Patent Application: US-34042406-A

Abstract:
the present invention provides compositions useful in preparing and / or serving as antitoxins against bacillus anthracis , the causative agent of anthrax . the present invention also provides polypeptides and polynucleotides relating to the capillary morphogenisis gene 2 , vectors containing the polynucleotides and polypeptides , and host cells containing related polynucleotide molecules , all used in association with the treatment of , or the research and development of treatments for anthrax . the present invention also relates to methods for identifying molecules that bind cmg - 2 and molecules that reduce the toxicity of anthrax toxin . finally , the present invention provides methods for treating human and non - human animals suffering from anthrax .

Description:
the present invention provides compositions useful in preparing or serving as an antitoxin against b . anthracis , the causative agent of anthrax . such compositions include complete and / or partial isolated polypeptides of the capillary morphogenesis gene 2 ( cmg - 2 ) protein , and isolated polynucleotides encoding such polypeptides . the applicants have identified the cmg - 2 protein as a second protein capable of binding to the b . anthracis protective antigen ( pa ). the cmg - 2 protein is closely related ( 40 % identity ) to the anthrax toxin receptor ( atr ) previously identified as a surface cell receptor used by pa to bind and internalize the anthrax toxins , thus contributing to the pathogenesis of b . anthracis . like atr , cmg - 2 binds pa on the cell surface and supports toxin entry . unlike atr , which is upregulated in tumor vasculature , cmg - 2 expression is dramatically upregulated in cultured human umbilical vein endothelial cells ( huves ) induced to undergo in vitro capillary morphogenesis . the cmg - 2 protein is encoded by capillary morphogenesis gene 2 , and has been previously shown to bind specifically to laminin and collagen type iv ( bell et al ., 2001 ). presented here as seq id no : 1 is a cdna clone isolated and determined by the applicants . the isolated cdna clone ( seq id no : 1 ) encodes a 489 amino acid polypeptide ( seq id no : 2 ) that contains a putative signal peptide ( sp ) at amino acids 1 - 33 , an integrin - like inserted domain ( 1 - domain ) at amino acids 44 - 214 and a type i transmembrane ( tm ) domain at amino acids 318 - 334 ( fig2 ). a blast search of the genbank database revealed that the first 476 amino acids matched a protein ( seq id no : 4 ) encoded by an uncharacterized cdna clone ( seq id no : 3 ) listed as genbank accession number ak091721 . the region encoding the last 13 amino acids of the cytoplasmic tail matched that of another cmg - 2 cdna clone ( genbank accession number ay040326 ). presumably this difference between the cdna clones is the result of alternative splicing of the primary cmg - 2 mrna transcript . the search also revealed that the isolated cmg - 2 ( seq id nos : 1 and 2 ) was identical to the cmg - 2 published by bell et al . ( 2001 ) ( seq id no : 5 and seq id no : 6 ) except for a 103 amino acid region ( amino acids 213 - 315 of seq id no : 2 ) not present in the bell et al . ( 2001 ) cmg - 2 protein . neither of these previously identified cmg - 2 clones and their proteins have heretofore been identified as a complete or partial anthrax toxin receptor . as illustrated in fig3 , the i - domain of the cmg - 2 protein is 56 % identical to that of atr / tem8 ( seq id no : 8 ). integrin i - domains are found in approximately one - half of all known integrin a - subunits and are the major ligand - binding region if present ( shimaoka et al ., 2002 ). integrin i - domains adopt the dinucleotide - binding ( rossmann ) fold with a central β - sheet containing six p - strands surrounded by six major α - helices and other short α - helices ( fig4 ). integrin i - domains also generally include a metal ion - dependent adhesion site ( midas ), comprised of five amino acid residues ( dxsxs . . . t . . . d ; where x can be any amino acid ), present at the top of the integrin i - domain . the midas motif and a bound divalent cation are generally important for ligand interaction as the ligand contributes a carboxylate side chain that acts as a sixth coordinating residue for the ion , thus greatly stabilizing the ligand - integrin interaction ( shimaoka et al ., 2002 ). it is believed that the midas motif of atr ( d50 , s52 , s54 . . . t118 . . . d150 ) and cmg - 2 ( d50 , s52 , s54 . . . t118 . . . d ( 148 or 152 ),) is critical for interaction with pa , and that such a motif may also be critical for other not yet identified anthrax toxin receptors . an isolated polynucleotide and an isolated polypeptide , as used herein , can be isolated from its natural environment or synthesized . complete purification is not required in either case . amino acid and nucleotide sequences flanking an isolated polypeptide or polynucleotide occurring in nature can , but need not , be absent from the isolated form . further , an isolated polynucleotide has a structure that is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes . the term includes , without limitation : ( a ) a nucleic acid molecule having a sequence of a naturally occurring genomic or extrachromosomal nucleic acid molecule but which is not flanked by the coding sequences that flank the sequence in its natural position ; ( b ) a nucleic acid molecule incorporated into a vector or into a prokaryote or eukaryote genome such that the resulting molecule is not identical to any naturally occurring vector or genomic dna ; ( c ) a separate molecule such as a cdna , a genomic fragment , a fragment produced by polymerase chain reaction ( pcr ™), or a restriction fragment ; and ( d ) a recombinant nucleotide sequence that is part of a hybrid gene , i . e ., a gene encoding a fusion protein . an isolated nucleic acid molecule can be modified or unmodified dna or rna , whether fully or partially single - stranded or double - stranded or even triple - stranded . a nucleic acid molecule may be chemically or enzymatically modified and may include so - called non - standard bases such as inosine . in addition to the full - length and partial cmg - 2 polypeptide sequences presented in seq id no : 2 , seq id no : 4 , and seq id no : 6 , other polypeptide fragments shorter than those sequences that retain pa - binding activity , and variants thereof , are also within the scope of the invention . the entire receptor is not required for utility ; rather , fragments that bind to pa are useful in the invention . a skilled artisan can readily assess whether a fragment binds to pa . a polypeptide is considered to bind to pa if the equilibrium dissociation constant of the binary complex is 10 micromolar or less . pa - binding to cmg - 2 ( or a fragment of cmg - 2 ) can be measured using a protein - protein binding method such as coimmunoprecipitation , affinity column analysis , elisa analysis , flow cytometry or fluorescence resonance energy transfer ( fret ), and surface plasmon resonance ( spr ). spr is particularly suited as it is highly sensitive and accurate , operable in real time , and consumes only minute amounts of protein . spr uses changes in refractive index to quantify macromolecular binding and dissociation to a ligand covalently tethered to a thin gold chip in a micro flow cell . besides the equilibrium dissociation constant ( kd ), on - and off - rate constants ( ka and kd ) can also be obtained . a biacore 2000 instrument ( pharmacia biotech ) can be used . for these measurements . typically , a protein is covalently tethered to a carboxymethyl dextran matrix bonded to the gold chip . binding of a proteinaceous ligand to the immobilized protein results in a quantifiable change in refractive index of the dextran / protein layer . spr can also be used to determine whether the interaction between pa and its receptor is sensitive to low ph , which is relevant to toxin endocytosis . this technique has been used to study protein - protein interactions in many systems , including the interactions of pa 63 with ef and lf . the invention also relates to polypeptides that are at least 80 %, preferably at least 90 %, more preferably at least 95 %, still more preferably at least 97 %, or most preferably at least 99 % identical to any aforementioned pa - binding polypeptide fragment , where pa - binding is maintained . as used herein , “ percent identity ” between amino acid or nucleic acid sequences is synonymous with “ percent homology ,” which can be determined using the algorithm of karlin and altschul ( 1990 ), modified by karlin and altschul ( 1993 ). such an algorithm is incorporated into the nblast and xblast programs of altschul et al . ( 1990 ). blast nucleotide searches are performed with the nblast program , score = 100 , wordlength = 12 , to obtain nucleotide sequences homologous to a nucleic acid molecule of the invention . blast protein searches are performed with the xblast program , score = 50 , wordlength = 3 , to obtain amino acid sequences homologous to a reference polypeptide ( e . g ., seq id no : 2 ). to obtain gapped alignments for comparison purposes , gapped blast is utilized as described in altschul et al . ( 1997 ). when utilizing blast and gapped blast programs , the default parameters of the respective programs ( e . g ., xblast and nblast ) are used . see www . ncbi . nlm . nih . gov . a variant can also include , e . g ., an internal deletion or insertion , a conservative or non - conservative substitution , or a combination of these variations from the sequence presented . soluble fragments are of great interest as these can competitively inhibit anthrax toxin binding to either cmg - 2 or atr and thereby protect cells from anthrax intoxication in vivo and in vitro . a fragment is soluble if it is not membrane - bound and is soluble in an aqueous fluid . the extracellular cmg - 2 domain is a soluble fragment of the cmg - 2 , as are fragments of that domain . even though the integrin i - domain is formally identified as extending from amino acid 44 to 214 in the extracellular domain , more or fewer natively adjacent amino acids can be included in the fragment without compromising solubility or pa - binding . for example , a pa - binding fragment having the sequence of seq id no : 2 or seq id no : 4 beginning at any amino acid in the range from 33 to 43 and ending at any amino acid in the range from 214 to 489 . a pa - binding fragment having the sequence of seq id no : 6 beginning at any amino acid in the range from 33 to 43 and ending at any amino acid in the range from 212 to 386 . a preferred soluble , pa - binding fragment extends from amino acid 44 to 214 of either seq id nos : 2 , 4 or 6 . another preferred soluble pa - binding fragment includes a fragment of cmg - 2 from amino acid 33 through amino acid 214 of either seq id nos : 2 , 4 or 6 . likewise , any polypeptide fragment of these preferred fragments that retains pa - binding activity is within the scope of the invention . preferably , such fragments include the midas motif comprised of five amino acid residues ( dxsxs . . . t . . . d ; where x can be any amino acid ). cmg - 2 in soluble form is effective in a monomeric form , as well as in multimeric forms such as dimeric , tetrameric , pentameric and higher oligomeric forms . pa - binding polypeptides can include , therefore , seq id no : 2 , seq id no : 4 , seq id no : 6 , a pa - binding fragment of seq id no : 2 , a pa - binding fragment of seq id no : 4 , a pa - binding fragment of seq id no : 6 , a pa - binding polypeptide at least 80 % identical to any of the foregoing fragments . the pa - binding polypeptides can also be provided as fusion proteins comprising any of the foregoing that can comprise still other non - natively adjacent amino acids for detecting , visualizing , isolating , or stabilizing the polypeptide . for example , pa binds to the fusion protein of egfp ( enhanced green fusion protein ) fused to the c - terminal residue of the cytoplasmic tail of the cmg - 2 cdna clone . likewise , isolated polynucleotides having an uninterrupted nucleic acid sequence encoding any of the aforementioned polypeptides and polypeptide fragments may be used to practice the present invention . the sequences that encode soluble , pa - binding polypeptide fragments of cmg - 2 are immediately apparent to the skilled artisan from the description of the relevant portions of the polypeptides , supra . an isolated nucleic acid containing the complement of any such polynucleotides , when used . to generate or develop anthrax antitoxins , is also within the scope of the present invention , as are polynucleotide and oligonucleotide fragments for use as molecular probes . the present invention also relates to an isolated polynucleotide and its complement , without regard to source , where the polynucleotide hybridizes under stringent or moderately stringent hybridization conditions to seq id no : 1 , seq id no : 3 , or seq id no : 5 , or to a fragment of any of the foregoing that encodes a soluble polypeptide that binds to pa . such a polypeptide can not include seq id no : 3 or seq id no : 5 . as used herein , stringent conditions involve hybridizing at 68 ° c . in 5 × ssc / 5 × denhardt &# 39 ; s solution / 1 . 0 % sds , and washing in 0 . 2 × ssc / 0 . 1 % sds +/− 100 μg / ml denatured salmon sperm dna , at room temperature . moderately stringent conditions include washing in the same buffer at 42 ° c . additional guidance regarding such conditions is readily available in the art , for example , by sambrook et al . ( 2001 ); ausubel et al . ( 1996 ). in a related aspect , any polynucleotide used to practice the present invention can be provided in a vector in a manner known to those skilled in the art . the vector can be a cloning vector or an expression vector . in an expression vector , the polypeptide - encoding polynucleotide is under the transcriptional control of one or more non - native expression control sequences , such as a promoter not natively adjacent to the polynucleotide , such that the encoded polypeptide can be produced when the vector is delivered into a compatible host cell that supports expression of an polypeptide encoded on a vector , for example by electroporation or transfection , or transcribed and translated in a cell - free transcription and translation system . such cell - based and cell - free systems are well known to the skilled artisan . cells comprising an insert - containing vector of the invention are themselves within the scope of the present invention , without regard to whether the vector is extrachromosomal or integrated in the genome . a skilled artisan in possession of the polypeptides and polynucleotides of the invention can also identify agents that can reduce or prevent the effect of anithrax toxin on a host having on the cell surface at least a portion of a receptor that binds pa . the effect altered can relate , for example , to ( 1 ) susceptibility of the host cell to anthrax toxin damage , ( 2 ) integration of cmg - 2 , atr or pa into the cell membrane , ( 3 ) binding between pa and cmg - 2 or atr , ( 4 ) pa heptamerization , ( 5 ) uptake of the pa / cmg - 2 complex or the pa / atr complex into cells , and ( 6 ) the translocation of toxin into host cell cytoplasm . the method includes separately exposing a plurality of putative agents in the presence of anthrax toxin to a plurality of cells , comparing the effect of anthrax toxin on the cells in the presence and absence of the agent , and identifying at least one agent that alters an effect of anthrax toxin on the cells . the skilled artisan can readily evaluate the typical effects of anthrax toxin and observe variations in those effects in the presence of a putative altering agent . for example , susceptibility to anthrax toxin damage can be evaluated by exposing host cells to anthrax toxin . integration of newly formed cmg - 2 into the host cell membrane can be evaluated by labeling newly synthesized proteins in the host cell and immunoprecipitating cmg - 2 from the cellular membrane fraction of the host cell . binding of wild - type cmg - 2 to pa can be evaluated with fluorescent labeled anti - pa antibody . pa heptamerization can be evaluated by several techbiques including native polyacrylamide gel electrophoresis , gel filtration , and western blotting . uptake of pa / atr or pa / cmg - 2 complex can be evaluated by binding pa to atr or cmg - 2 at 4 ° c ., increasing the temperature to 37 ° c . to allow endocytosis , shifting the temperature back to 4 ° c ., and incubating cells with fluorescent labeled anti - pa antibodies . toxin translocation into the host cell cytoplasm can be evaluated as described in wesche et al . ( 1998 ), which is incorporated herein by reference as if set forth in its entirety . the agents screened can be , for example , a high molecular weight molecule such as a polypeptide ( including , e . g ., a mutant anthrax toxin , a soluble cmg - 2 , a monoclonal or polyclonal antibody to cmg - 2 ; pa , or an pa / cmg - 2 complex ), a polysaccharide , a lipid , a nucleic acid , a low molecular weight organic or inorganic molecule , or the like . antibodies can be produced by administering to a non - human animal an immunogenic , pa - binding fragment of a polypeptide which can be , e . g ., seq id no : 2 , seq id no : 4 , seq id no : 6 , a polypeptide at least 80 % identical to any of the foregoing and a fusion protein comprising any of the foregoing , and then obtaining the desired antibodies using known methods . chemical libraries for screening putative agents , including peptide libraries , are readily available to the skilled artisan . examples include those from asinex ( i . e . the combined wisdom library of 24 , 000 manually synthesized organic molecules ) and from chembridge corporation ( i . e ., the diverse ™ library of 50 , 000 manually synthesized chemical compounds ; the screen - set ™ library of 24 , 000 manually synthesized chemical compounds ; the cns - set ™ library of 11 , 000 compounds ; the cherry - pick ™ library of up to 300 , 000 compounds ) and linear library , multimeric library and cyclic library ( tecnogen , italy ). once an agent with desired activity is identified , a library of derivatives of that agent can be screened for better agents . phage display is also a suitable approach for finding novel inhibitors of the interaction between pa and both cmg - 2 and atr . another aspect of the present invention relates to cmg - 2 ligands other than pa , laminine and collagen type iv , and methods for identifying such other ligands . to identify these other ligands , a polypeptide that contains a cmg - 2 integrin i - domain , preferably an entire extracellular domain , can be provided in soluble or tethered form , e . g ., in a chromatographic column . preferably , the i - domain of cmg - 2 can be provided as a fusion protein that also contains rabbit igg constant region , a gst domain or a hexahistidine tag . this fusion protein can be immobilized on a chromatographic column using known methods . a cell extract can be passed over the column . a ligand is identified when binding is observed between the i - domain and a compound present in the cell extract . the identified ligand can be used in methods for identifying agents that alter an effect of anthrax toxin , to identify an agent that selectively inhibits pa - receptor binding . it is also desirable to use the other ligands and cmg - 2 in comparative high throughput screening methods for identifying small molecules that do not interfere with natural ligand binding to cmg - 2 , but which do prevent or reduce binding of cmg - 2 or other receptors to anthrax toxin . the present invention also relates to reducing cellular damage caused by anthrax toxin , which can be achieved by administering an agent for reducing cmg - 2 levels , inhibiting the binding between cmg - 2 and pa , or by reducing downstream cmg - 2 activity after binding to pa . for example , an antisense oligonucleotide can reduce or prevent expression of cmg - 2 using delivery methods known to the skilled artisan , thus reducing the cellular cmg - 2 level . a cmg - 2 anthrax binding inhibition agent can competitively inhibit the binding between cmg - 2 and pa and / or pa and other anthrax toxin receptors , such as atr . dominant negative cmg - 2s can block downstream cmg - 2 activities required for anthrax toxin toxicity . such agents can be administered to a human or non - human animal , preferably in a standard pharmaceutical carrier , in an amount effective to reduce or eliminate anthrax toxicity . a 20 - 25 mer antisense oligonucleotide can be directed against the 5 ′ end of cmg - 2 mrna with phosphorothioate derivatives on the last three base pairs on the 3 ′ end and the 5 ′ end to enhance the half life and stability of the oligonucleotides . a carrier for an antisense oligonucleotide can be used . an example of a suitable carver is cationic liposomes . for example , an oligonucleotide can be mixed with cationic liposomes prepared by mixing 1 - alpha dioleylphatidylcelthanolamine with dimethldioctadecylammomum bromide in a ratio of 5 : 2 in 1 ml of chloroform . the solvent will be evaporated and the lipids resuspended by sonication in 10 ml of saline . another way to use an antisense oligonucleotide is to engineer it into a vector so that the vector can produce an antisense crna that blocks the translation of the mrnas encoding for cmg - 2 . similarly , rnai techniques , which are now being applied to mammalian systems , are also suited for inhibiting cmg - 2 expression . ( zamore , 2001 , incorporated herein by reference as if set forth in its entirety ). the present invention also relates to a method for detecting cmg - 2 mrna or cmg - 2 protein in a sample . such detection can be readily accomplished by using oligonucleotide or polynucleotide probes for cmg - 2 mrna , or antibodies for the cmg - 2 protein . in a related aspect , the antibodies made and identified as being able to bind to cmg - 2 can also be used to separate cmg - 2 from a sample . the invention also relates to the use of cell lines containing or not containing cmg - 2 for testing anthrax toxicity , and methods for making such cell lines . methods for developing such cell lines are well known by the skilled artisan . for example , generating a cell line that does not express cmg - 2 may be feasible using mutagenesis and screening , or homologous recombination . such cell lines and their use are deemed to be within the scope of the present invention . the invention also provides molecules and methods for specifically targeting and killing cells of interest by delivering , e . g ., anthrax toxin or lf to the cell . it is anticipated that soluble cmg - 2 molecules can be coupled to a ligand or to a single chain antibody selected for targeting to the cell of interest ( e . g . i a ligand that binds a receptor presented on a tumor cell surface ). the coupling may be accomplished by producing a fusion protein that encodes both the cmg - 2 binding portion and the ligand or single chain antibody molecule . the ligand or single chain antibody domains simply serve to attach the toxin to cells with the cognate surface markers . the toxin or factor may be preloaded onto the cmg - 2 portion before exposing the coupled molecules to the targeted cells . this is similar in principle to that previously described for retroviral targeting using soluble retroviral receptor - ligand bridge proteins and retroviral receptor - single chain antibody bridge proteins ( snitkovsky and young , 1998 ; boerger et al ., 1999 ; snitkovsky et al ., 2000 ; snitkovsky et al ., 2001 ), each incorporated herein by reference as if set forth in its entirety . the invention will be more fully understood upon consideration of the following non - limiting examples . to test cmg - 2 function and to facilitate protein detection , a fusion protein was generated in which egfp was fused to the c - terninal residue of the cytoplasmic tail of the hs003 cmg - 2 cdna clone ( seq id no : 1 ). this strategy was used because it had previously been shown that a similarly constructed atr - egfp fusion protein was competent for pa - binding and intoxication . the gene encoding cmg - 2 - egfp was expressed from a retroviral vector in cho - r1 . 1 cells that lack anthrax toxin receptors as described in bradley et al ., supra , incorporated herein by reference . these cells bound pa ( fig5 a ) and were highly susceptible to cell killing by pa and lfn - dta ( fig5 b ). therefore , cmg - 2 is a bona fide anthrax toxin receptor . cmg2 - 488 ( genbank accession no . ak091721 ) is different from cmg2 - 489 ( genbank accession no . ay233452 ) in its c - terminal 12 amino acids . although cmg - 489 is only one amino longer in total length , the c - terminal 13 amino acids of cmg2 - 489 have been replaced with 12 different amino acids that are derived from the c - terminus of cmg2 - 488 . both of these cmg2 protein isoforms are encoded by naturally occurring mrna splice variants . cmg2 - 488 has been shown by rt - pcr analysis to be expressed in most tissues ( 15 of 16 human tissues tested ). since cmg2 - 488 can act as a toxin receptor , this broad expression makes it likely to play a role in in vivo anthrax intoxication . when similarly constructed and produced soluble atr and cmg2 i domain proteins ( satr and scmg2 respectively ) were tested as inhibitors ( receptor decoys ) of intoxication in a cell culture model , scmg2 was 10 - fold more potent than satr in protecting cultured cells from death ( fig6 ). this may indicate that the cmg2 - pa binding interaction is stronger than that of atr - pa , but this remains to be formally proven in binding affinity studies . nevertheless , this difference in relative potency suggests that scmg2 is a better candidate for use as an in vivo receptor - based antitoxin . all of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure . while the compositions and methods of this invention have been described in terms of preferred embodiments , it will be apparent to those of skill in the art that variations may be applied to the compositions and / or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept , spirit and scope of the invention . more specifically , it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved . all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit , scope and concept of the invention as defined by the appended claims . the following references , to the extent that they provide exemplary procedural or other details supplementary to those set forth herein , are specifically incorporated herein by reference . altschul et al ., nucleic acids res ., 25 : 3389 - 3402 , 1997 . altschul et al ., j . mol . biol ., 215 : 403 - 410 , 1990 . ausubel et al ., in : current protocols in molecular biology , john , wiley & amp ; 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