We have identified the membranotropic regions of proteins C and E of DENV virus by performing an exhaustive study of membrane rupture induced by two C and E-derived peptide libraries on model membranes having different phospholipid compositions as well as its ability to modulate the DEPE L(β)-L(α) and L(α)-H(II) phospholipid phase transitions. Protein C presents one hydrophobic leakage-prone region coincidental with a proposed membrane interacting domain, whereas protein E presents five membrane-rupture zones coincidental with different significant zones of the protein, i.e., the fusion peptide, a proline-rich sequence, a sequence containing a hydrophobic pocket as well as the stem and transmembrane domains of the protein. The identification of these membrane-active segments supports their role in viral membrane fusion, formation of the replication complex and morphogenesis and therefore attractive targets for development of new anti-viral compounds.
Determination of the membrane spanning domains of highly hydrophobic proteins from its primary structure, i.e., sequence, is cumbersome. However, transmembrane topology is better correlated with protein secondary structure than with the primary one. In this work we have determined the number and location of the transmembrane domains of the highly hydrophobic hepatitis C virus NS4B protein by studying the water-to-bilayer and water-to-interface transfer free energies of thirty-one different hepatitis C virus strains assuming that NS4B forms an α-helical wheel. Additionally, we have studied the effect of a peptide library encompassing the full length of the NS4B protein hepatitis C virus strain 1a_H77 on the phase transitions of DEPE through the use of differential scanning calorimetry. Our findings show that NS4B protein has five transmembrane domains and, as previously suggested, three interfacial segments. One of these segments, segment AH2, could behave similarly to viral pre-transmembrane segments, which would partition into and interact with the membrane and be responsible for the fluctuation of the protein between different topologies and therefore possible locations.
Dengue virus (DENV) C protein is essential for viral assembly. DENV C protein associates with intracellular membranes through a conserved hydrophobic domain and accumulates around endoplasmic reticulum-derived lipid droplets which could provide a platform for capsid formation during assembly. In a previous work we described a region in DENV C protein which induced a nearly complete membrane rupture of several membrane model systems, which was coincident with the theoretically predicted highly hydrophobic region of the protein. In this work we have carried out a study of the binding to and interaction with model biomembranes of a peptide corresponding to this DENV C region, DENV2C6. We show that DENV2C6 partitions into phospholipid membranes, is capable of rupturing membranes even at very low peptide-to-lipid ratios and its membrane-activity is modulated by lipid composition. These results identify an important region in the DENV C protein which might be directly implicated in the DENV life cycle through the modulation of membrane structure.
HCV NS4B, a highly hydrophobic protein involved in the alteration of the intracellular host membranes forming the replication complex, plays a critical role in the HCV life cycle. NS4B is a multifunctional membrane protein that possesses different regions where diverse and significant functions are located. One of these important regions is the AH2 segment, which besides being highly conserved has been shown to play a significant role in NS4B functioning. We have carried out an in-depth biophysical study aimed at the elucidation of the capacity of this region to interact, modulate and disrupt membranes, as well as to study the structural and dynamic features relevant for that disruption. We show that a peptide derived from this region, NS4BAH2, is capable of specifically binding phosphatidyl inositol phosphates with high affinity, and its interfacial properties suggest that this segment could behave similarly to a pre-transmembrane domain partitioning into and interacting with the membrane depending on the membrane composition and/or other proteins. Moreover, NS4BAH2 is capable of rupturing membranes even at very low peptide-to-lipid ratios and its membrane-activity is modulated by lipid composition. NS4BAH2 is located in a shallow position in the membrane but it is able to affect the lipid environment from the membrane surface down to the hydrophobic core. The NS4B region where peptide NS4BAH2 resides might have an essential role in the membrane replication and/or assembly of the viral particle through the modulation of the membrane structure and hence the replication complex.
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