Cholesterol-binding viral proteins in virus entry and morphogenesis

Abstract

Up to now less than a handful of viral cholesterol-binding proteins have been characterized, in HIV, influenza virus and Semliki Forest virus. These are proteins with roles in virus entry or morphogenesis. In the case of the HIV fusion protein gp41 cholesterol binding is attributed to a cholesterol recognition consensus (CRAC) motif in a flexible domain of the ectodomain preceding the trans-membrane segment. This specific CRAC sequence mediates gp41 binding to a cholesterol affinity column. Mutations in this motif arrest virus fusion at the hemifusion stage and modify the ability of the isolated CRAC peptide to induce segregation of cholesterol in artificial membranes.Influenza A virus M2 protein co-purifies with cholesterol. Its proton translocation activity, responsible for virus uncoating, is not cholesterol-dependent, and the transmembrane channel appears too short for integral raft insertion. Cholesterol binding may be mediated by CRAC motifs in the flexible post-TM domain, which harbours three determinants of binding to membrane rafts. Mutation of the CRAC motif of the WSN strain attenuates virulence for mice. Its affinity to the raft-non-raft interface is predicted to target M2 protein to the periphery of lipid raft microdomains, the sites of virus assembly. Its influence on the morphology of budding virus implicates M2 as factor in virus fission at the raft boundary. Moreover, M2 is an essential factor in sorting the segmented genome into virus particles, indicating that M2 also has a role in priming the outgrowth of virus buds.SFV E1 protein is the first viral type-II fusion protein demonstrated to directly bind cholesterol when the fusion peptide loop locks into the target membrane. Cholesterol binding is modulated by another, proximal loop, which is also important during virus budding and as a host range determinant, as shown by mutational studies.

Fig. 3.1

Conformational transitions of HIV protein gp41 during the fusion cascade. A Release of the metastable state of the gp120-gp41 complex by binding to the primary and secondary receptors, CD4 and CKR. The membrane-proximal region MPR (pre-TM) is exposed adjacent to the virus envelope. The MPR-distal sequence occludes the fusion peptide. B gp120 trimers refold into extended α-helical structure and harpoon the fusion peptide into the target membrane. Coil-to-amphipathic helix transition of the MPR-distal sequence enables immersion of the pre-TM in the membrane interfacial zone. C Extended α-helices zip into a six-helix bundle (6HB) and clamp virus and cell membrane, causing (D) hemifusion and, by pulling the pre-TM into the trimer of hairpins, (E) fusion pore opening. Model of Bellamy-McIntyre et al. (2007), Figure 8 (modified), with permission from the American Society for Biochemistry and Molecular Biology

Fig. 3.1

Conformational transitions of HIV protein gp41 during the fusion cascade. A Release of the metastable state of the gp120-gp41 complex by binding to the primary and secondary receptors, CD4 and CKR. The membrane-proximal region MPR (pre-TM) is exposed adjacent to the virus envelope. The MPR-distal sequence occludes the fusion peptide. B gp120 trimers refold into extended α-helical structure and harpoon the fusion peptide into the target membrane. Coil-to-amphipathic helix transition of the MPR-distal sequence enables immersion of the pre-TM in the membrane interfacial zone. C Extended α-helices zip into a six-helix bundle (6HB) and clamp virus and cell membrane, causing (D) hemifusion and, by pulling the pre-TM into the trimer of hairpins, (E) fusion pore opening. Model of Bellamy-McIntyre et al. (2007), Figure 8 (modified), with permission from the American Society for Biochemistry and Molecular Biology

Fig. 3.2

Structural and functional domains of influenza A M2 protein. Influenza A strains: WS – WSN/33 (H1N1) – NCBI accession L25818.1; Ud – Udorn/307/72 – NCBI accession J02167.1 (H3N2); We – Weybridge/27 (H7N7) – EMBL accession AX006731.1. Bold print: post-TM. Bold printed, underlined residues interact with M1 protein

Fig. 3.2

Structural and functional domains of influenza A M2 protein. Influenza A strains: WS – WSN/33 (H1N1) – NCBI accession L25818.1; Ud – Udorn/307/72 – NCBI accession J02167.1 (H3N2); We – Weybridge/27 (H7N7) – EMBL accession AX006731.1. Bold print: post-TM. Bold printed, underlined residues interact with M1 protein

Fig. 3.3

3D-model of the tetrameric M2 transmembrane and post-TM structure (Schnell and Chou, 2008). The four helices forming the ion channel are in the upper left, the four post-TM helices in the lower right. Key residues are indicated: S50 marks the position of the palmitoylated C50 of the wild-type sequence. L46 is the first and Y52 the central residue of a common M2 CRAC motif. The position normally occupied by a basic residue is mutated in the Udorn strain (E56) (cp. Table 3.1, Fig. 3.2) (Redrawn after MMDB ID: 62125; PDB ID: 2RLF.)

Fig. 3.3

3D-model of the tetrameric M2 transmembrane and post-TM structure (Schnell and Chou, 2008). The four helices forming the ion channel are in the upper left, the four post-TM helices in the lower right. Key residues are indicated: S50 marks the position of the palmitoylated C50 of the wild-type sequence. L46 is the first and Y52 the central residue of a common M2 CRAC motif. The position normally occupied by a basic residue is mutated in the Udorn strain (E56) (cp. Table 3.1, Fig. 3.2) (Redrawn after MMDB ID: 62125; PDB ID: 2RLF.)

Fig. 3.4

Peripheral raft association of the M2 tetramer. (a) Cross-section of the membrane showing the TM and post-TM of two of the four subunits of the tetramer. TM is surrounded by non-raft membrane while post-TM connects to raft membrane via the palmitate bound to C50 (C50p) and other raft-targeting sequence elements. (b) Tetramer viewed from the endodomain. Subunits bridge separate rafts. (c) Merger of rafts, trapping the tetramer in small patch of non-raft membrane within raft domain. From Schroeder et al. (2005), with permission from Springer Publishers

Fig. 3.4

Peripheral raft association of the M2 tetramer. (a) Cross-section of the membrane showing the TM and post-TM of two of the four subunits of the tetramer. TM is surrounded by non-raft membrane while post-TM connects to raft membrane via the palmitate bound to C50 (C50p) and other raft-targeting sequence elements. (b) Tetramer viewed from the endodomain. Subunits bridge separate rafts. (c) Merger of rafts, trapping the tetramer in small patch of non-raft membrane within raft domain. From Schroeder et al. (2005), with permission from Springer Publishers