SOLUBLE FRAGMENTS OF INFLUENZA VIRUS PB2 PROTEIN CAPABLE OF BINDING RNA-CAP

Abstract

des an upper portion having a mouth defining an opening into the container. A shoulder region extends from the upper portion. A sidewall portion extends between the shoulder region and a base portion. The base portion closes off an end of the container. A vacuum panel region defined in part by at least two vacuum panels. Each of the vacuum panels are movable to accommodate vacuum forces generated within the container resulting from heating and cooling of its contents. The vacuum panel region occupies an area outboard of the sidewall portion.

Description

TECHNICAL FIELD

[0001]This disclosure generally relates to plastic containers for retaining a commodity, and in particular a liquid commodity. More specifically, this disclosure relates to a plastic container having a vacuum panel region defined on the plastic container in an area distinct from a sidewall having a label panel area.

BACKGROUND

[0002]As a result of environmental and other concerns, plastic containers, more specifically polyester and even more specifically polyethylene terephthalate (PET) containers are now being used more than ever to package numerous commodities previously supplied in glass containers. Manufacturers and fillers, as well as consumers, have recognized that PET containers are lightweight, inexpensive, recyclable and manufacturable in large quantities.

[0003]Blow-molded plastic containers have become commonplace in packaging numerous commodities. PET is a crystallizable polymer, meaning that it is available in an amorphous form or a semi-crystalline form. The ability of a PET container to maintain its material integrity relates to the percentage of the PET container in crystalline form, also known as the "crystallinity" of the PET container. The following equation defines the percentage of crystallinity as a volume fraction:

% Crystallinity = ( ρ - ρ a ρ c - ρ a ) × 100 ##EQU00001##

where ρ is the density of the PET material; ρ_a is the density of pure amorphous PET material (1.333 g/cc); and ρ_c is the density of pure crystalline material (1.455 g/cc).

[0004]Container manufacturers use mechanical processing and thermal processing to increase the PET polymer crystallinity of a container. Mechanical processing involves orienting the amorphous material to achieve strain hardening. This processing commonly involves stretching an injection molded PET preform along a longitudinal axis and expanding the PET preform along a transverse or radial axis to form a PET container. The combination promotes what manufacturers define as biaxial orientation of the molecular structure in the container. Manufacturers of PET containers currently use mechanical processing to produce PET containers having approximately 20% crystallinity in the container's sidewall.

[0005]Thermal processing involves heating the material (either amorphous or semi-crystalline) to promote crystal growth. On amorphous material, thermal processing of PET material results in a spherulitic morphology that interferes with the transmission of light. In other words, the resulting crystalline material is opaque, and thus, generally undesirable. Used after mechanical processing, however, thermal processing results in higher crystallinity and excellent clarity for those portions of the container having biaxial molecular orientation. The thermal processing of an oriented PET container, which is known as heat setting, typically includes blow molding a PET preform against a mold heated to a temperature of approximately 250° F.-350° F. (approximately 121° C.-177° C.), and holding the blown container against the heated mold for approximately two (2) to five (5) seconds. Manufacturers of PET juice bottles, which must be hot-filled at approximately 185° F. (85° C.), currently use heat setting to produce PET bottles having an overall crystallinity in the range of approximately 25%-35%.

[0006]After being hot-filled, the heat-set containers may be capped and allowed to reside at generally the filling temperature for approximately five (5) minutes at which point the container, along with the product, is then actively cooled prior to transferring to labeling, packaging, and shipping operations. The cooling reduces the volume of the liquid in the container. This product shrinkage phenomenon results in the creation of a vacuum within the container. Generally, vacuum pressures within the container range from 1-380 mm Hg less than atmospheric pressure (i.e., 759 mm Hg-380 mm Hg). If not controlled or otherwise accommodated, these vacuum pressures result in deformation of the container, which leads to either an aesthetically unacceptable container or one that is unstable. Hot-fillable plastic containers must provide sufficient flexure to compensate for the changes of pressure and temperature, while maintaining structural integrity and aesthetic appearance. Typically, the industry accommodates vacuum related pressures with sidewall structures or vacuum panels formed within the sidewall of the container. Such vacuum panels generally distort inwardly under vacuum pressures in a controlled manner to eliminate undesirable deformation.

[0007]While such vacuum panels allow containers to withstand the rigors of a hot-fill procedure, the panels have limitations and drawbacks. First, such panels formed within the sidewall of a container do not create a generally smooth glass-like appearance. Second, packagers often apply a wrap-around or sleeve label to the container over these panels. The appearance of these labels over the vacuum panels is such that the label often becomes wrinkled and not smooth. Additionally, one grasping the container generally feels the vacuum panels beneath the label and often pushes the label into various panel crevasses and recesses.

SUMMARY

[0008]A plastic container includes an upper portion having a mouth defining an opening into the container. A shoulder region extends from the upper portion. A sidewall portion extends between the shoulder region and a base portion. The base portion closes off an end of the container. A vacuum panel region is defined in part by at least two vacuum panels. Each of the vacuum panels are movable to accommodate vacuum forces generated within the container resulting from heating and cooling of its contents. The vacuum panel region occupies an area outboard of the sidewall portion.

[0009]According to additional features, the vacuum panels each define a plane that is substantially parallel to a longitudinal axis of the plastic container. The vacuum panels can be generally rectangular shaped. In one example, the vacuum panels include three pair of vacuum panels. Each vacuum panel opposes a corresponding vacuum panel. The sidewall portion includes a series of horizontal ribs that substantially circumscribe a perimeter of the sidewall portion.

[0010]According to another example, the vacuum panel region can comprise a first vacuum panel region and a second vacuum panel region. The sidewall portion is formed intermediate of the first and second vacuum panel regions. Both of the first and second vacuum panel regions define three pair of vacuum panels.

[0011]Additional benefits and advantages of the present disclosure will become apparent to those skilled in the art to which the present disclosure relates from the subsequent description and the appended claims, taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 is a perspective view of a plastic container constructed in accordance with the teachings of the present disclosure.

[0013]FIG. 2 is a side elevational view of the plastic container of FIG. 1.

[0014]FIG. 3 is a top view of the plastic container of FIG. 1.

[0015]FIG. 4 is a cross-sectional view of the plastic container taken along line 4-4 of FIG. 1.

[0016]FIG. 5 is a perspective view of a plastic container constructed in accordance with additional teachings of the present disclosure.

[0017]FIG. 6 is a side elevational view of the plastic container of FIG. 5.

[0018]FIG. 7 is a top view of the plastic container of FIG. 5; and

[0019]FIG. 8 is a cross-sectional view of the plastic container taken along line 8-8 of FIG. 5.

DETAILED DESCRIPTION

[0020]The following description is merely exemplary in nature, and is in no way intended to limit the disclosure or its application or uses.

[0021]With reference to FIGS. 1-4, a plastic, e.g. polyethylene terephthalate (PET), hot-fillable container according to the present teachings is shown and generally identified at reference number 10. As shown in FIG. 2, the plastic container 10 has an overall height H₁ of about 190.3 mm (7.49 inches). The height H₁ may be selected so that the plastic container 10 fits on the shelves of a supermarket or store. In this particular embodiment, the plastic container 10 has a volume capacity of about 20 fl. oz. (591 cc). Those of ordinary skill in the art would appreciate that the following teachings are applicable to other containers, such as containers having different shapes, which may have different dimensions and volume capacities. It is also contemplated that other modifications can be made depending on the specific application and environmental requirements.

[0022]The plastic container 10 according to the present teachings defines a body 12 and includes an upper portion 14 having a finish 16. Integrally formed with the finish 16 and extending downward therefrom is a shoulder region 20. The shoulder region 20 merges into and provides a transition between the finish 16 and a sidewall portion 22. The sidewall portion 22 extends downward from the shoulder region 20 to a vacuum panel region 26. The vacuum panel region 26 merges into a base portion 28 having a base 30. A neck 32 may also be included having an extremely short height, that is, becoming a short extension from the finish 16, or an elongated height, extending between the finish 16 and the shoulder region 20. The plastic container 10 has been designed to retain a commodity. The commodity may be in any form such as a solid or liquid product. In one example, a liquid commodity may be introduced into the plastic container 10 during a thermal process, typically a hot-fill process. For hot-fill bottling applications, bottlers generally fill the plastic container 10 with a liquid or product at an elevated temperature between approximately 155° F. to 205° F. (approximately 68° C. to 96° C.) and seal the plastic container 10 with a cap (not illustrated) before cooling. In addition, the plastic container 10 may be suitable for other high-temperature pasteurization or retort filling processes or other thermal processes as well. In another example, the commodity may be introduced into the plastic container 10 under ambient temperatures.

[0023]The finish 16 of the plastic container 10 includes a portion defining an aperture or mouth 36, and a threaded region 38 having threads 40. The finish 16 can also define a support ring 42. The support ring 42 may be used to carry or orient a preform (the precursor to the plastic container 10, not illustrated) through and at various stages of manufacture. For example, the preform may be carried by the support ring 42, the support ring 42 may be used to aid in positioning the preform in the mold, or an end consumer may use the support ring 42 to carry the plastic container 10 once manufactured.

[0024]The aperture 36 allows the plastic container 10 to receive a commodity while the threaded region 38 provides a means for attachment of a similarly threaded closure or cap (not illustrated). Alternatives may include other suitable devices that engage the finish 16 of the plastic container 10. Accordingly, the closure or cap (not illustrated) engages the finish 16 to preferably provide a hermetical seal of the plastic container 10. The closure or cap (not illustrated) is preferably of a plastic or metal material conventional to the closure industry and suitable for subsequent thermal processing, including high temperature pasteurization and retort.

[0025]The sidewall portion 22 includes a series of horizontal ribs 44. The horizontal ribs 44 substantially circumscribe the entire perimeter of the sidewall portion 22 of the plastic container 10. The horizontal ribs 44 extend continuously in a longitudinal direction from the shoulder region 20 to the vacuum panel region 26. According to one example, the sidewall portion 22 can define a width W₅. The width W₅ can be approximately 60 mm (2.36 inches). The base 30 functions to close off the base portion 28 of the plastic container 10 and, together with the finish 16, the shoulder region 20, the sidewall portion 22, and the vacuum panel region 26, to retain the commodity. The base portion 28 generally defines an outer surface having a thread detail 48 formed therearound. The thread detail 48 can assist in providing structural integrity to the base portion 28 as well as provide an ornamental appeal to the plastic container 10. Additionally, the thread detail 48 may facilitate attachment of a secondary container or closure.

[0026]The vacuum panel region 26 is generally defined between lateral surfaces 50 at a stepped-out portion 52 of the plastic container 10. The vacuum panel region 26 defines a plurality of vacuum panels 56 generally extending on respective planes that are parallel to a central longitudinal axis 60 of the plastic container 10. According to one example, the stepped-out portion 52 can define a width W₁ between opposing vacuum panels 56. The width W₁ can be approximately 85 mm (3.35 inches). Preferably, the width W₁ may be at least 10% greater than the width W₅. More preferably, the width W₁ may be about 20%-40% greater than the width W₅.

[0027]As illustrated in FIGS. 1-4, the vacuum panels 56 may be generally rectangular in shape. It is appreciated that the vacuum panels 56 may define other geometrical configurations as well. Accordingly, the plastic container 10 illustrated in the FIGS. 1-4 has six (6) vacuum panels 56. The inventors however equally contemplate that more than or less than six (6) vacuum panels 56 can be provided. By way of example, the vacuum panel region 26 can also be formed on the plastic container 10 having two (2), three (3), four (4), five (5), seven (7) or eight (8) vacuum panels. As illustrated, the present teachings facilitate the orientation of vacuum panels 56 in a horizontal direction relative to the central longitudinal axis 60 of the plastic container 10. Surrounding the vacuum panels 56 are horizontal and vertical connecting walls 62 and 64, respectively. Each horizontal connecting wall 62 is generally defined between the vacuum panel 56 and respective lateral surfaces 50. The horizontal connecting walls 62 define a generally arcuate profile in horizontal cross-section (see FIG. 4). Each vertical connecting wall 64 is defined between adjacent vacuum panels 56.

[0028]Optionally, each horizontal connecting wall 62 may define a distinctly identifiable structure between the lateral surfaces 50 and an underlying surface 66 of vacuum panels 56. The horizontal connecting walls 62 provide strength to the transition between the lateral surfaces 50 and the underlying surface 66 of the vacuum panels 56. The resulting localized strength increases the resistance to creasing and denting in the vacuum panel region 26 and the plastic container 10 as a whole.

[0029]A label panel area 70 is defined at the sidewall portion 22. The label panel area 70 therefore occupies a distinct portion of the plastic container 10 relative to the vacuum panel region 26. As is commonly known and understood by container manufacturers skilled in the art, a label (not shown) may be applied to the sidewall portion 22 (label panel area 70) using methods that are well known to those skilled in the art, including shrink-wrap labeling and adhesive methods. As applied, the label may extend around the entire body 12 or be limited to a partial circumference of the sidewall portion 22.

[0030]Upon filling, capping, sealing and cooling, as illustrated in FIG. 4 in phantom, the horizontal connecting walls 62 each act as a hinge that aids in the allowance of the underlying surface 66 of vacuum panels 56 to be pulled radially inward, toward the central longitudinal axis 60 of the plastic container 10, displacing volume, as a result of vacuum forces. In this position, the underlying surface 66 of vacuum panels 56, in cross section, illustrated in FIG. 4 in phantom, forms a generally concave surface 66'. The configuration of the sidewall portion 22 and the vacuum panel region 26, allow the vacuum reaction to be absorbed in a controlled manner by the vacuum panels 56 without substantial disruption to the label panel area 70 or a remainder of the plastic container 10.

[0031]As illustrated in FIG. 2, the vacuum panels 56 have a width W₂. In one example, for the plastic container 10 having a nominal capacity of approximately 16.9 fl. oz. (500 cc), the width W₂ may be about 43.81 mm (1.72 inches). A height H₂ defined at an outermost edge of the vacuum panels 56 may be about 27.16 mm (1.07 inches). The height H₂ may vary slightly across the width W₂ of the vacuum panels 56. A height H₃ defined from the shoulder region 20 to a transition between the sidewall portion 22 and the vacuum panel region 26 may be about 74.33 mm (2.93 inches). A height H₄ of the finish 16 may be about 19.71 mm (0.76 inch). A height H₅ of the base portion 28 may be about 48.08 mm (1.89 inches).

[0032]With reference to FIGS. 5-8, a plastic, e.g. polyethylene terephthalate (PET), hot-fillable container according to the present teachings is shown and generally identified at reference number 110. As shown in FIG. 6, the plastic container 110 has an overall height H₆ of about 262.92 mm (10.35 inches). The height H₆ may be selected so that the plastic container 110 fits on the shelves of a supermarket or store. Again, it is contemplated that other modifications can be made depending on the specific application.

[0033]The plastic container 110 according to the present teachings defines a body 112 and includes an upper portion 114 having a finish 116. Integrally formed with the finish 116 and extending downward therefrom is a shoulder region 120. The shoulder region 120 merges into and provides a transition between the finish 116 and a first vacuum panel region 118. The first vacuum panel region 118 merges into a sidewall portion 122. The sidewall portion 122 extends downward from the first vacuum panel region 118 to a second vacuum panel region 126. The second vacuum panel region 126 can transition into a base portion 128 having a base 130. A neck 132 may also be included having an extremely short height, that is, becoming a short extension from the finish 116, or an elongated height, extending between the finish 116 and the shoulder region 120. The plastic container 110 has been designed to retain a commodity. The commodity may be in any form such as a solid or liquid product. In one example, a liquid commodity may be introduced into the plastic container 110 during a thermal process, typically a hot-fill process, such as described above. In another example, the commodity may be introduced into the plastic container 110 under ambient temperatures.

[0034]The finish 116 of the plastic container 110 includes a portion defining an aperture or mouth 136, and a threaded region 138 having threads 140. The finish 116 can also define a support ring 142. The support ring 142 may be used to carry or orient a preform (the precursor to the plastic container 110, not illustrated) through and at various stages of manufacture. For example, the preform may be carried by the support ring 142, the support ring 142 may be used to aid in positioning the preform in the mold, or an end consumer may use the support ring 142 to carry the plastic container 110 once manufactured.

[0035]The aperture 136 allows the plastic container 110 to receive a commodity while the threaded region 138 provides a means for attachment of a similarly threaded closure or cap (not illustrated). Accordingly, the closure or cap (not illustrated) engages the finish 116 to preferably provide a hermetical seal of the plastic container 110. The closure or cap (not illustrated) is preferably of a plastic or metal material conventional to the closure industry and suitable for subsequent thermal processing, including high temperature pasteurization and retort.

[0036]The sidewall portion 122 includes a series of horizontal ribs 144. The horizontal ribs 144 circumscribe the entire perimeter of the sidewall portion 122 of the plastic container 110. The horizontal ribs 144 extend continuously in a longitudinal direction from the first vacuum panel region 118 to the second vacuum panel region 126. According to one example, the sidewall portion 122 can define a width W₆. The width W₆ can be approximately 50.8 mm (2.0 inches). The base 130 functions to close off the base portion 128 of the plastic container 110 and, together with the finish 116, the shoulder region 120, the sidewall portion 122, and the first and second vacuum panel regions 118 and 126, respectively, to retain the commodity.

[0037]The first and second vacuum panel regions 118 and 126 are generally defined at first and second stepped-out portions 152 and 154, respectively, of the plastic container 110. The figures and the following description are directed toward first and second vacuum panel regions that are substantially equivalent in formation, however, they may be formed differently from each other. The first and second vacuum panel regions 118 and 126 each define a plurality of vacuum panels 156 and 158, respectively, generally extending on respective planes that are parallel to a central longitudinal axis 160 of the plastic container 110. According to one example, the stepped-out portions 152 and 154 can define a width W₃ between opposing vacuum panels 156 (and likewise, opposing vacuum panels 158). The width W₃ can be approximately 67.06 mm (2.64 inches). As in the previous example, preferably, the width W₃ may be at least 10% greater than the width W₆. More preferably, the width W₃ may be about 20%-40% greater than the width W₆.

[0038]As illustrated in FIGS. 5-8, the vacuum panels 156 and 158 may be generally rectangular in shape. It is appreciated that the vacuum panels 156 and 158 may define other geometrical configurations as well. Accordingly, the plastic container 110 illustrated in the FIGS. 5-8 has six (6) vacuum panels 156 defined on the first vacuum panel region 118, and six (6) vacuum panels 158 defined on the second vacuum panel region 126. The inventors however equally contemplate that more than or less than six (6) vacuum panels 156 and 158 can be provided. By way of example, one or both of the first and second vacuum panel regions 118 and 126 can also be formed on the plastic container 110 having two (2), three (3), four (4), five (5), seven (7) or eight (8) vacuum panels. As illustrated, the present teachings facilitate the orientation of vacuum panels 156 and 158 in a horizontal direction relative to the central longitudinal axis 160 of the plastic container 110.

[0039]Surrounding the vacuum panels 156 are horizontal and vertical connecting walls 162 and 164, respectively. Each horizontal connecting wall 162 is generally defined between the vacuum panel 156 and an adjacent radial surface 165. The horizontal connecting walls 162 define a generally arcuate profile in horizontal cross-section (see FIG. 8). Each vertical connecting wall 164 is defined between adjacent vacuum panels 156.

[0040]Surrounding the vacuum panels 158 are horizontal and vertical connecting walls 167 and 168, respectively. Each horizontal connecting wall 167 is generally defined between the vacuum panel 158 and an adjacent radial surface 169. The horizontal connecting walls 167 define a generally arcuate profile in horizontal cross-section (see FIG. 8). Each vertical connecting wall 168 is defined between adjacent vacuum panels 158.

[0041]Optionally, each horizontal connecting wall 162 and 167 may define a distinctly identifiable structure between the adjacent radial surfaces 165 and 169 and an underlying surface 171 and 172 of vacuum panels 156 and 158, respectively. The horizontal connecting walls 162 and 167 provide strength to the transition between the adjacent radial surfaces 165 and 169 and the underlying surfaces 171 and 172. The resulting localized strength increases the resistance to creasing and denting in the first and second vacuum panel regions 118 and 126, and the plastic container 110 as a whole.

[0042]A label panel area 180 is defined at the sidewall portion 122. The label panel area 180 therefore occupies a distinct portion of the plastic container 110 relative to the first and second vacuum panel regions 118 and 126. In this example, the label panel area 180 is defined between the first and second vacuum panel regions 118 and 126. As is commonly known and understood by container manufacturers skilled in the art, a label (not shown) may be applied to the sidewall portion 122 (label panel area 180) using methods that are well known to those skilled in the art, including shrink-wrap labeling and adhesive methods. As applied, the label may extend around the entire body 112 or be limited to a partial circumference of the sidewall portion 122.

[0043]Upon filling, capping, sealing and cooling, as illustrated in FIG. 8 in phantom, the horizontal connecting walls 162 and 167 each act as a hinge that aids in the allowance of the underlying surface 171 and 172 of vacuum panels 156 and 158 to be pulled radially inward, toward the central longitudinal axis 160 of the plastic container 110, displacing volume, as a result of vacuum forces. In this position, the underlying surface 171 and 172 of vacuum panels 156 and 158, in cross section, illustrated in FIG. 8 in phantom, form a generally concave surface 171' and 172', respectively. The configuration of the sidewall portion 122 and the first and second vacuum panel regions 118 and 126, allow the vacuum reaction to be absorbed in a controlled manner by the vacuum panels 156 and 158 without substantial disruption to the label panel area 180 or a remainder of the plastic container 110.

[0044]As illustrated in FIG. 6, the vacuum panels 156 and 158 have a width W₄. In one example, for the plastic container 110 having a nominal capacity of approximately 16.9 fl. oz. (500 cc), the width W₄ may be about 34.63 mm (1.36 inches). A height H₇ defined at an outermost edge of vacuum panels 156 and 158 may be about 21.16 mm (0.83 inch). The height H₇ may vary slightly across the width W₄ of the vacuum panels 156 and 158. A height H₈ defined by the sidewall portion 122 (label panel area 180) may be about 76.29 mm (3.00 inches). A height H₉ of the finish 116 may be about 18.62 mm (0.73 inch). A height H₁₀ of the second vacuum panel region 126 and the base portion 128 may be about 74.81 mm (2.95 inches).

[0045]While the above description constitutes the present disclosure, it will be appreciated that the disclosure is susceptible to modification, variation and change without departing from the proper scope and fair meaning of the accompanying claims.

Sequence CWU 1

271759PRTInfluenza A virusCHAIN(1)..(759)PB2 1Met Glu Arg Ile Lys Glu Leu Arg Asn Leu Met Ser Gln Ser Arg Thr1 5 10 15Arg Glu Ile Leu Thr Lys Thr Thr Val Asp His Met Ala Ile Ile Lys 20 25 30Lys Tyr Thr Ser Gly Arg Gln Glu Lys Asn Pro Ser Leu Arg Met Lys 35 40 45Trp Met Met Ala Met Lys Tyr Pro Ile Thr Ala Asp Lys Arg Ile Thr 50 55 60Glu Met Val Pro Glu Arg Asn Glu Gln Gly Gln Thr Leu Trp Ser Lys65 70 75 80Met Ser Asp Ala Gly Ser Asp Arg Val Met Val Ser Pro Leu Ala Val 85 90 95Thr Trp Trp Asn Arg Asn Gly Pro Val Thr Ser Thr Val His Tyr Pro 100 105 110Lys Val Tyr Lys Thr Tyr Phe Asp Lys Val Glu Arg Leu Lys His Gly 115 120 125Thr Phe Gly Pro Val His Phe Arg Asn Gln Val Lys Ile Arg Arg Arg 130 135 140Val Asp Ile Asn Pro Gly His Ala Asp Leu Ser Ala Lys Glu Ala Gln145 150 155 160Asp Val Ile Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg Ile 165 170 175Leu Thr Ser Glu Ser Gln Leu Thr Ile Thr Lys Glu Lys Lys Glu Glu 180 185 190Leu Gln Asp Cys Lys Ile Ser Pro Leu Met Val Ala Tyr Met Leu Glu 195 200 205Arg Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Ala Gly Gly Thr 210 215 220Ser Ser Val Tyr Ile Glu Val Leu His Leu Thr Gln Gly Thr Cys Trp225 230 235 240Glu Gln Met Tyr Thr Pro Gly Gly Glu Val Arg Asn Asp Asp Ile Asp 245 250 255Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg Ala Ser Val 260 265 270Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Gln 275 280 285Ile Gly Gly Thr Arg Met Val Asp Ile Leu Arg Gln Asn Pro Thr Glu 290 295 300Glu Gln Ala Val Asp Ile Cys Lys Ala Ala Met Gly Leu Arg Ile Ser305 310 315 320Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser 325 330 335Ser Ile Lys Arg Glu Glu Glu Val Leu Thr Gly Asn Leu Gln Thr Leu 340 345 350Lys Ile Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Lys 355 360 365Arg Ala Thr Ala Ile Leu Arg Lys Ala Thr Arg Arg Leu Val Gln Leu 370 375 380Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val385 390 395 400Ala Met Val Phe Ser Gln Glu Asp Cys Met Ile Lys Ala Val Arg Gly 405 410 415Asp Leu Asn Phe Val Asn Arg Ala Asn Gln Arg Leu Asn Pro Met His 420 425 430Gln Leu Leu Arg His Phe Gln Lys Asp Ala Lys Val Leu Phe Gln Asn 435 440 445Trp Gly Ile Glu His Ile Asp Asn Val Met Gly Met Val Gly Val Leu 450 455 460Pro Asp Met Thr Pro Ser Thr Glu Met Ser Met Arg Gly Ile Arg Val465 470 475 480Ser Lys Met Gly Val Asp Glu Tyr Ser Ser Thr Glu Arg Val Val Val 485 490 495Ser Ile Asp Arg Phe Leu Arg Val Arg Asp Gln Arg Gly Asn Val Leu 500 505 510Leu Ser Pro Glu Glu Val Ser Glu Thr His Gly Thr Glu Arg Leu Thr 515 520 525Ile Thr Tyr Ser Ser Ser Met Met Trp Glu Ile Asn Gly Pro Glu Ser 530 535 540Val Leu Val Asn Thr Tyr Gln Trp Ile Ile Arg Asn Trp Glu Thr Val545 550 555 560Lys Ile Gln Trp Ser Gln Asn Pro Thr Met Leu Tyr Asn Lys Met Glu 565 570 575Phe Glu Pro Phe Gln Ser Leu Val Pro Lys Ala Ile Arg Gly Gln Tyr 580 585 590Ser Gly Phe Val Arg Thr Leu Phe Gln Gln Met Arg Asp Val Leu Gly 595 600 605Thr Phe Asp Thr Thr Gln Ile Ile Lys Leu Leu Pro Phe Ala Ala Ala 610 615 620Pro Pro Lys Gln Ser Arg Met Gln Phe Ser Ser Leu Thr Val Asn Val625 630 635 640Arg Gly Ser Gly Met Arg Ile Leu Val Arg Gly Asn Ser Pro Val Phe 645 650 655Asn Tyr Asn Lys Thr Thr Lys Arg Leu Thr Ile Leu Gly Lys Asp Ala 660 665 670Gly Thr Leu Ile Glu Asp Pro Asp Glu Ser Thr Ser Gly Val Glu Ser 675 680 685Ala Val Leu Arg Gly Phe Leu Ile Leu Gly Lys Glu Asp Arg Arg Tyr 690 695 700Gly Pro Ala Leu Ser Ile Asn Glu Leu Ser Asn Leu Ala Lys Gly Glu705 710 715 720Lys Ala Asn Val Leu Ile Gly Gln Gly Asp Val Val Leu Val Met Lys 725 730 735Arg Lys Arg Asp Ser Ser Ile Leu Thr Asp Ser Gln Thr Ala Thr Lys 740 745 750Arg Ile Arg Met Ala Ile Asn 7552770PRTInfluenza B virusCHAIN(1)..(770)PB2 2Met Thr Leu Ala Lys Ile Glu Leu Leu Lys Gln Leu Leu Arg Asp Asn1 5 10 15Glu Ala Lys Thr Val Leu Lys Gln Thr Thr Val Asp Gln Tyr Asn Ile 20 25 30Ile Arg Lys Phe Asn Thr Ser Arg Ile Glu Lys Asn Pro Ser Leu Arg 35 40 45Met Lys Trp Ala Met Cys Ser Asn Phe Pro Leu Ala Leu Thr Lys Gly 50 55 60Asp Met Ala Asn Arg Ile Pro Leu Glu Tyr Lys Gly Ile Gln Leu Lys65 70 75 80Thr Asn Ala Glu Asp Ile Gly Thr Lys Gly Gln Met Cys Ser Ile Ala 85 90 95Ala Val Thr Trp Trp Asn Thr Tyr Gly Pro Ile Gly Asp Thr Glu Gly 100 105 110Phe Glu Lys Val Tyr Glu Ser Phe Phe Leu Arg Lys Met Arg Leu Asp 115 120 125Asn Ala Thr Trp Gly Arg Ile Thr Phe Gly Pro Val Glu Arg Val Arg 130 135 140Lys Arg Val Leu Leu Asn Pro Leu Thr Lys Glu Met Pro Pro Asp Glu145 150 155 160Ala Ser Asn Val Ile Met Glu Ile Leu Phe Pro Lys Glu Ala Gly Ile 165 170 175Pro Arg Glu Ser Thr Trp Ile His Arg Glu Leu Ile Lys Glu Lys Arg 180 185 190Glu Lys Leu Lys Gly Thr Met Ile Thr Pro Ile Val Leu Ala Tyr Met 195 200 205Leu Glu Arg Glu Leu Val Ala Arg Arg Arg Phe Leu Pro Val Ala Gly 210 215 220Ala Thr Ser Ala Glu Phe Ile Glu Met Leu His Cys Leu Gln Gly Glu225 230 235 240Asn Trp Arg Gln Ile Tyr His Pro Gly Gly Asn Lys Leu Thr Glu Ser 245 250 255Arg Ser Gln Ser Met Ile Val Ala Cys Arg Lys Ile Ile Arg Arg Ser 260 265 270Ile Val Ala Ser Asn Pro Leu Glu Leu Ala Val Glu Ile Ala Asn Lys 275 280 285Thr Val Ile Asp Thr Glu Pro Leu Lys Ser Cys Leu Thr Ala Ile Asp 290 295 300Gly Gly Asp Val Ala Cys Asp Ile Ile Arg Ala Ala Leu Gly Leu Lys305 310 315 320Ile Arg Gln Arg Gln Arg Phe Gly Arg Leu Glu Leu Lys Arg Ile Ser 325 330 335Gly Arg Gly Phe Lys Asn Asp Glu Glu Ile Leu Ile Gly Asn Gly Thr 340 345 350Ile Gln Lys Ile Gly Ile Trp Asp Gly Glu Glu Glu Phe His Val Arg 355 360 365Cys Gly Glu Cys Arg Gly Ile Leu Lys Lys Ser Lys Met Arg Met Glu 370 375 380Lys Leu Leu Ile Asn Ser Ala Lys Lys Glu Asp Met Lys Asp Leu Ile385 390 395 400Ile Leu Cys Met Val Phe Ser Gln Asp Thr Arg Met Phe Gln Gly Val 405 410 415Arg Gly Glu Ile Asn Phe Leu Asn Arg Ala Gly Gln Leu Leu Ser Pro 420 425 430Met Tyr Gln Leu Gln Arg Tyr Phe Leu Asn Arg Ser Asn Asp Leu Phe 435 440 445Asp Gln Trp Gly Tyr Glu Glu Ser Pro Lys Ala Ser Glu Leu His Gly 450 455 460Ile Asn Glu Leu Met Asn Ala Ser Asp Tyr Thr Leu Lys Gly Val Val465 470 475 480Val Thr Lys Asn Val Ile Asp Asp Phe Ser Ser Thr Glu Thr Glu Lys 485 490 495Val Ser Ile Thr Lys Asn Leu Ser Leu Ile Lys Arg Thr Gly Glu Val 500 505 510Ile Met Gly Ala Asn Asp Val Ser Glu Leu Glu Ser Gln Ala Gln Leu 515 520 525Met Ile Thr Tyr Asp Thr Pro Lys Met Trp Glu Met Gly Thr Thr Lys 530 535 540Glu Leu Val Gln Asn Thr Tyr Gln Trp Val Leu Lys Asn Leu Val Thr545 550 555 560Leu Lys Ala Gln Phe Leu Leu Gly Lys Glu Asp Met Phe Gln Trp Asp 565 570 575Ala Phe Glu Ala Phe Glu Ser Ile Ile Pro Gln Lys Met Ala Gly Gln 580 585 590Tyr Ser Gly Phe Ala Arg Ala Val Leu Lys Gln Met Arg Asp Gln Glu 595 600 605Val Met Lys Thr Asp Gln Phe Ile Lys Leu Leu Pro Phe Cys Phe Ser 610 615 620Pro Pro Lys Leu Arg Ser Asn Gly Glu Pro Tyr Gln Phe Leu Arg Leu625 630 635 640Val Leu Lys Gly Gly Gly Glu Asn Phe Ile Glu Val Arg Lys Gly Ser 645 650 655Pro Leu Phe Ser Tyr Asn Pro Gln Thr Glu Val Leu Thr Ile Cys Gly 660 665 670Arg Met Met Ser Leu Lys Gly Lys Ile Glu Asp Glu Glu Arg Asn Arg 675 680 685Ser Met Gly Asn Ala Val Leu Ala Gly Phe Leu Val Ser Gly Lys Tyr 690 695 700Asp Pro Asp Leu Gly Asp Phe Lys Thr Ile Glu Glu Leu Glu Lys Leu705 710 715 720Lys Pro Gly Glu Lys Ala Asn Ile Leu Leu Tyr Gln Gly Lys Pro Val 725 730 735Lys Val Val Lys Arg Lys Arg Tyr Ser Ala Leu Ser Asn Asp Ile Ser 740 745 750Gln Gly Ile Lys Arg Gln Arg Met Thr Val Glu Ser Met Gly Trp Ala 755 760 765Leu Ser 7703774PRTInfluenza C virusCHAIN(1)..(774)PB2 3Met Ser Phe Leu Leu Thr Ile Ala Lys Glu Tyr Lys Arg Leu Cys Gln1 5 10 15Asp Ala Lys Ala Ala Gln Met Met Thr Val Gly Thr Val Ser Asn Tyr 20 25 30Thr Thr Phe Lys Lys Trp Thr Thr Ser Arg Lys Glu Lys Asn Pro Ser 35 40 45Leu Arg Met Arg Trp Ala Met Ser Ser Lys Phe Pro Ile Ile Ala Asn 50 55 60Lys Arg Met Leu Glu Glu Ala Gln Ile Pro Lys Glu His Asn Asn Val65 70 75 80Ala Leu Trp Glu Asp Thr Glu Asp Val Ser Lys Arg Asp His Val Leu 85 90 95Ala Ser Ala Ser Cys Ile Asn Tyr Trp Asn Phe Cys Gly Pro Cys Val 100 105 110Asn Asn Ser Glu Val Ile Lys Glu Val Tyr Lys Ser Arg Phe Gly Arg 115 120 125Leu Glu Arg Arg Lys Glu Ile Met Trp Lys Glu Leu Arg Phe Thr Leu 130 135 140Val Asp Arg Gln Arg Arg Arg Val Asp Thr Gln Pro Val Glu Gln Arg145 150 155 160Leu Arg Thr Gly Glu Ile Lys Asp Leu Gln Met Trp Thr Leu Phe Glu 165 170 175Asp Glu Ala Pro Leu Ala Ser Lys Phe Ile Leu Asp Asn Tyr Gly Leu 180 185 190Val Lys Glu Met Arg Ser Lys Phe Ala Asn Lys Pro Leu Asn Lys Glu 195 200 205Val Val Ala His Met Leu Glu Lys Gln Phe Asn Pro Glu Ser Arg Phe 210 215 220Leu Pro Val Phe Gly Ala Ile Arg Pro Glu Arg Met Glu Leu Ile His225 230 235 240Ala Leu Gly Gly Glu Thr Trp Ile Gln Glu Ala Asn Thr Ala Gly Ile 245 250 255Ser Asn Val Asp Gln Arg Lys Asn Asp Met Arg Ala Val Cys Arg Lys 260 265 270Val Cys Leu Ala Ala Asn Ala Ser Ile Met Asn Ala Lys Ser Lys Leu 275 280 285Val Glu Tyr Ile Lys Ser Thr Ser Met Arg Ile Gly Glu Thr Glu Arg 290 295 300Lys Leu Glu Glu Leu Ile Leu Glu Thr Asp Asp Val Ser Pro Glu Val305 310 315 320Thr Leu Cys Lys Ser Ala Leu Gly Gly Pro Leu Gly Lys Thr Leu Ser 325 330 335Phe Gly Pro Met Leu Leu Lys Lys Ile Ser Gly Ser Gly Val Lys Val 340 345 350Lys Asp Thr Val Tyr Ile Gln Gly Val Arg Ala Val Gln Phe Glu Tyr 355 360 365Trp Ser Glu Gln Glu Glu Phe Tyr Gly Glu Tyr Lys Ser Ala Thr Ala 370 375 380Leu Phe Ser Arg Lys Glu Arg Ser Leu Glu Trp Ile Thr Ile Gly Gly385 390 395 400Gly Ile Asn Glu Asp Arg Lys Arg Leu Leu Ala Met Cys Met Ile Phe 405 410 415Cys Arg Asp Gly Asp Tyr Phe Lys Asp Ala Pro Ala Thr Ile Thr Met 420 425 430Ala Asp Leu Ser Thr Lys Leu Gly Arg Glu Ile Pro Tyr Gln Tyr Val 435 440 445Met Met Asn Trp Ile Gln Lys Ser Glu Asp Asn Leu Glu Ala Leu Leu 450 455 460Tyr Ser Arg Gly Ile Val Glu Thr Asn Pro Gly Lys Met Gly Ser Ser465 470 475 480Met Gly Ile Asp Gly Ser Lys Arg Ala Ile Lys Ser Leu Arg Ala Val 485 490 495Thr Ile Gln Ser Gly Lys Ile Asp Met Pro Glu Ser Lys Glu Lys Ile 500 505 510His Leu Glu Leu Ser Asp Asn Leu Glu Ala Phe Asp Ser Ser Gly Arg 515 520 525Ile Val Ala Thr Ile Leu Asp Leu Pro Ser Asp Lys Lys Val Thr Phe 530 535 540Gln Asp Val Ser Phe Gln His Pro Asp Leu Ala Val Leu Arg Asp Glu545 550 555 560Lys Thr Ala Ile Thr Lys Gly Tyr Glu Ala Leu Ile Lys Arg Leu Gly 565 570 575Thr Gly Asp Asn Asp Ile Pro Ser Leu Ile Ala Lys Lys Asp Tyr Leu 580 585 590Ser Leu Tyr Asn Leu Pro Glu Val Lys Leu Met Ala Pro Leu Ile Arg 595 600 605Pro Asn Arg Lys Gly Val Tyr Ser Arg Val Ala Arg Lys Leu Val Ser 610 615 620Thr Gln Val Thr Thr Gly His Tyr Ser Leu His Glu Leu Ile Lys Val625 630 635 640Leu Pro Phe Thr Tyr Phe Ala Pro Lys Gln Gly Met Phe Glu Gly Arg 645 650 655Leu Phe Phe Ser Asn Asp Ser Phe Val Glu Pro Gly Val Asn Asn Asn 660 665 670Val Phe Ser Trp Ser Lys Ala Asp Ser Ser Lys Ile Tyr Cys His Gly 675 680 685Ile Ala Ile Arg Val Pro Leu Val Val Gly Asp Glu His Met Asp Thr 690 695 700Ser Leu Ala Leu Leu Glu Gly Phe Ser Val Cys Glu Asn Asp Pro Arg705 710 715 720Ala Pro Met Val Thr Arg Gln Asp Leu Ile Asp Val Gly Phe Gly Gln 725 730 735Lys Val Arg Leu Phe Val Gly Gln Gly Ser Val Arg Thr Phe Lys Arg 740 745 750Thr Ala Ser Gln Arg Ala Ala Ser Ser Asp Val Asn Lys Asn Val Lys 755 760 765Lys Ile Lys Met Ser Asn 7704262PRTInfluenza A virusCHAIN(1)..(262)PB2 235 to 496 4Thr Gln Gly Thr Cys Trp Glu Gln Met Tyr Thr Pro Gly Gly Glu Val1 5 10 15Arg Asn Asp Asp Ile Asp Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile 20 25 30Val Arg Arg Ala Ser Val Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu 35 40 45Met Cys His Ser Thr Gln Ile Gly Gly Thr Arg Met Val Asp Ile Leu 50 55 60Arg Gln Asn Pro Thr Glu Glu Gln Ala Val Asp Ile Cys Lys Ala Ala65 70 75 80Met Gly Leu Arg Ile Ser Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe 85 90 95Lys Arg Thr Ser Gly Ser Ser Ile Lys Arg Glu Glu Glu Val Leu Thr 100 105 110Gly Asn Leu Gln Thr Leu Lys Ile Arg Val His Glu Gly Tyr Glu Glu 115 120 125Phe Thr Met Val Gly Lys Arg Ala Thr Ala Ile Leu Arg Lys Ala Thr 130 135 140Arg Arg Leu Val Gln Leu Ile Val Ser Gly Arg

Asp Glu Gln Ser Ile145 150 155 160Ala Glu Ala Ile Ile Val Ala Met Val Phe Ser Gln Glu Asp Cys Met 165 170 175Ile Lys Ala Val Arg Gly Asp Leu Asn Phe Val Asn Arg Ala Asn Gln 180 185 190Arg Leu Asn Pro Met His Gln Leu Leu Arg His Phe Gln Lys Asp Ala 195 200 205Lys Val Leu Phe Gln Asn Trp Gly Ile Glu His Ile Asp Asn Val Met 210 215 220Gly Met Val Gly Val Leu Pro Asp Met Thr Pro Ser Thr Glu Met Ser225 230 235 240Met Arg Gly Ile Arg Val Ser Lys Met Gly Val Asp Glu Tyr Ser Ser 245 250 255Thr Glu Arg Val Val Val 2605243PRTInfluenza A virusCHAIN(1)..(243)PB2 241 to 483 5Glu Gln Met Tyr Thr Pro Gly Gly Glu Val Arg Asn Asp Asp Ile Asp1 5 10 15Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg Ala Ser Val 20 25 30Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Gln 35 40 45Ile Gly Gly Thr Arg Met Val Asp Ile Leu Arg Gln Asn Pro Thr Glu 50 55 60Glu Gln Ala Val Asp Ile Cys Lys Ala Ala Met Gly Leu Arg Ile Ser65 70 75 80Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser 85 90 95Ser Ile Lys Arg Glu Glu Glu Val Leu Thr Gly Asn Leu Gln Thr Leu 100 105 110Lys Ile Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Lys 115 120 125Arg Ala Thr Ala Ile Leu Arg Lys Ala Thr Arg Arg Leu Val Gln Leu 130 135 140Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val145 150 155 160Ala Met Val Phe Ser Gln Glu Asp Cys Met Ile Lys Ala Val Arg Gly 165 170 175Asp Leu Asn Phe Val Asn Arg Ala Asn Gln Arg Leu Asn Pro Met His 180 185 190Gln Leu Leu Arg His Phe Gln Lys Asp Ala Lys Val Leu Phe Gln Asn 195 200 205Trp Gly Ile Glu His Ile Asp Asn Val Met Gly Met Val Gly Val Leu 210 215 220Pro Asp Met Thr Pro Ser Thr Glu Met Ser Met Arg Gly Ile Arg Val225 230 235 240Ser Lys Met6216PRTInfluenza A virusCHAIN(1)..(216)PB2 268 to 483 6Arg Arg Ala Ser Val Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met1 5 10 15Cys His Ser Thr Gln Ile Gly Gly Thr Arg Met Val Asp Ile Leu Arg 20 25 30Gln Asn Pro Thr Glu Glu Gln Ala Val Asp Ile Cys Lys Ala Ala Met 35 40 45Gly Leu Arg Ile Ser Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys 50 55 60Arg Thr Ser Gly Ser Ser Ile Lys Arg Glu Glu Glu Val Leu Thr Gly65 70 75 80Asn Leu Gln Thr Leu Lys Ile Arg Val His Glu Gly Tyr Glu Glu Phe 85 90 95Thr Met Val Gly Lys Arg Ala Thr Ala Ile Leu Arg Lys Ala Thr Arg 100 105 110Arg Leu Val Gln Leu Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala 115 120 125Glu Ala Ile Ile Val Ala Met Val Phe Ser Gln Glu Asp Cys Met Ile 130 135 140Lys Ala Val Arg Gly Asp Leu Asn Phe Val Asn Arg Ala Asn Gln Arg145 150 155 160Leu Asn Pro Met His Gln Leu Leu Arg His Phe Gln Lys Asp Ala Lys 165 170 175Val Leu Phe Gln Asn Trp Gly Ile Glu His Ile Asp Asn Val Met Gly 180 185 190Met Val Gly Val Leu Pro Asp Met Thr Pro Ser Thr Glu Met Ser Met 195 200 205Arg Gly Ile Arg Val Ser Lys Met 210 2157204PRTInfluenza A virusCHAIN(1)..(204)PB2 277 to 480 7Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Gln Ile Gly Gly Thr1 5 10 15Arg Met Val Asp Ile Leu Arg Gln Asn Pro Thr Glu Glu Gln Ala Val 20 25 30Asp Ile Cys Lys Ala Ala Met Gly Leu Arg Ile Ser Ser Ser Phe Ser 35 40 45Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser Ser Ile Lys Arg 50 55 60Glu Glu Glu Val Leu Thr Gly Asn Leu Gln Thr Leu Lys Ile Arg Val65 70 75 80His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Lys Arg Ala Thr Ala 85 90 95Ile Leu Arg Lys Ala Thr Arg Arg Leu Val Gln Leu Ile Val Ser Gly 100 105 110Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val Ala Met Val Phe 115 120 125Ser Gln Glu Asp Cys Met Ile Lys Ala Val Arg Gly Asp Leu Asn Phe 130 135 140Val Asn Arg Ala Asn Gln Arg Leu Asn Pro Met His Gln Leu Leu Arg145 150 155 160His Phe Gln Lys Asp Ala Lys Val Leu Phe Gln Asn Trp Gly Ile Glu 165 170 175His Ile Asp Asn Val Met Gly Met Val Gly Val Leu Pro Asp Met Thr 180 185 190Pro Ser Thr Glu Met Ser Met Arg Gly Ile Arg Val 195 2008202PRTInfluenza A virusCHAIN(1)..(202)PB2 281 to 482 8Leu Glu Met Cys His Ser Thr Gln Ile Gly Gly Thr Arg Met Val Asp1 5 10 15Ile Leu Arg Gln Asn Pro Thr Glu Glu Gln Ala Val Asp Ile Cys Lys 20 25 30Ala Ala Met Gly Leu Arg Ile Ser Ser Ser Phe Ser Phe Gly Gly Phe 35 40 45Thr Phe Lys Arg Thr Ser Gly Ser Ser Ile Lys Arg Glu Glu Glu Val 50 55 60Leu Thr Gly Asn Leu Gln Thr Leu Lys Ile Arg Val His Glu Gly Tyr65 70 75 80Glu Glu Phe Thr Met Val Gly Lys Arg Ala Thr Ala Ile Leu Arg Lys 85 90 95Ala Thr Arg Arg Leu Val Gln Leu Ile Val Ser Gly Arg Asp Glu Gln 100 105 110Ser Ile Ala Glu Ala Ile Ile Val Ala Met Val Phe Ser Gln Glu Asp 115 120 125Cys Met Ile Lys Ala Val Arg Gly Asp Leu Asn Phe Val Asn Arg Ala 130 135 140Asn Gln Arg Leu Asn Pro Met His Gln Leu Leu Arg His Phe Gln Lys145 150 155 160Asp Ala Lys Val Leu Phe Gln Asn Trp Gly Ile Glu His Ile Asp Asn 165 170 175Val Met Gly Met Val Gly Val Leu Pro Asp Met Thr Pro Ser Thr Glu 180 185 190Met Ser Met Arg Gly Ile Arg Val Ser Lys 195 2009194PRTInfluenza A virusCHAIN(1)..(194)PB2 290 to 483 9Gly Gly Thr Arg Met Val Asp Ile Leu Arg Gln Asn Pro Thr Glu Glu1 5 10 15Gln Ala Val Asp Ile Cys Lys Ala Ala Met Gly Leu Arg Ile Ser Ser 20 25 30Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser Ser 35 40 45Ile Lys Arg Glu Glu Glu Val Leu Thr Gly Asn Leu Gln Thr Leu Lys 50 55 60Ile Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Lys Arg65 70 75 80Ala Thr Ala Ile Leu Arg Lys Ala Thr Arg Arg Leu Val Gln Leu Ile 85 90 95Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val Ala 100 105 110Met Val Phe Ser Gln Glu Asp Cys Met Ile Lys Ala Val Arg Gly Asp 115 120 125Leu Asn Phe Val Asn Arg Ala Asn Gln Arg Leu Asn Pro Met His Gln 130 135 140Leu Leu Arg His Phe Gln Lys Asp Ala Lys Val Leu Phe Gln Asn Trp145 150 155 160Gly Ile Glu His Ile Asp Asn Val Met Gly Met Val Gly Val Leu Pro 165 170 175Asp Met Thr Pro Ser Thr Glu Met Ser Met Arg Gly Ile Arg Val Ser 180 185 190Lys Met10188PRTInfluenza A virusCHAIN(1)..(188)PB2 295 to 482 10Val Asp Ile Leu Arg Gln Asn Pro Thr Glu Glu Gln Ala Val Asp Ile1 5 10 15Cys Lys Ala Ala Met Gly Leu Arg Ile Ser Ser Ser Phe Ser Phe Gly 20 25 30Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser Ser Ile Lys Arg Glu Glu 35 40 45Glu Val Leu Thr Gly Asn Leu Gln Thr Leu Lys Ile Arg Val His Glu 50 55 60Gly Tyr Glu Glu Phe Thr Met Val Gly Lys Arg Ala Thr Ala Ile Leu65 70 75 80Arg Lys Ala Thr Arg Arg Leu Val Gln Leu Ile Val Ser Gly Arg Asp 85 90 95Glu Gln Ser Ile Ala Glu Ala Ile Ile Val Ala Met Val Phe Ser Gln 100 105 110Glu Asp Cys Met Ile Lys Ala Val Arg Gly Asp Leu Asn Phe Val Asn 115 120 125Arg Ala Asn Gln Arg Leu Asn Pro Met His Gln Leu Leu Arg His Phe 130 135 140Gln Lys Asp Ala Lys Val Leu Phe Gln Asn Trp Gly Ile Glu His Ile145 150 155 160Asp Asn Val Met Gly Met Val Gly Val Leu Pro Asp Met Thr Pro Ser 165 170 175Thr Glu Met Ser Met Arg Gly Ile Arg Val Ser Lys 180 18511166PRTInfluenza A virusCHAIN(1)..(166)PB2 318 to 483 R389K 11Arg Ile Ser Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr1 5 10 15Ser Gly Ser Ser Ile Lys Arg Glu Glu Glu Val Leu Thr Gly Asn Leu 20 25 30Gln Thr Leu Lys Ile Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met 35 40 45Val Gly Lys Arg Ala Thr Ala Ile Leu Arg Lys Ala Thr Arg Arg Leu 50 55 60Val Gln Leu Ile Val Ser Gly Lys Asp Glu Gln Ser Ile Ala Glu Ala65 70 75 80Ile Ile Val Ala Met Val Phe Ser Gln Glu Asp Cys Met Ile Lys Ala 85 90 95Val Arg Gly Asp Leu Asn Phe Val Asn Arg Ala Asn Gln Arg Leu Asn 100 105 110Pro Met His Gln Leu Leu Arg His Phe Gln Lys Asp Ala Lys Val Leu 115 120 125Phe Gln Asn Trp Gly Ile Glu His Ile Asp Asn Val Met Gly Met Val 130 135 140Gly Val Leu Pro Asp Met Thr Pro Ser Thr Glu Met Ser Met Arg Gly145 150 155 160Ile Arg Val Ser Lys Met 16512164PRTInfluenza A virusCHAIN(1)..(164)PB2 320 to 483 12Ser Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly1 5 10 15Ser Ser Ile Lys Arg Glu Glu Glu Val Leu Thr Gly Asn Leu Gln Thr 20 25 30Leu Lys Ile Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly 35 40 45Lys Arg Ala Thr Ala Ile Leu Arg Lys Ala Thr Arg Arg Leu Val Gln 50 55 60Leu Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile65 70 75 80Val Ala Met Val Phe Ser Gln Glu Asp Cys Met Ile Lys Ala Val Arg 85 90 95Gly Asp Leu Asn Phe Val Asn Arg Ala Asn Gln Arg Leu Asn Pro Met 100 105 110His Gln Leu Leu Arg His Phe Gln Lys Asp Ala Lys Val Leu Phe Gln 115 120 125Asn Trp Gly Ile Glu His Ile Asp Asn Val Met Gly Met Val Gly Val 130 135 140Leu Pro Asp Met Thr Pro Ser Thr Glu Met Ser Met Arg Gly Ile Arg145 150 155 160Val Ser Lys Met1382PRTInfluenza A virusCHAIN(1)..(82)PB2 323 to 404 13Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser Ser Ile1 5 10 15Lys Arg Glu Glu Glu Val Leu Thr Gly Asn Leu Gln Thr Leu Lys Ile 20 25 30Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Lys Arg Ala 35 40 45Thr Ala Ile Leu Arg Lys Ala Thr Arg Arg Leu Val Gln Leu Ile Val 50 55 60Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val Ala Met65 70 75 80Val Phe1411PRTInfluenza A virusCHAIN(1)..(11)PB2 320 to 330 14Ser Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe1 5 101511PRTInfluenza A virusCHAIN(1)..(11)PB2 330 to 340 15Phe Lys Arg Thr Ser Gly Ser Ser Ile Lys Arg1 5 101611PRTInfluenza A virusCHAIN(1)..(11)PB2 340 to 350 16Arg Glu Glu Glu Val Leu Thr Gly Asn Leu Gln1 5 101711PRTInfluenza A virusCHAIN(1)..(11)PB2 350 to 360 17Gln Thr Leu Lys Ile Arg Val His Glu Gly Tyr1 5 101811PRTInfluenza A virusCHAIN(1)..(11)PB2 360 to 370 18Tyr Glu Glu Phe Thr Met Val Gly Lys Arg Ala1 5 101911PRTInfluenza A virusCHAIN(1)..(11)PB2 370 to 380 19Ala Thr Ala Ile Leu Arg Lys Ala Thr Arg Arg1 5 102011PRTInfluenza A virusCHAIN(1)..(11)PB2 380 to 390 20Arg Leu Val Gln Leu Ile Val Ser Gly Arg Asp1 5 102111PRTInfluenza A virusCHAIN(1)..(11)PB2 390 to 400 21Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val1 5 102211PRTInfluenza A virusCHAIN(1)..(11)PB2 400 to 410 22Val Ala Met Val Phe Ser Gln Glu Asp Cys Met1 5 10232277DNAInfluenza A virusCDS(1)..(2277)PB2 WT 23atggaaagaa taaaagaact acggaacctg atgtcacagt ctcgcactcg cgagatacta 60acaaaaacca cagtggacca catggccata attaagaaat acacatcagg gagacaggaa 120aagaacccgt cacttaggat gaaatggatg atggcaatga aatatccaat tacagctgac 180aagaggataa cagaaatggt tcctgagaga aatgagcaag gacaaaccct atggagtaaa 240atgagtgatg ccgggtcaga tcgagtgatg gtatcacctt tggcagtgac atggtggaat 300agaaatggac cagtgacaag tacggttcat tatccaaaag tctacaagac ttattttgat 360aaagtcgaaa ggttaaaaca tggaaccttt ggccctgtcc attttagaaa ccaagtcaaa 420atacgccgaa gagttgacat aaaccctggt catgcagacc tcagtgccaa ggaggcacaa 480gatgtaatca tggaagttgt tttccccaat gaagtggggg ccaggatact aacgtcggaa 540tcacaattga caataaccaa agagaaaaaa gaagaactcc aagattgcaa aatttctcct 600ttgatggttg catacatgtt agagagagaa cttgtccgaa aaacgagatt tctcccagtt 660gctggtggaa caagcagtgt gtacattgaa gtgttacact tgactcaagg aacgtgttgg 720gaacagatgt acactccagg tggagaagtg aggaatgacg atattgacca aagcctaatt 780attgcagcca ggaacatagt gagaagagca tcagtatcag cagatccact agcatcttta 840ttggagatgt gccacagcac acaaattggc gggacaagga tggtggacat tcttagacag 900aacccgacgg aagaacaagc tgtggatata tgcaaggctg caatgggact gagaatcagc 960tcatccttca gttttggtgg gttcacattt aagagaacaa gcgggtcatc aatcaaaaga 1020gaggaagaag tgcttacggg caatctccaa acattgaaaa taagggtgca tgaagggtac 1080gaggagttca caatggtggg gaaaagggca acagctatac tcagaaaagc aaccaggaga 1140ttggttcagc ttatagtgag tggaagggac gagcagtcaa tagccgaagc gataattgta 1200gccatggtgt tttcacaaga ggattgcatg ataaaagcag ttagaggtga cctgaatttc 1260gttaacaggg caaatcagcg gttgaatccc atgcaccaac ttttaaggca ttttcagaaa 1320gatgcgaaag tgctttttca gaattgggga attgaacata ttgacaatgt gatgggaatg 1380gttggagtat taccagacat gactccaagc acagagatgt caatgagagg aataagagtc 1440agcaaaatgg gcgtggatga atactccagc acagagaggg tagtggttag cattgatcgg 1500tttttgagag ttcgagacca acgtgggaat gtattactat ctcctgagga ggtcagtgaa 1560acacatggga cagagagact gacaataact tactcatcgt caatgatgtg ggagattaat 1620ggccctgagt cagtgttggt caatacctat caatggatca tcagaaattg ggaaactgtt 1680aaaattcaat ggtctcagaa tcctacaatg ttgtacaaca aaatggaatt tgagccattt 1740cagtctttag ttcctaaggc cattagaggc caatacagtg gatttgtcag aactctattc 1800caacaaatga gggatgtact tgggacattt gataccaccc agataataaa gcttctcccc 1860tttgcagccg ccccaccaaa gcaaagtaga atgcagttct cttcattgac tgtgaatgtg 1920aggggatcag ggatgagaat acttgtaagg ggcaattctc ctgtattcaa ctacaacaag 1980accactaaaa gactaacaat tctcggaaaa gatgctggca ctttaattga agacccagat 2040gaaagcacat ccggagtgga gtccgctgtt ttgagaggat ttctcattct aggtaaggaa 2100gatagaagat acggaccagc attaagcatc aatgaactga gcaaccttgc aaaaggagaa 2160aaggctaatg tgctaattgg gcaaggagac gtggtgttgg taatgaaacg aaaacgggac 2220tctagcatac ttactgacag ccagacagcg accaaaagaa ttcggatggc catcaat 2277242277DNAInfluenza A virusCDS(1)..(2277)PB2 Arg389Lys 24atggaaagaa taaaagaact acggaacctg atgtcacagt ctcgcactcg cgagatacta 60acaaaaacca cagtggacca catggccata attaagaaat acacatcagg gagacaggaa 120aagaacccgt cacttaggat gaaatggatg atggcaatga aatatccaat tacagctgac 180aagaggataa cagaaatggt tcctgagaga aatgagcaag gacaaaccct atggagtaaa 240atgagtgatg ccgggtcaga tcgagtgatg gtatcacctt tggcagtgac atggtggaat 300agaaatggac cagtgacaag tacggttcat tatccaaaag tctacaagac ttattttgat 360aaagtcgaaa ggttaaaaca tggaaccttt ggccctgtcc attttagaaa ccaagtcaaa 420atacgccgaa gagttgacat aaaccctggt

catgcagacc tcagtgccaa ggaggcacaa 480gatgtaatca tggaagttgt tttccccaat gaagtggggg ccaggatact aacgtcggaa 540tcacaattga caataaccaa agagaaaaaa gaagaactcc aagattgcaa aatttctcct 600ttgatggttg catacatgtt agagagagaa cttgtccgaa aaacgagatt tctcccagtt 660gctggtggaa caagcagtgt gtacattgaa gtgttacact tgactcaagg aacgtgttgg 720gaacagatgt acactccagg tggagaagtg aggaatgacg atattgacca aagcctaatt 780attgcagcca ggaacatagt gagaagagca tcagtatcag cagatccact agcatcttta 840ttggagatgt gccacagcac acaaattggc gggacaagga tggtggacat tcttagacag 900aacccgacgg aagaacaagc tgtggatata tgcaaggctg caatgggact gagaatcagc 960tcatccttca gttttggtgg gttcacattt aagagaacaa gcgggtcatc aatcaaaaga 1020gaggaagaag tgcttacggg caatctccaa acattgaaaa taagggtgca tgaagggtac 1080gaggagttca caatggtggg gaaaagggca acagctatac tcagaaaagc aaccaggaga 1140ttggttcagc ttatagtgag tggaaaggac gagcagtcaa tagccgaagc gataattgta 1200gccatggtgt tttcacaaga ggattgcatg ataaaagcag ttagaggtga cctgaatttc 1260gttaacaggg caaatcagcg gttgaatccc atgcaccaac ttttaaggca ttttcagaaa 1320gatgcgaaag tgctttttca gaattgggga attgaacata ttgacaatgt gatgggaatg 1380gttggagtat taccagacat gactccaagc acagagatgt caatgagagg aataagagtc 1440agcaaaatgg gcgtggatga atactccagc acagagaggg tagtggttag cattgatcgg 1500tttttgagag ttcgagacca acgtgggaat gtattactat ctcctgagga ggtcagtgaa 1560acacatggga cagagagact gacaataact tactcatcgt caatgatgtg ggagattaat 1620ggccctgagt cagtgttggt caatacctat caatggatca tcagaaattg ggaaactgtt 1680aaaattcaat ggtctcagaa tcctacaatg ttgtacaaca aaatggaatt tgagccattt 1740cagtctttag ttcctaaggc cattagaggc caatacagtg gatttgtcag aactctattc 1800caacaaatga gggatgtact tgggacattt gataccaccc agataataaa gcttctcccc 1860tttgcagccg ccccaccaaa gcaaagtaga atgcagttct cttcattgac tgtgaatgtg 1920aggggatcag ggatgagaat acttgtaagg ggcaattctc ctgtattcaa ctacaacaag 1980accactaaaa gactaacaat tctcggaaaa gatgctggca ctttaattga agacccagat 2040gaaagcacat ccggagtgga gtccgctgtt ttgagaggat ttctcattct aggtaaggaa 2100gatagaagat acggaccagc attaagcatc aatgaactga gcaaccttgc aaaaggagaa 2160aaggctaatg tgctaattgg gcaaggagac gtggtgttgg taatgaaacg aaaacgggac 2220tctagcatac ttactgacag ccagacagcg accaaaagaa ttcggatggc catcaat 2277252277DNAInfluenza A virusCDS(1)..(2274)PB2 synthetic gene, optimized for E.coli codon usage 25atggaacgca ttaaagaact gcgcaacctg atgagccaga gccgtacccg tgaaattctg 60accaaaacca ccgtggatca catggcgatc atcaaaaaat ataccagcgg ccgtcaggaa 120aaaaatccga gcctgcgcat gaaatggatg atggcgatga aatatccgat caccgcggat 180aaacgcatta ccgaaatggt gccggaacgt aatgaacagg gccagaccct gtggagcaaa 240atgagcgatg cgggtagcga tcgtgttatg gttagcccgc tggcggttac ctggtggaat 300cgtaatggtc cggttaccag caccgtgcat tatccgaaag tgtacaaaac ctatttcgat 360aaagtggaac gcctgaaaca tggcaccttt ggcccggtgc attttcgtaa ccaggtgaaa 420attcgtcgtc gcgtggatat taatccgggt catgcggatc tgagcgcgaa agaagcgcaa 480gacgttatca tggaagttgt tttcccgaac gaagttggtg cgcgtattct gaccagcgaa 540agccagctga ccatcaccaa agaaaaaaaa gaagaactgc aggattgcaa aattagcccg 600ctgatggtgg cgtatatgct ggaacgtgaa ctggttcgta aaacccgttt tctgccggtt 660gcgggtggca ccagcagcgt gtatattgaa gtgctgcatc tgacccaggg cacctgttgg 720gaacagatgt ataccccggg tggtgaagtg cgtaacgatg atatcgatca gagcctgatt 780attgcggcgc gtaacattgt tcgtcgtgcg agcgttagcg cggatccgct ggcgagcctg 840ctggaaatgt gtcatagcac ccagattggt ggcacccgta tggttgatat tctgcgtcag 900aacccgaccg aagaacaggc ggtggatatt tgtaaagcgg cgatgggtct gcgtattagc 960agcagcttta gctttggcgg ctttaccttt aaacgtacca gcggcagcag cattaaacgt 1020gaagaagaag tgctgaccgg taatctgcag accctgaaaa ttcgtgtgca cgaaggctat 1080gaagaattca cgatggttgg caaacgtgcg accgcgattc tgcgtaaagc gacccgtcgt 1140ctggttcagc tgattgtgag cggcaaagat gaacagagca ttgcggaagc gattatcgtt 1200gcgatggttt tcagccagga agattgcatg attaaagcgg ttcgtggcga tctgaacttt 1260gtgaaccgtg cgaaccagcg tctgaatccg atgcaccagc tgctgcgcca ttttcagaaa 1320gatgcgaaag tgctgtttca gaactggggc atcgaacata tcgataacgt gatgggcatg 1380gttggtgttc tgccggatat gaccccgagc accgaaatga gcatgcgtgg tattcgtgtg 1440agcaaaatgg gcgtggatga atacagcagc accgaacgtg tggttgtgag cattgatcgt 1500tttctgcgtg tgcgtgatca gcgtggtaat gttctgctga gcccggagga ggttagcgaa 1560acgcacggca ccgaacgtct gaccattacc tatagcagca gcatgatgtg ggagattaac 1620ggtccggaaa gcgtgctggt gaacacctat cagtggatca ttcgcaactg ggaaaccgtg 1680aaaattcagt ggagccagaa tccgaccatg ctgtacaaca aaatggaatt cgaaccgttt 1740cagagcctgg ttccgaaagc gattcgtggt cagtatagcg gttttgtgcg taccctgttt 1800cagcagatgc gtgatgttct gggcaccttt gataccaccc agatcattaa actgctgccg 1860tttgcggcgg caccgccgaa acagagccgt atgcagttta gcagcctgac cgtgaatgtt 1920cgtggtagcg gcatgcgtat tctggttcgt ggtaacagcc cggtgttcaa ctataacaaa 1980accaccaaac gcctgaccat tctgggtaaa gatgcgggca ccctgattga agatccggat 2040gaaagcacca gcggcgttga aagcgcggtt ctgcgcggtt ttctgattct gggcaaggag 2100gatcgtcgtt acggtccggc gctgagcatt aacgaactga gcaatctggc gaaaggcgaa 2160aaagcgaacg ttctgattgg tcagggtgat gtggtgctgg tgatgaaacg taaacgcgat 2220agcagcattc tgaccgatag ccagaccgcg accaaacgta ttcgtatggc gattaat 2277262313DNAInfluenza B virusCDS(1)..(2313)PB2 26atgacattgg ctaaaattga attgttaaaa caactgttaa gggacaatga agccaaaaca 60gtattgaaac aaacaacggt agaccaatat aacataataa gaaaattcaa tacatcaaga 120attgaaaaga acccttcatt aaggatgaag tgggcaatgt gttctaattt tcccttggct 180ttgaccaagg gtgacatggc aaacagaatc cccttggaat acaagggaat acaacttaaa 240acaaatgctg aagacatagg aaccaaaggc caaatgtgct caatagcagc agttacctgg 300tggaatacat atggaccaat aggagatact gaaggtttcg aaaaagtcta cgaaagcttt 360tttctcagaa agatgagact tgacaatgcc acttggggcc gaataacttt tggcccagtt 420gaaagagtaa gaaaaagggt actgctaaac cctctcacta aggaaatgcc tccagatgaa 480gcaagtaatg tgataatgga aatattgttc cctaaggaag caggaatacc aagagaatct 540acttggatac atagggaact gataaaagaa aaaagagaaa aattgaaagg aacgatgata 600actcccattg tactggcata catgcttgag agggaattgg ttgccaggag aaggttcctg 660ccggtggcag gagcaacatc agctgagttc atagaaatgc tacactgctt acaaggtgaa 720aattggagac aaatatacca cccgggaggg aataaactaa ctgaatctag gtctcaatcg 780atgattgtgg cttgtagaaa gataatcaga agatcaatag tcgcatcaaa cccattggag 840ctggctgtag aaattgcaaa caagactgtg atagatactg aacctttaaa atcatgtctg 900acagccatag acggaggtga tgtcgcctgt gacataataa gagctgcatt aggactaaag 960atcagacaaa gacaaagatt tggacgactt gaactaaaaa ggatatcagg aagaggattc 1020aaaaatgatg aagaaatatt aatcgggaac ggaacaatac agaagattgg aatatgggac 1080ggagaagagg agttccatgt aagatgtggt gaatgcaggg gaatattaaa aaagagcaaa 1140atgagaatgg aaaaactact aataaattca gctaaaaagg aagacatgaa agatttaata 1200atcttgtgca tggtattttc tcaagacact aggatgttcc aaggagtaag gggagaaata 1260aattttctta atagagcagg ccaactttta tctccaatgt accaactcca aagatatttt 1320ttgaatagaa gtaacgatct ctttgatcaa tgggggtatg aggaatcacc caaagcaagt 1380gagctacatg ggataaatga attaatgaat gcatctgact acactttgaa aggggttgta 1440gtaacaaaaa atgtgattga tgattttagt tctactgaaa cagaaaaagt atctataaca 1500aaaaatctta gtttaataaa aagaactggg gaagtcataa tgggggccaa tgacgtaagt 1560gaattagaat cacaagctca gctaatgata acatatgata cacctaagat gtgggagatg 1620ggaacaacca aggaactggt gcaaaacacc tatcaatggg tgctaaaaaa tttggtaaca 1680ctgaaggctc agtttcttct aggaaaagaa gacatgttcc aatgggatgc atttgaagca 1740tttgaaagca taatccccca gaagatggct ggccaataca gtggatttgc aagagcagtg 1800ctcaaacaaa tgagagacca agaggtcatg aaaactgacc agttcataaa gttgttgccc 1860ttttgtttct caccaccaaa gttaaggagc aatggggagc cttatcagtt cttgaggctt 1920gtattgaagg gaggaggaga aaatttcatc gaagtaagga aagggtctcc tctattctct 1980tacaatccac aaacagaagt cctaactata tgcggcagaa tgatgtcatt aaaagggaaa 2040attgaagatg aagaaaggaa tagatcaatg gggaatgcag tgttggcggg ttttcttgtt 2100agtggcaagt atgacccaga tcttggagat ttcaaaacta ttgaagaact tgaaaagcta 2160aaaccggggg agaaagcaaa catcttactt tatcaaggaa agcccgttaa agtagttaaa 2220aggaaaagat atagtgcttt atccaatgac atttcacaag gaattaagag acaaagaatg 2280acagttgagt ccatggggtg ggccttgagc taa 2313272325DNAInfluenza C virusCDS(1)..(2325)PB2 27atgtcttttc tattgacaat agcaaaggaa tacaaaagac tatgccaaga tgctaaggca 60gctcaaatga tgacagtagg aactgtatca aactacacta cgttcaagaa atggactaca 120tcaaggaagg aaaagaatcc ttcactaaga atgagatggg caatgagcag caaattccca 180ataatagcta acaagagaat gctggaagaa gctcaaattc ctaaagaaca caacaatgta 240gccctttggg aagacacaga agatgtttca aaaagggatc atgttcttgc aagcgcctct 300tgtataaatt attggaattt ttgtggacct tgtgtcaaca attcagaagt gatcaaagaa 360gtttataaat ctagatttgg aagattagaa agaaggaaag aaataatgtg gaaagaactt 420agatttacat tagttgatag acaacgaaga agagttgaca ctcagcctgt agaacaaaga 480ttgagaactg gagaaattaa agacttgcaa atgtggactt tgttcgaaga tgaagctcct 540cttgctagca aatttatttt agacaattat ggtctagtca aagaaatgag atcaaagttt 600gcaaacaaac ctctgaataa agaagtagtt gcacacatgt tagaaaaaca attcaatccg 660gaaagtagat tcttgcctgt tttcggagct ataaggccag aaagaatgga attgatccat 720gcattaggag gagaaacttg gatacaagaa gctaacactg cagggatttc caatgttgat 780caaaggaaaa atgatatgag agcagtatgt aggaaagttt gtcttgcagc aaatgcaagt 840ataatgaacg ccaaaagcaa actggttgag tatataaaaa gtacaagtat gagaattgga 900gaaacagaaa gaaagcttga agaacttata cttgaaaccg atgatgtctc acctgaagta 960acattatgta aatctgcttt aggaggacca ttaggaaaaa ctctatcttt tgggcccatg 1020ctactcaaga aaatttctgg ttccggagta aaagttaaag atacagtata tatccaaggt 1080gtcagagcag tacaatttga atactggagt gagcaagaag aattctatgg agaatataag 1140tcagccaccg ctttattcag cagaaaggaa agatcactag aatggattac aataggagga 1200ggaataaatg aagacagaaa gagacttcta gctatgtgca tgatattttg cagagatgga 1260gattatttta aagacgcccc tgcaacaata acaatggcag atttaagtac gaagttagga 1320agagaaattc catatcaata tgtgatgatg aattggatac aaaaatcaga agataatctc 1380gaagccttat tatacagtag gggaattgta gaaaccaatc caggaaaaat ggggagctca 1440atgggaattg atggttccaa aagagcaatt aaatctttaa gggctgtcac aatacaatca 1500ggaaagattg acatgccaga atcaaaagaa aaaattcacc ttgagctctc tgataatctt 1560gaagcatttg attcatcagg aagaattgtt gcaacaattt tagaccttcc tagtgacaaa 1620aaggtaacat ttcaggatgt aagctttcaa catcctgatc tggcagtatt gagagatgag 1680aaaacggcca taacaaaagg gtatgaagcg ctaatcaaaa ggctaggaac aggggacaat 1740gatattcctt ccttaattgc aaagaaggat tatttgtctc tttataattt accagaagta 1800aaattaatgg ctcccttaat cagacccaat agaaaaggag tttattccag agttgctaga 1860aaattagtgt ctacacaagt tactactgga cattattcat tacatgaatt gataaaggtc 1920ttacccttta cttatttcgc cccaaaacag ggaatgtttg aaggaaggct tttctttagc 1980aacgatagct ttgttgagcc tggagtaaat aacaatgtat tttcttggag taaggctgac 2040agttctaaaa tatattgtca tggaatagcg ataagggtac ctttagttgt tggagatgaa 2100cacatggaca cttcgttagc actattagaa gggtttagtg tttgtgaaaa cgaccccaga 2160gcaccaatgg taacaagaca agatttaatt gatgtgggat ttgggcaaaa agttagactc 2220ttcgtaggcc aagggagcgt tagaaccttc aagcgaactg cctcacaaag ggctgcatca 2280agcgatgtaa ataagaatgt gaaaaagata aagatgtcta actaa 2325

Claims

1. A soluble polypeptide fragment, wherein said polypeptide fragment is (i) derived from the Influenza virus RNA-dependent RNA polymerase subunit PB2 or variant thereof and (ii) capable of binding to a RNA cap or analog thereof.

2. The polypeptide fragment of claim 1, wherein said polypeptide fragment is purified to an extent to be suitable for crystallization.

3. The polypeptide fragment of claim 1, wherein the Influenza virus RNA-dependent RNA polymerase subunit PB2 is from an Influenza A, B, or C virus or variant thereof.

4. The polypeptide fragment of claim 1, wherein(i) the N-terminus is identical to or corresponds to amino acid position 220 or higher and the C-terminus is identical to or corresponds to amino acid position 510 or lower of the amino acid sequence of PB2 according to SEQ ID NO: 1,(ii) the N-terminus is identical to or corresponds to amino acid position 222 or higher and the C-terminus is identical to or corresponds to amino acid position 511 or lower of the amino acid sequence of PB2 according to SEQ ID NO: 2, or(iii) wherein the N-terminus is identical to or corresponds to amino acid position 227 or higher and the C-terminus is identical to or corresponds to amino acid position 528 or lower of the amino acid sequence of PB2 according to SEQ ID NO: 3and variants thereof, which retain the ability to associate with an RNA cap or analog thereof.

5. The polypeptide fragment of claim 4, wherein said polypeptide fragment has or corresponds to an amino acid sequence selected from the group of amino acid sequences according to SEQ ID NO: 4 to 13 and variants thereof, which retain the ability to associate with an RNA cap or analog thereof.

6. A complex comprising the polypeptide fragment of claim 1 and a RNA cap or analog thereof.

7. The complex of claim 6, wherein the cap analog is selected from the group consisting of m⁷G, m⁷GMP, m⁷GTP, m⁷GpppG, m⁷GpppGm, m⁷GpppA, m⁷GpppAm, m⁷GpppC, m⁷GpppCm, m⁷GpppU, and m⁷GpppUm.

8. The complex of claim 6, wherein said polypeptide fragment consists of an amino acid sequence according to SEQ ID NO: 11 and said cap analog is m⁷GTP, having the structure defined by the structure coordinates as shown in FIG. 18.

9. The complex of claim 8, wherein said complex has a crystalline form with space group C222i and unit cell dimensions of a=9.2 nm, b=9.4 nm; c=22.0 nm (.+-.0.3 nm)

10. The complex of claim 8, wherein the crystal diffracts X-rays to a resolution of 3.0 A or higher, preferably 2.4 A or higher.

11. An isolated polynucleotide coding for an isolated polypeptide of claim 1.

12. A recombinant vector comprising said isolated polynucleotide of claim 11.

13. A recombinant host cell comprising said isolated polynucleotide of claim 11.

14. A method for identifying compounds which associate with all or part of the RNA cap binding pocket of PB2 or the binding pocket of a PB2 polypeptide variant, comprising the steps of (a) constructing a computer model of said binding pocket defined by the structure coordinates of the complex of claim 8 as shown in FIG. 18;(b) selecting a potential binding compound by a method selected from the group consisting of:(i) assembling molecular fragments into said compound, (ii) selecting a compound from a small molecule database, and (iii) de novo ligand design of said compound;(c) employing computational means to perform a fitting program operation between computer models of the said compound and the said binding pocket in order to provide an energy-minimized configuration of the said compound in the binding pocket; and(d) evaluating the results of said fitting operation to quantify the association between the said compound and the binding pocket model, whereby evaluating the ability of said compound to associate with the said binding pocket.

15. The method of claim 14, wherein said binding pocket comprises amino acids Phe323, His357, and Phe404 of PB2 according to SEQ ID NO: 1 or amino acids corresponding thereto.

16. The method of claim 15, wherein said binding pocket further comprises amino acids Phe325, Phe330, and Phe363 of PB2 according to SEQ ID NO: 1 or amino acids corresponding thereto.

17. The method of claim 15, wherein said binding pocket further comprises amino acids Glu361, and Lys376 of PB 2 according to SEQ ID NO: 1 or amino acids corresponding thereto.

18. The method of claim 15, wherein said binding pocket further comprises amino acids Ser320, Arg332, Ser337, and Gln406

19. The method of claim 15, wherein said binding pocket further comprises amino acids Lys339, Arg355, Asn429, and His432 of PB2 according to SEQ ID NO: 1 or amino acids corresponding thereto.

20. The method of claim 14, wherein said binding pocket is defined by the structure coordinates of PB2 SEQ ID NO: 1 amino acids Phe323, His357, and Phe404 according to FIG. 18.

21. The method of claim 20, wherein said binding pocket is further defined by the structure coordinates of PB2 SEQ ID NO: 1 amino acids Phe325, Phe330, and Phe363 according to FIG. 18.

22. The method of claim 20, wherein said binding pocket is further defined by the structure coordinates of PB2 SEQ ID NO: 1 amino acids Glu361, and Lys376 according to FIG. 18.

23. The method of claim 20, wherein said binding pocket is further defined by the structure coordinates of PB2 SEQ ID NO: 1 amino acids Ser320, Arg332, Ser337, and Gln406 according to FIG. 18.

24. The method of claim 20, wherein said binding pocket is further defined by the structure coordinates of PB2 SEQ ID NO: 1 amino acids Lys339, Arg355, Asn429, and His432 according to FIG. 18.

25. The method of claim 20, wherein the binding pocket of a PB2 polypeptide variant has a root mean square deviation from the backbone atoms of the amino acids Phe323, His357, and Phe404 of said binding pocket of not more than 2.5 A.

26. The method of claim 14 comprising the further step of (e) synthesizing said compound and optionally formulating said compound or a pharmaceutically acceptable salt thereof with one or more pharmaceutically acceptable excipient(s) and/or carrier(s).

27. The method of claim 26 comprising the further step of (f) contacting said compound, said polypeptide fragment of claim 1 and a RNA cap or analog thereof to determine the ability of said compound to inhibit binding between said PB2 polypeptide fragment and said RNA cap or analog thereof.

28. A compound identifiable by the method of claim 14, under the provision that the compound is not m⁷G, m⁷GMP, m⁷GTP, m⁷GpppG, m⁷GpppGm, m⁷GpppA, m⁷GpppAm, m⁷GpppC, m⁷GpppCm, m⁷GpppU, m⁷GpppUm, 2-Amino-7-benzyl-9-(4-hydroxy-butyl)-1,9-dihydro-purin-6-one, T-705 or m⁷Gppp(N)_1-15, wherein N is A, Am, G, Gm, C, Cm, U or Um and is able to bind to the RNA cap binding pocket of PB2 or variant thereof.

29. A compound identifiable by the method of claim 14, under the provision that the compound is not m⁷G, m⁷GMP, m⁷GTP, m⁷GpppG, m⁷GpppGm, m⁷GpppA, m⁷GpppAm, m⁷GpppC, m⁷GpppCm, m⁷GpppU, m⁷GpppUm, 2-Amino-7-benzyl-9-(4-hydroxy-butyl)-1,9-dihydro-purin-6-one, T-705 or m⁷ Gppp(N)i.)₅, wherein N is A, Am, G, Gm, C₅ Cm, U or Um and is able to inhibit binding between the PB2 polypeptide, variant thereof or fragment thereof and the RNA cap or analog thereof.

30. A method for identifying compounds which associate with the RNA cap binding pocket of PB2 or binding pockets of PB2 polypeptide variants, comprising the steps of (i) contacting said polypeptide fragment of claim 1 with a test compound and (ii) analyzing the ability of said test compound to bind to PB2.

31. The method of claim 30, comprising the further step of adding a RNA cap or analog thereof.

32. The method of claim 31, wherein the ability of said test compound to bind to PB2 or a variant thereof in presence of said RNA cap or analog thereof or the ability of said test compound to inhibit binding of said RNA cap or analog thereof to PB2 or a variant thereof is analyzed.

33. The method of claim 31, wherein said RNA cap or analog thereof is added prior, concomitantly, or after addition of said test compound.

34. The method of claim 30 performed in a high-throughput setting.

35. The method of claim 30, wherein said test compound is a small molecule.

36. The method of claim 30, wherein said test compound is a peptide or protein.

37. The method of any of claims 26, 27, or 30 to 36, wherein said method further comprises the step of formulating said compound or a pharmaceutically acceptable salt thereof with one or more pharmaceutically acceptable excipient(s) and/or carrier(s).

38. A pharmaceutical composition producible according to the method of claim 27.

39. A compound identifiable by the method of any of claims 30 to 37, under the provision that the compound is not m⁷G, m⁷GMP, m⁷GTP, m⁷GpppG, m⁷GpppGm, m⁷GpppA, m⁷GpppAm, m⁷GpppC, m⁷GpppCm, m⁷GpppU, m⁷GpppUm, 2-Amino-7-benzyl-9-(4-hydroxy-butyl)-1,9-dihydro-purin-6-one, T-705 or m⁷Gppp(N)i.i₅, wherein N is A, Am, G, Gm, C, Cm, U or Um and is able to bind to the PB2 polypeptide, variant thereof or fragment thereof.

40. A compound identifiable by the method of any of claims 29 to 36, under the provision that the compound is not m⁷G, m⁷GMP, m⁷GTP, m⁷GpppG, m⁷GpppGm, m⁷GpppA, m⁷GpppAm, m⁷GpppC, m⁷GpppCm, m⁷GpppU, m⁷GpppUm, 2-Amino-7-benzyl-9-(4-hydroxy-butyl)-1,9-dihydro-purin-6-one, T-705 or m⁷ Gppp(N)i.i₅, wherein N is A, Am, G₅ Gm, C, Cm, U or Um, and is able to inhibit binding between the PB2 polypeptide, variant thereof or fragment thereof and the RNA cap or analog thereof.

41. An antibody directed against the RNA cap binding domain of PB2.

42. The antibody of claim 41, wherein said antibody recognizes a polypeptide fragment selected from the group of polypeptides defined by SEQ ID NO: 14 to 22.

43. Use of a compound according to claim 28, 29, 39, or 40, a pharmaceutical composition according to claim 36, or an antibody according to claim 41 or 42 for the manufacture of a medicament for treating, ameliorating, or preventing disease conditions caused by viral infections with negative-sense ssRNA viruses.

44. The use of claim 43, wherein said disease condition is caused by viral infections of the Mononegavirales order comprising the Bornaviridae, Filoviridae, Paramyxoviridae, and Rhabdoviridae families.

45. The use of claim 43, wherein said disease condition is caused by the Orthomyxoviridae, Arenaviridae, or Bunyaviridae families.

46. The use of claim 43, wherein said disease condition is caused by a virus selected from the group consisting of Borna disease virus, Marburg virus, Ebola virus, Sendai virus, Mumps virus, Measles virus, Human respiratory syncytial virus, Turkey rhinotracheitis virus, Vesicular stomatitis Indiana virus, Nipah virus, Henda virus, Rabies virus, Bovine ephemeral fever virus, Infectious hematopoietic necrosis virus, Thogoto virus, Influenza A virus, Influenza B virus, Influenza C virus, Hantaan virus, Crimean-congo hemorrhagic fever virus, Rift Valley fever virus, and La Crosse virus.

47. The use of claim 43, wherein said disease condition is caused by a virus selected from the group consisting of Influenza A virus, Influenza B virus, Influenza C virus, Thogoto virus, and Hantaan virus.

48. A pharmaceutical composition producible according to the method of claim 37.

Images