Proc. Natl. Acad. Sci. USA Vol. 85, pp. 7094-7098, October 1988 Biochemistry Conserved and residues of the avian myeloblastosis virus nucleocapsid are essential for viral replication but are not "zinc-binding fingers" (single-site substitution/nucleic acid binding/circular dichroism) JOYCE E. JENTOFT*, LISA M. SMITH, XIANGDONG Fu, MARK JOHNSON, AND JONATHAN LEIS Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 Communicated by Harland G. Wood, June 6, 1988 (receivedfor review March 22, 1988)

ABSTRACT The nucleocapsid protein from the Rous sar- showing the conserved residues to be essential to virus coma virus has two regions of sequence with the motif Cys- replication. However, our biochemical evidence argues Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Gly-His-Xaa-Xaa-Xaa-Xaa-Cys. against the hypothesis (9) that the ppl2 protein is a metallo- All retrovirus nucleocapsid contain one or two ofthese protein. motifs, and they represent the only conserved sequences among these proteins. Sequence analysis of nucleocapsid from avian MATERIALS AND METHODS myeloblastosis virus shows that it also contains two Cys-His sequences and, in fact, differs from the Rous sarcoma nucleo- Isolation and Sequencing of the DNA for NC. A pBR322 capsid protein only in three residues near the carboxyl termi- clone (HI91) containing the entire AMV gag gene was nus. The hypothesized role of the conserved and obtained from M. Baluda (10). A 0.91-kilobase (kb) Pst I as zinc ligands was tested experimentally. No tightly fragment containing the entire NC coding region was isolated bound metal ions were detected for avian myeloblastosis from HI91 and subcloned into the Pst I site of vector nucleocapsid protein, and the molar amount of zinc in virions M13mp19 in both orientations. Both strands of the NC gene was less by a factor of 50 than that ofthe nucleocapsid protein. were then sequenced by the Sanger method (11) using the Added Zn2+ did not significantly affect nucleocapsid binding synthetic sequencing primers 5'-CTACACTTGTGGATC- to poly(ethenoadenylic acid) or its secondary structure, as CC-3' for the plus strand and 5'-CTACACTGTTTAGCGTT- determined from circular dichroism. Nevertheless, the con- 3' for the minus strand. served cysteine and histidine residues of the Rous sarcoma Purification of AMV NC. In the preparations used for CD (Prague-C strain) nucleocapsid protein are essential for fully and binding measurements, the NC was isolated from the functional virus, as shown by the fact that single-site substitu- supernatant of detergent-extracted virus that was passed tions of five of the six conserved cysteines and either of the two through a DEAE-ceilulose column (12) and then chromato- histidine residues blocked viral replication. graphed on CM-Sephadex C-50 (13). All buffers contained 20 mM 2-phosphoglycerol to help stabilize the phosphorylated Retrovirus nucleocapsid (NC) proteins are members of a form ofthe NC. Purified protein was dialyzed against distilled larger class of nucleic acid-binding proteins with "Cys-His" and deionized water and stored at - 200C as a lyophilized boxes (1). All of the NC proteins that have been sequenced powder. For metal analysis, two alternative purification contain at least one sequence with the motif Cys-Xaa-Xaa- schemes were also used. In one case, the NC was purified Cys-Xaa-Xaa-Xaa-Gly-His-Xaa-Xaa-Xaa-Xaa-Cys, where from AMV (obtained from Molecular Genetics Resources, the first two cysteines and the histidine are invariant for nine Tampa, FL) by chloroform/methanol/salt extraction fol- Cys-His boxes from NC proteins from five viral strains lowed by solubilization of the protein in the presence of infecting four different species (2). The Cys-His motif also Triton X-100 and chromatography on CM-Sephadex C-50 represents the only conserved sequence noted for retroviral (12). In another case, the NC was isolated from AMV as NC proteins, implying that it plays a key structural and/or described (12) but with solubilization done without deter- functional role in these proteins. gents to avoid any contamination of protein with divalent ppl2 is the major NC protein of the avian retroviruses, cations (14). including avian myeloblastosis virus (AMV) and Rous sar- The AMV preparation used to isolate the NC protein coma virus, Prague-C strain (RSV). About 2500 copies (3, 4) contains an unknown amount of helper virus (myeloblastosis of NC are tightly bound to genomic RNA within the viral associated virus 1 and 2). Because the NC protein from RSV core, resulting in approximately one NC for every seven or and that from AMV are very similar, we assume the sequence eight nucleotides. of the helper virus NC is highly homologous as well. This The NC of RSV and AMV has two of the Cys-His assumption is reasonable since the 1H NMR spectra of the sequences (ref. 5 and this report). As part of our ongoing aromatic region from the AMV NC protein shows only a characterization ofthe function and structure ofthe NC from single set of resonances for each of the aromatic residues in both AMV and RSV (6-8), we undertook an investigation of the AMV NC (data not shown). its properties due to the residues in the Cys-His sequences. Plasma Emission Spectroscopy. The NC was dissolved in We report the primary sequence ofAMV NC, the distribution distilled deionized water, and its concentration was estimated of its secondary structural elements, an analysis of its metal using an AO-170J value of 0.84 (15). The NC samples covering content, and the results of site-directed mutagenesis exper- from 10 to 90 ,ug/ml (ppm) were made by serial dilution. The iments on conserved cysteine and histidine residues in RSV distilled deionized water used for dialysis served as a control. NC. This communication provides experimental evidence Abbreviations: NC, nucleocapsid; AMV, avian myeloblastosis vi- The publication costs of this article were defrayed in part by page charge rus; RSV, Rous sarcoma virus, Prague-C strain; poly(EA), payment. This article must therefore be hereby marked "advertisement" poly(1,N6-ethenoadenylic acid). in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 7094 Downloaded by guest on September 26, 2021 Biochemistry: Jentoft et al. Proc. Natl. Acad. Sci. USA 85 (1988) 7095 AMV purified by isopycnic sucrose-density-gradient centrif- transcriptase activity using exogenous poly(G) as the tem- ugation was also subjected to plasma emission analysis. The plate and oligo(dC12_18) as the primer (22). samples were shipped in dry ice to the Plasma Emission Spectroscopy Laboratory at the Institute of Ecology, Uni- RESULTS versity of Georgia (Athens), where analyses were performed on a Mark II Jarrell-Ash 965 Atom-Comp plasma emission Primary Sequence of AMV NC. The sequence of the NC spectrometer (Thermo Jarrel-Ash, Franklin, MD) equipped protein from AMV was deduced from the nucleic acid with a single-channel scanning monochrometer. This spec- sequence as described. The 88- sequence is trometer can detect parts-per-billion levels of 40 fixed ele- compared with the sequence for the NC from RSV (5, 23) in ments per scan, including the biologically important elements Fig. 1. Each NC protein contains two Cys-His sequences as Al, Ca, Co, Mg, Mn, Ni, P, K, Na, and Zn. indicated by the underlined regions. The conserved cysteines The ability of the NC to bind exogenous Zn2+ was and histidines are in boldface. The 12 amino acids that examined by incubating the protein, isolated without expo- connect the two Cys-His sequences include both Ser-40, sure to chloroform/methanol, with a 5-fold molar excess of which is the site ofin vivo phosphorylation (6), and the Zn2+ for 10 min at room temperature in 0.1 M sodium that have been proposed to form part of the nucleic acid- phosphate buffer, pH 7.0. The NC was then twice dialyzed binding site (8, 21). against a 1000-fold excess ofdistilled deionized water for 2 hr, The AMV and RSV sequences for the NC are identical and then overnight, all at 40C. Samples of NC at 10, 30, and except for two changes at the carboxyl-terminal end of the 90 ppm were subjected to plasma emission analysis. Samples polypeptide chain. In the RSV NC sequence (5, 23), Arg-73 of the final dialysate and the distilled deionized water were is replaced by a and the dipeptide Leu-81-Ser-82 is analyzed as control solutions. replaced by the tripeptide Pro-81-Gly-82-Pro-83. These Circular Dichroism. CD spectra were obtained from 190 to changes are unlikely to produce changes in secondary struc- 230 nm using a Jasco J-40-A recording spectropolarimeter. ture, in agreement with our previous observations that the The raw data was digitized and converted into molar ellip- two proteins have identical biochemical properties (6-8). ticity. This implies that the NC proteins from these viruses have Fluorescence-Binding Assay. Nucleic acid binding by the similar tertiary structures. NC was measured by enhancement of poly(l,N6-ethenoa- Secondary Structure Analysis of AMY NC. Previously, the denylic acid) [poly(EA)] (Pharmacia) fluorescence (16). Mea- Chou-Fasman algorithm was used to predict the secondary surements were made on a Perkin-Elmer LS-3 spectrofluo- structural elements of RSV NC (8). This analysis suggested rimeter, equipped with a xenon lamp, 10-nm fixed slits, and that the NC had more random coil than a-helix or P-sheet a microprocessor-controlled photomultiplier gain. The exci- structure. We have now obtained the CD spectra between 190 tation A was 309 nm, and fluorescence was measured at an and 230 nm for the AMV NC with and without added Zn2+ emission A of 400 nm. The initial conditions for titration at pH 7.5 (Fig. 2). The average percentage distribution of a experiments were a volume of 1.000 ml containing 4.61 AM helical, p sheet, and random structure for the AMV NC was poly(eA) bases, 40 mM Mes/HCl, 80 mM NaCl, pH 5.6, at calculated from the molar ellipticities (24) to be 7 ± 2% room temperature. During a binding experiment successive a-helix, 22 ± 4% 8-sheet, and 74 ± 9% random coil. The additions were made of concentrated NC dissolved in the calculated distribution of secondary structural elements was buffered poly(eA) solution with the relative fluorescence statistically indistinguishable for the NC with and without obtained after each addition. Under these conditions the Zn2" (3 mol/mol). CD data obtained above 200 nm are most relative fluorescence was stable with time, so that the NC accurate for estimating a-helical content (25); we conclude concentration was not altered by adsorption to the cuvette that the NC contains little or no a-helical structure. These (17); no correction for dilution was necessary under these analyses further indicate that the NC is composed of no more conditions. The relative fluorescence values were corrected than one-quarter to one-third p-strands, whereas the remain- for inner filter effects (18). Self-absorption was insignificant. der ofthe protein is random coil. The CD spectrum ofthe NC Synthesis of Oligodeoxynucleotides. Oligodeoxynucleotides protein has no detectable features in the near UV range of230 were synthesized on a solid support by the phosphoramidite to 350 nm (data not shown). method (19) using an Applied Biosystems (Foster City, CA) Metal Analysis of the NC. Samples of the NC, obtained by model 350A DNA synthesizer and were purified by electropho- several variations of the isolation procedure (see Materials resis on 20o polyacrylamide gels. The oligodeoxynucleotides and Methods), were screened for bound metal ions. None of used to change Cys-21 -* Ser was 5'-GAGGGCTCTCGTA- the NC solutions were dialyzed against a metal chelator CACTTGT-3'; Cys-24 -> Ser, 5'-GCTACACTTCCG- before analysis. Although Zn2+ was considered the metal ion GATCCCCG-3'; His-29 -- Ile, 5'-TCCCCGGGAATTTATC- most likely to bind the NC, plasma emission analysis was AGGC-3'; Cys-34 -- Ser, 5'-AGGCGCAGTCGCCGAAAA- used to assay NC solutions for the presence of 40 elements, AA-3'; Cys-47 -- Ser, 5'-GTGAGCGATCGCAGTTGTGT-3'; Cys-50-* Ser, 5'-GTCAGTTGTCGAACGGGAT-3'; His-55 AVVN RQRDGQ TGSGGRARGL CYTCGSPGHY Asn, 5'-GGGATGGGAAACAACGCTA-3'; and Cys-60 10 20 30 Ser, 5'-CTAAACAGTCGAGGAAGC-3'. These oligodeoxy- (P) QAQCPKKRKS GNSRERQRLC NGMGHNAKQC nucleotides were phosphorylated in vitro at their 5' ends by T4 40 kinase and ATP before their use for site-directed mutagenesis. 50 60 used for were labeled R LS AMV Oligonucleotides hybridization probes RKRDGNQGQR PG GLSSGPW EPPAVS-COO- with 32P by the same reaction except that [_32P]ATP (7,000 70 K 80 PG P RSV Ci/mmol; 1 Ci = 37 GBq) was used. Site-Directed Mutagenesis. The site-directed mutagenesis FIG. 1. Comparison of the amino acid sequences of RSV and reactions were done on single-stranded M13mpl9pl2 as AMV NC using the one-letter amino acid codes. The amino acid described (20, 21). The 2.1-kb Sac II-HindIII fragments sequence for the RSV NC was deduced from the nucleotide sequence containing point mutations were separately subcloned into (5). The AMV NC gene was isolated and sequenced as described. Cys-His regions are underlined; the conserved cysteine, , and the Sac II-Hpa I sites of pATV8-K for assaying replication histidine residues are shown in boldface; Ser-40 is indicated as the of mutant viruses. Cell culture and DEAE-dextran-mediated phosphorylated residue; and the amino acids (AMV residues 73 and DNA transfection were as described (20). The virus particles 81, 82) that differ for the AMV and RSV proteins are shown above released from transfected cells were assayed for reverse and below, respectively, the main sequence line. Downloaded by guest on September 26, 2021 7096 Biochemistry: Jentoft et al. Proc. Natl. Acad. Sci. USA 85 (1988) bind nucleic acids was determined from the NC-induced enhancement ofthe relative fluorescence ofpoly(EA) (16). At 0 low ionic strength, the binding of NC to poly(EA) is stoichi- 0 ometric with and without added Zn2+ (data not shown). The x site size derived from this plot was five nucleotides, in agreement with previous estimates (17). Binding studies were .0 a done in the presence of 80 mM NaCl at pH 5.6, resulting in MQ -1 the binding data shown in Fig. 3A. The change in relative m 0 fluorescence is plotted as a function offree NC concentration in the inset to Fig. 3A. A similar change in relative fluores- as cence with added NC was seen with and without added Zn2 -2 + I (3 mol/mol). The binding data were analyzed on a Scatchard 1 85 195 205 215 225 235 plot, shown in Fig. 3B, using the model of McGhee and von Wavelength (nm) Hippel (27), which considers the polynucleotide as a one- FIG. 2. The molar ellipticity of the AMV NC as a function of A. dimensional homogeneous lattice and takes into account edge CD spectra between 190 and 230 nm were obtained for AMV NC with and lattice effects as well as cooperativity. Binding is posi- (m) and without (a) a 3-fold molar excess of Zn2 , the spectra were tively cooperative, as indicated by the concave plot of vIL digitized, and the data were converted to molar ellipticities. The solutions contained 21 juM NC in 50 mM phosphate/0.1 M NaCl at pH 7.5.

including all of the first-row transition metals and other 00- transition metals that have been identified in metalloproteins. ci No other metals were detected at levels as high as those for C.) Zn2 . Table 1 shows the ratio of Zn2 + to NC for solutions of C. a)0 NC with and without C.) exogenous Zn2+ added before dialysis a) and for the whole virus. NC obtained by the alternate u) methods of purification described contained comparable a: levels ofZn2+ (data not shown). No NC preparations contained ai) stoichiometric amounts of Zn2 . More importantly, the virus cci particles themselves contain <2% of the zinc that would be required if each NC were to contain a single bound Zn2 . The small amount of Zn2+ that was detected is just sufficient for each reverse transcriptase molecule to contain two bound o Zn2 +, a result anticipated from the known requirement of this Total [NC], AM enzyme for Zn2 + (26). These data indicate that the NC does not 0I contain bound Zn2+ in the virion and that the Zn2+ was not lost 40 from the protein during preparation. While the NC does not contain intrinsic Zn2+, it does have a weak affinity for added Zn2+ (Table 1), probably due to the presence offree sulflhydryl groups. Effect of Added Zinc on NC Binding to Poly(EA). The ability of the NC, in the absence and presence of added Zn2+, to x Table 1. Results of plasma emission analysis of AMV j NC protein Zn2+/NC, mol/mol NC, AM Whole virus* 0.02 210 0.02 420 0.03 530 Isolated NCt 0.18 1.0 v 0.20 1.1 FIG. 3. AMV NC binding to poly(EA) with and without a 3-fold 0.12, 0.29 3.2 molar excess of Zn2+ in 80 mM NaCl/40 mM Mes/HCl at pH 5.6. 0.13 4.4 (A) The relative poly(EA) fluorescence as a function of added NC 0.30 9.5 protein. z, Data for NC without Zn2; *, data with Zn2". The two Isolated NC plus added Zn2+t sets ofdata are not distinguishable statistically. The lines through the 3.16, 3.13 1.1 data sets have no theoretical significance. (Inset) Change in relative 3.20, 3.19 3.2 fluorescence as a function offree NC. Concentration offree NC was 3.22, 3.13 9.5 calculated from the difference between total and bound NC, where 0.86 9.5§ the amount ofbound NC was obtained by assuming the change in the 0.76 poly(EA) fluorescence to be proportional to the amount of NC bound 0.42, 0.55, 9.5¶ and that each NC enhanced the fluorescence offive nucleotides. (B) *Concentration estimated from Coomassie staining. Scatchard plot of the data in A. The solid lines represent bracketing tDetergent extracted. fits to the data using the approach ofMcGhee and von Hippel (27, 28) t5-Fold excess Zn2+ added before dialysis. where end and lattice effects are taken into account and where n, the §The above 9.5 uM NC sample after additional dialysis vs. H20. site size, is taken to be 5. For line 1 the cooperativity parameter W MThe starting sample for § after additional dialysis vs. 0.050-0.20 mM is 20 and Ka is 1.1 x 10-' M, while for line 2, w is 25 and Ka is 0.9 1,10-o-phenanthroline. x 10-5M. Downloaded by guest on September 26, 2021 Biochemistry: Jentoft et al. Proc. Natl. Acad. Sci. USA 85 (1988) 7097

versus v. The solid lines represent fits to the data using 5 equation 15 of McGhee and von Hippel (27, as corrected in

ref. 28) for a site size n of 5. This value of n establishes the 4 4 intercept on the v axis as 0.2 (1/n) (27, 28). Line 1 represents cnw

the theoretical curve for a value of the cooperativity param- 1c 3 eter, w, of 20 and a Ka of 1.1 x 105 M-1, whereas line 2 represents the theoretical curve for a value of w of 25 and a Ka of 0.9 x 105 M-1. Similar theoretical curves could be generated for an n of 4, co of 20, and a Ka of 0.6 x 105 M-1, Uj orforan n of 6, w of35, and aKaof 1.6 x 105 M-1 (datanot Cn shown). Thus, the range of theoretical parameters that give w rise to reasonable fits to the experimental points are a value 0 8 10 12 14 16 18 of n between 4 and 6, a value of co between 20 and 35, and a DAYS AFTER TRANSFECTION value of Ka between 0.6 and 1.6 x 105 M-1. The data points that lie at FIG. 4. Replication of RSV containing wild-type and mutant NC above the theoretical line low concentrations ofNC proteins. The plasmid pATV8-K containing wild-type RSV DNA and reflect the existence of an additional process, such as the mutant plasmids containing point mutations in the Cys-His boxes NC-induced loss of residual poly(EA) structure. of the NC were introduced into chicken embryo fibroblasts by Site-Directed Mutagenesis of Cysteines and Histidines in DEAE-dextran-mediated DNA transfection as described. Cells RSV NC. Because the Cys-His sequences ofAMV NC do not transfected with wild-type plasmid DNA (z) produced virus after 8 tightly bind Zn2+ or other metal ions, the reason for the to 10 days as detected by reverse transcriptase activity in the cell conservation of this motif in NC proteins is undefined. To culture supernatant, whereas cells transfected with mutant plasmid determine whether the conserved cysteine and histidine DNAs (v) showed no detectable virus production. residues of the NC are important to the biological function of the virus, five of the six cysteine residues and both histidine DISCUSSION residues of the RSV NC were mutated separately by site- The substitution ofamino acids at the conserved cysteine and directed mutagenesis. As shown in Table 2, we made a radical histidine residues of the RSV NC, as summarized in Table 2, substitution of for His-29, a conservative substi- resulted in each case in the loss of infectious progeny virus. tution of for His-55, and structurally conservative Thus, each substitution alters the biological function of NC, substitutions of for cysteines-21, -24, -34, -50, and -60 emphasizing that these conserved residues in both Cys-His of the NC. Previously we have used this approach to study sequences are essential for protein function. The requirement the effect of amino acid changes on the biological function of for the NC protein in the production of infectious virus the NC in nearby residues 36, 37, 39, 40, and 43 (20, 21). particles had been shown in an earlier mutation study (29). Viral DNAs carrying these point mutations were intro- Replication and transformation-defective viruses were also duced into chicken embryo fibroblasts by the DEAE-dextran produced by deletion and duplication mutations involving the transfection method (20). The release of infectious virus Cys-His regions ofRSV NC (30). Depending on the mutation, particles from the cells was monitored by assaying for reverse defective virus particles were released from cells that lacked transcriptase activity in cell supernatants. In a typical trans- viral RNA or had an abnormal ratio between 70S (dimeric) fection experiment with wild-type proviral DNA (Kpn I- and 35S (monomeric) viral RNA. Finally, double-point amino digested pATV8K), the transformed cellular phenotype be- acid changes at positions 36 and 37 resulted in the loss of came apparent, and the release of virus particles from cells high-affinity RNA binding of the protein and a RNA pack- was detected 7 days post-transfection (Fig. 4). In contrast, aging defect in vivo (21), whereas single-point mutations at when DNA containing any ofthe base changes in the Cys-His non-Cys-His positions 36, 37, 39, 40, and 43 had no effect (20, sequences in the NC was transfected, no virus production 21). Our present results with single amino acid substitutions could be detected as measured by the absence of reverse further demonstrate that replication competence depends transcriptase activity (Fig. 4 and Table 2). The supernatants upon the highly conserved cysteine and histidine residues. of transfected cells were assayed for reverse transcriptase The suggestion that the 14 residue Cys-His sequences of activity for as long as six weeks post-transfection without AMV, RSV, and other retroviral NC proteins provide the detection ofactivity (data not shown). In addition, these cells ligands for binding Zn2+ (9) has been widely accepted (31) lacked the transformation morphology readily apparent in despite the absence of confirming experimental data. The wild-type transfected cells. Each of the seven single-site spacing of the cysteine and histidine residues is conserved mutations, therefore, was lethal for subsequent viral repli- among all viral NC proteins (9) but differs from those in the cation. "zinc-binding fingers" of the transcription factors. This study now shows that the AMV NC protein does not contain Table 2. Summary of point mutations made on the Cys-His intrinsically bound Zn2+ or other metal ions and that while boxes of RSV the NC can bind Zn2+ weakly, its presence does not affect Residue Substitution Virus growth* the secondary structure of the NC or its binding to nucleic acid. Nor is there sufficient Zn2+ present in virions to bind Wild type No Yes to more than 1% of the NC present. Because the virion is Cys-21 Ser No pinched off from the host cell, it is likely that Zn2+ is not Cys-24 Ser No bound to the NC (or the Pr76 precursor polypeptide) in vivo. His-29 Ile No In addition to the data presented here, the absence of bound Cys-34 Ser No Zn2 + in isolated NC is indicated by our previous finding that Cys-47 Ser NDt the NMR parameters of both histidine residues of the AMV Cys-50 Ser No NC were very close to those of solvent-exposed histidines His-55 Asn No (32), an observation inconsistent with the presence of a metal Cys-60 Ser No ion bound through the histidine. Karpel et al. (17) have *Virus growth is determined by the reverse transcriptase activity reported that the analogous murine leukemia virus plO NC associated with released viral particles. protein (which contains a single Cys-His sequence) binds tND, not determined. poly(eA) after extensive modification of its three cysteine Downloaded by guest on September 26, 2021 7098 Biochemistry: Jentoft et al. Proc. Natl. Acad. Sci. USA 85 (1988) residues. Moreover, murine leukemia virus NC that con- 1. Sanchez-Pescador, R., Power, M. D., Barr, P. J., Steimer, tained oxidized cysteines had a binding constant for nucleic K. S.,-Stempien, M. M., Brown-Shimer, S. L., Gee, W. W., acids that was qualitatively similar to that of'unmodified Renard, A., Randolph, A., Levy, J. A., Dina, D. & Luciw, protein. Because the cysteine residues of murine leukemia P. A. (1985) Science 227, 484-492. 2. Fuetterer, J. & Hohn, T. (1987) Trends Biochem. Sci. 12, 92- virus NC were quantitatively modified, this NC contains no 95. disulfide bonds, and its cysteines are neither metal-binding 3. Fleissner, E. (1971) J. Virol. 8, 778-785. ligands nor required for binding to nucleic acid. This implies 4. Davis, N. L. & Rueckert, R. R. (1972) J. Virol. 10, 1010-1020. that' the cysteines in murine leukemia virus NC play a 5. Schwartz, D. E., Tizard, R. & Gilbert, W. (1983) Cell 32, 853- structural role, a finding entirely consistent with the conclu- 869. sions of this report. 6. Leis, J., Johnson, S., Collins, L. S. & Traugh, J. A. (1984) J. An understanding ofthe role ofthese essential cysteine and Biol. Chem. 259, 7726-7732. histidine residues in the NC protein requires some insight into 7. Leis, J. & Jentoft, J. (1983) J. Virol. 48, 361-369. its structure. Because the AMV NC can be obtained in 8. Fu, X., Philips, N., Jentoft, J., Leis, J., Tuazon, P. & Traugh, J. (1985) J. 'Biol. Chem. 260, 9941-9947. quantity more readily than the RSV protein, it is important to 9. Berg, J. M. (1986) Science 232, 485-487. know that their respective primary structures are very nearly 10. Souza, L. M., Briskin, M. J., Hillyard, R. L. & Baluda, M. A. the same. The secondary structure based on the CD data (1980) J. Virol. 36, 325-336. indicates that the AMV NC protein contains more than 11. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. two-thirds random structure and less than one-third a-helix Acad. Sci. USA 74, 5463-5467. and P-sheet. However, the protein assumes a stable folded 12. Houts, G. E., Miyagi, M., Ellis, C., Beard, D. & Beard, J. W. configuration in solution, based on chemical-modification (1979) J. Virot. 29, 517-522. results. For example, only three offive lysines (8) and one of 13. Johnson, S., Veigl, M., Vanaman, T. & Leis, J. (1983) J. Virol. six cysteines (L.M.S. and J.E.J., unpublished data) react 45, 876-881. readily with specific modifying reagents. The protein is also 14. Herman, A. C. (1975) Ph.D. Thesis (Duke University, Durham, extremely stable to denaturation: it was difficult to find NC). residues or all of the 15. Katz, R. A., Fu, X., Skalka, A. M. & Leis, J. (1986) Gene 50, conditions whereby all of the lysine 361-369. cysteine residues could be modified. 16. Kowalczykowski, S. C., Lonberg, N., Newport, J. W. & von These data permit us to eliminate several structural pat- Hippel, P. H. (1981) J. Mol. Biol. 145, 75-104. terns commonly seen for small proteins (33) as possible 17. Karpel, R. L., Henderson, L. E. & Oroszlan, S. (1987) J. Biol. folding patterns for the avian NC protein. The CD results Chem. 262, 4961-4967. eliminate the helical bundle and the (3 sandwich as structural 18. Helene, C., Brun, F. & Yaniv, M. (1969) Biochem. Biophys. patterns, and the plasma emission results eliminate the Res. Commun. 37, 393-398. possibility that the structure is organized by metal-binding. A 19. deHaseth, P., Goldman, R., Cech, C. & Caruthers, M. (1983) fourth pattern, that ofa protein with little secondary structure Nucleic Acids Res. 11, 3773-3787. was an 20. Fu, X., Tuazon, P. T., Traugh, J. A. & Leis, J. (1988) J. Biol. but with multiple disulfide bonds, intriguing possibil- Chem. 263, 2134-2139. ity because all cysteine residues ofthe NC are in the Cys-His 21. Fu, X., Katz, R. A., Skalka, A. M. & Leis, J. (1988a) J. Biol. sequences. However, when the primary amino acid sequence Chem. 263, 2140-2145. of NC was reported (23), no mention was made of disulfide 22. Cobrinik, D., Katz, R., Terry, R., Skalka, A. M. & Leis, J. linkages, and our preliminary data indicate that no more than (1987) J. Virol. 61, 1999-2008. a single disulfide bond exists (L.M.S. and J.E.J., unpublished 23. Misono, K., Farida, S., Leis, J. & Vanaman, J. (1980) Fed. data). Thus, based on folding patterns, the AMV/RSV NC Proc. Fed. Am. Soc. Exp. Biol. 36, 1611 (abstr.). protein may represent a fifth, and as yet undescribed, class 24. Chen, Y.-H., Yang, J. T. & Chau, K. H. (1974) Biochemistry of small proteins. 13, 3350-3359. that the from AMV 25. Chang, C. T., Wu, C.-S. C. & Yang, J. T. (1978) Anal. Bio- Finally, it should be noted NC protein chem. 91, 13-31. binds to nucleic acid with positive cooperativity. Protein- 26. Auld, D. S., Kawaguchi, H., Livingston, D. M. & Vallee, protein'interactions thus contribute to NC binding to nucleic B. L. (1974) Biochem. Biophys. Res. Commun. 57, 967-972. acid. The details ofthese binding interactions require further 27. McGhee, J. D. & von Hippel, P. H. (1974) J. Mol. Biol. 86, investigation. 469-489. 28. McGhee, J. D. & von Hippel, P. H. (1976) J. Mol. Biol. 103, Virus supernatants were a gift from Dr. Edward Houts (Molecular 679. Genetics Resources, Tampa, FL). We thank Josephine Secnik for aid 29. Voynow, S. L. & Coffin, J. M. (1985) J. Virol. 55, 79-85. in isolating the AMV NC protein and in performing the CD studies. 30. Meric, C. & Spahr, P.-F. (1986) J. Virol. 60, 450-459. We are also grateful to Dr. M. Baluda for providing the M191 31. Klug, A. & Rhodes, D. (1987) Trends Biochem. Sci. 12, 464- plasmid. This work was supported by Grant GM 36948 from the 469. National Institute ofGeneral Medical Sciences, and Grant CA 38046 32. Bundi, A. & Wuthrich, K. (1979) Biopolymers 18, 285-298. from the U.S. Public Health Service. 33. Richardson, J. (1981) Adv. Protein Chem. 34, 168-339. Downloaded by guest on September 26, 2021