DBMS/TSV, BP 85X, 38041 Grenoble Cedex, France A peptide containing the CCHH motif, the putative zinc-binding sequence of influenza virus M1 protein, was found to bind zinc in a one-to-one complex with the characteristics of a typical zinc-binding peptide. Intact influenza virus also contained zinc and we show that this zinc is bound to the M1 protein in the virus. However, only a small proportion of M1 contained zinc: 4% in virus and 6 to 9 % in isolated protein. One strain, B/Yamagata/16/88, consistently contained more zinc: 15 to 20% both in virus and in isolated protein. We also determined the RNA binding and transcription in- hibition activities of various M1 proteins and found that the zinc content of M1 had no influence on either activity. We suggest that the zinc in M1 has a structural role in the virion other than nucleic acid binding. Introduction Influenza viruses are enveloped viruses with a segmented negative strand RNA genome. Each vRNA segment is complexed with the major RNA-binding protein nucleo- protein (NP) and carries a copy of the polymerase complex. These structures, called ribonucleoprotein particles (RNPs), are independent active transcription units. The total genome consists of eight RNPs which are contained within a shell of M1 protein which lines the inside of the viral lipid bilayer. Embedded in this lipid bilayer are the viral glycoproteins which are essential for entry into and release from the host cell. The structure of the virion suggests an important role for the M 1 protein. M 1 may interact with the cytoplasmic tails of the glycoproteins, the lipid membrane and the RNPs and so form the 'glue' between them. Interaction of M 1 with lipid has been shown in intact virus using light-activated cross-linking (Gregoriades & Frangione, 1981) and also in vitro using isolated M1 and liposomes (Bucher et al., 1980). Contact between M1 and the glycoproteins has not yet been shown but interaction of M1 with RNP is suggested by the fact that purified M1, when added to transcribing RNPs, inhibits transcription (Zvonarjev & Ghendon, 1980; Ye et al., 1989). hi vitro interaction of M 1 with RNA has been shown with filter- binding assays and a blotting technique (Wakefield & Brownlee, 1989; Ye et al., 1989). The primary sequence of all influenza A and B M1 proteins contains the putative zinc-binding sequence CysXaa~-CysXaa 7 His-Xaa~ His (CCHH motif) (residues 148 to 162 of A/PR/8/34 M1 protein; Allen et al., 1980), first remarked upon by Wakefield & Brownlee (1989), which could be involved in RNA binding. This CCHH motif is situated in a part of the M1 sequence that may be involved in transcription inhibition (Ye et al., 1989). Zinc-binding sequences have been implicated in specific protein-nucleic acid interactions and protein dimerization domains (see references in Discussion). In this paper we describe the zinc-binding activity of a 27 amino acid peptide containing the CCHH motif and have determined the zinc content of intact influenza virus and purified M 1. We further show that the zinc content of M1 does not influence the in vitro RNA binding and transcription inhibition activities of M1. Methods Virus and M1 protein. InfluenzavirusesA/PR/8/34, B/Beijing/1/87 and B/Yamagata/16/88 were grown in embryonated hen's eggs and obtained in purified form from Pasteur-Mrrieux, Marcy L'Etoile, France. M1 protein was isolated from spikeless virus. Spikes were removed by bromelain digestion (Brand & Skehel, 1972) which was stopped by addition of 100 mM-iodoacetamide. Spikeless virus was then purified by pelleting through 14% sucrosein PBS. The virus was then disrupted with 1% Triton X-I00 in PBS and centrifugedon a 10 to 30 % continuousglycerol gradientin PBS (SW41rotor, 36000 r.p.m., 4 °C, 16 h). M1 was collectedfrom the upper fractions of the gradient and stored immediatelyat -20 °C. The purity was judged to be higher than 90% by PAGE (Fig. 1). Virus protein concentrationswere determinedby the Lowry method (Lowry et al., 1951) using BSA as a standard. The concentration of isolated M 1 protein was determinedwith the Lowry and the Bradford 0001-1904 © 1994 SGM