Free Download Bashan 200cc Service Manual Programs For Low Income

Besides the penetration protein VP5, we also obtained full atomic models for all three core proteins of the ISVP: VP1, VP3 and VP6. Our final model of the ISVP contains six conformers of four proteins with a total of 4990 residues: two VP3 (VP3A: residues 188-1211 of 1214; VP3B: residues 15-1214 of 1214), two VP6 (VP6A and VP6B, both residues 2-412 of 412), one VP1 (residues 1-1299 of 1299) and one VP5 (residues 2-646 of 648, ). (For the inner shell protein VP3A, the N-terminal 187 residuals are disordered, probably as a result of their interaction with the genome or with other proteins inside the “core” shell.). Primed Membrane Penetration ProteinFor our primed, membrane penetration protein VP5, the ten quasiequivalent subunits in an asymmetric unit are almost identical, so we averaged their densities to further improve map quality. The atomic model of primed VP5, based on this averaged VP5 density map, has a “Z” shape. It can be divided into three domains: a jelly-roll domain at the tip of the Z, a base domain at the bottom, and a linker domain joining the two.

• Free Download Bashan 200cc Service Manual Programs For Low Income Seniors
• Free Download Bashan 200cc Service Manual Programs For Low Income People

The jelly-roll domain (residues 287-484, ), at the outer surface, mainly consists of a β hairpin on the top, a five-stranded β sheet in the middle, and a four-stranded β sheet at the bottom. The base domain (residues 2-242, ) at the bottom consists of four α helices and several long loops. The linker domain (residues 243-286 and 485-648, ) is composed of six α helices and several short loops.The VP5 protein of aquareovirus shares a high degree of sequence similarity (38.4%) to the μ1 protein of orthoreovirus and their structures in their virions (dormant state) were found to be very similar.

In contrast, we find a conformational difference between the primed and dormant states of the penetration protein by comparing our primed VP5 protein of aquareovirus to the dormant μ1 protein in complex with the protection protein σ3 of orthoreovirus. In vivo, the conformational change is probably induced by removal of the aquareovirus protection protein VP7 during the residence of the virus in the endosome. In our study, priming was accomplished by proteolytic removal of VP7 (see the Experimental Procedures).

We find different conformations between the dormant and primed proteins for some of the loops in the helix-rich region and similar conformations for the remainder of that region. We also find different conformations between the two proteins in the β hairpin and the middle five-stranded β sheet in the tip region of the jelly-roll domain but similar conformations in the bottom four-stranded β sheet of that region.

Indeed, in orthoreovirus, the correct initial folding of the membrane penetration protein μ1 requires protection protein σ3 (; ). Thus, it is not surprising that removal of the protection protein would permit a conformational change of the penetration protein as part of the priming process for cell entry. Direct Observation of a Myristoyl Group in the Primed Membrane Penetration ProteinBiochemical data has shown that there is a myristoyl group linked to the N-terminal amino acid of reovirus μ1 protein. In the cryo-EM density map of our primed VP5 protein, a rod-shaped density extends from the N-terminal end, specifically from Gly2 into a pocket in the base domain of all ten VP5 molecules of each asymmetric unit of the ISVP ( and ).

The rod-like density, without side-chain features, can be modeled as a myristoyl group. The continuity of the myristoyl group density with Gly2 indicates that it is covalently linked to the N-terminus of the VP5 protein ( and ).

(Met1 is known to be removed by amino-peptidase prior to adding of the myristoyl group by a N-myristoyl transferase.). Proteolytic Cleavage SiteModulating the activity of the myristoyl insertion finger, the ϕ-δ proteolytic cleavage site, as distinct from the autocleavage site, is present in the linker domain of the aquareovirus and orthoreovirus penetration proteins, loop 551-560 for the former and loop 579-588 for the latter (; ). This proteolytic cleavage increases pore-forming activity by the myristoylated N terminus of the penetration protein during membrane penetration and releases the ϕ fragment that may be involved in recruiting virus cores to newly penetrated membrane pores. In addition, the ϕ fragment released into the cytoplasm can induce apoptosis, which is required for the maximum growth of progeny viruses (; ). This proteolytic cleavage was proposed to occur before the autocleavage (; ). In contrast, our cryo-EM structure shows that this loop is intact in the primed penetration protein VP5 of the ISVP, thus indicating that the ϕ-δ proteolytic cleavage occurs at a later step after the autocleavage.

Implications into the Processes of Cell Entry of Non-enveloped VirusOur results demonstrate that after the conversion from the dormant to the primed state the myristoyl groups remain embedded in the hydrophobic pockets of the penetration protein. As a membrane insertion “finger”, the buried myristoyl group must be released from the pocket during perforation of the membrane (; ). Our result indicates that the release of the myristoyl group from the pocket occurs during a later conversion of ISVP to ISVP. This step was previously proposed to be coupled to the autocleavage of the penetration protein (;; ).

Instead, our results reveal that the processes of autocleavage (during dormant to primed ISVP) and release of the myristoyl group (during primed ISVP to ISVP.) are not coupled. Structural Differences among VP5 Homologs Suggest Adaptations for Infection at Different TemperaturesThe aquatic environment in which aquareoviruses establish infection differs significantly in temperature from that encountered by mammalian orthoreovirus. Comparison of our atomic structure for aquareovirus with that for orthoreovirus reveals a possible structural basis for their adaptation to these different environments. Consistent with sequence alignment results (; ), our atomic structure of aquareovirus VP5 does not have two major structural segments found in the orthoreovirus penetration protein, a helix (residues 72-96) and a hub structure (residues 675-708). These segments in orthoreovirus interact with neighboring penetration trimers to further stabilize the lattice of the penetration proteins. Because the μ1 lattice is disrupted in the orthoreovirus ISVP. particle , this stabilization should apply to the ISVP, thus providing an extra energy barrier for the ISVP-to-ISVP.

Free Download Bashan 200cc Service Manual Programs For Low Income Seniors

conversion and requiring a higher conversion temperature. The absence of these stabilization segments in aquareovirus may be related to its adaptation to infect cold-blooded, aquatic animals at a lower temperature. Indeed, unlike orthoreovirus, which only infects cells at relatively high temperature (e.g. ≥32°C) , aquareovirus can establish active infection at 4°C and 2.5°C. Virus Propagation and PurificationGrass carp aquareovirus (GCRV) was propagated in CIK cell cultures as described previously. The virus particles were recovered from the infected cell-culture supernatants by low-speed centrifugation (36000 rpm, SW28 rotor) for 20 min to remove cell debris, and ultracentrifugation (26,000 rpm, SW28 rotor) for 2 hr to pellet virus.

To prepare ISVP, the pelleted virions of GCRV were suspended very carefully in virion buffer (150 mM NaCl, 10 mM MgCl 2, 10 mM Tris, pH 7.5) at a concentration of 2.5mg/ml and then digested with 200 mg/ml of α-chymotrypsin (Bovine Pancreas, Sigma) at 28 °C for 15 min, when the digestion was stopped by cooling the digestion mixtures to 0°C. Further C sCl gradient (20%-50%) purification of intact virion, ISVP and core were performed as described previously (;; ). ISVP particles collected from gradients were dissolved in10 mM phosphate-buffered saline and checked by negative-stain transmission electron microscopy to confirm the presence of highly purified ISVP. We thank Stan Schein for editing our manuscript, Xiaokang Zhang for assistance in programming, Xuekui Yu for advice in sample purification, Hongrong Liu and Lingpeng Cheng for assistance in film development, and Connie Huang for film digitization. This project is supported in part by grants from the National Institutes of Health (NIH; and to Z.H.Z.) and Abraxis BioScience. We acknowledge the use of the cryo-EM facility at the Electron Imaging Center for NanoMachines supported by NIH (1S10RR23057 to Z.H.Z.).

Free Download Bashan 200cc Service Manual Programs For Low Income People

This project is also supported by the National Basic Research Program of China (973 Program, #2009CB118701 to Q.F.), the National Natural Scientific Foundation of China (#30671615 and #30871940 to Q.F.) and the Chinese Academy of Sciences (KSCX2-YW-N-021 to Q.F.).