Role of Conserved Histidine Residues in the Low-pH Dependence of the Semliki Forest Virus Fusion Protein

Abstract

A wide variety of enveloped viruses infects cells by taking advantage of the low pH in the endocytic pathway to trigger virus-membrane fusion. For alphaviruses such as Semliki Forest virus (SFV), acidic pH initiates a series of conformational changes in the heterodimeric virus envelope proteins E1 and E2. Low pH dissociates the E2/E1 dimer, releasing the membrane fusion protein E1. E1 inserts into the target membrane and refolds to a trimeric hairpin conformation, thus driving the fusion reaction. The means by which E1 senses and responds to low pH is unclear, and protonation of conserved E1 histidine residues has been proposed as a possible mechanism. We tested the role of four conserved histidines by mutagenesis of the wild-type (wt) SFV infectious clone to create virus mutants with E1 H3A, H125A, H331A, and H331A/H333A mutations. The H125A, H331A, and H331A/H333A mutants had growth properties similar to those of wt SFV and showed modest change or no change in the pH dependence of virus-membrane fusion. By contrast, the E1 H3A mutation produced impaired virus growth and a markedly more acidic pH requirement for virus-membrane fusion. The dissociation of the H3A heterodimer and the membrane insertion of the mutant E1 protein were comparable to those of the wt in efficiency and pH dependence. However, the formation of the H3A homotrimer required a much lower pH and showed reduced efficiency. Together, these results and the location of H3 suggest that this residue acts to regulate the low-pH-dependent refolding of E1 during membrane fusion.Enveloped viruses infect cells by fusing their membrane with that of the target cell through the action of transmembrane proteins in the virus envelope (15, 47). These membrane fusion proteins, although differing in structure among different enveloped viruses, nonetheless act through a common mechanism. Following an initial triggering event, the fusion protein interacts with the target membrane via a hydrophobic fusion peptide(s) and refolds into a hairpin-like conformation with the transmembrane domain and fusion peptide at the same end of the molecule. To date, the postfusion structures of all virus fusion proteins are trimeric hairpins. The triggering events for virus membrane fusion include virus-receptor/coreceptor interactions, exposure to a mildly acidic pH, and a combination of these processes. The critical triggering events can occur at the plasma membrane or within the low-pH environment of the endocytic pathway. While there has been remarkable progress in our understanding of the structures of virus membrane fusion proteins, the mechanism of triggering and the process of conversion from the prefusion conformation to the postfusion conformation are not understood.Semliki Forest virus (SFV) is a member of the alphaviruses, a genus of small, enveloped, plus-strand RNA viruses (25). SFV infects cells through a low-pH-triggered fusion reaction mediated by the E1 transmembrane protein (19). E1 is an elongated molecule containing three domains (DI, DII, and DIII) composed p...

Crystal structure of glycoprotein E2 from bovine viral diarrhea virus

“…Histidine side chains have pK a values in the range of 6-6.4 and therefore typically become protonated during endosomal acidification. The increase in positive charge resulting from histidine protonation is an important part of the pH sensing mechanism of many viruses (24)(25)(26)(27)(28)(29).…”

Critical Role of Leucine-Valine Change in Distinct Low pH Requirements for Membrane Fusion between Two Related Retrovirus Envelopes

“…More recent studies on influenza and other viruses suggest that ionic residues present in the viral fusion proteins can affect the stability of 6HB and therefore pH-dependent fusion (42)(43)(44). In particular, histidine (His) residues have been shown to be crucial for fusion activation of a number of pH-dependent viruses covering all three classes of viral fusion proteins (45)(46)(47)(48)(49)(50)(51). Given that the pH threshold of JSRV for membrane fusion is pH 6.3 (27), which is very close to the side chain pK a of His (52), and that the ectodomain of TM dictates the pH threshold as shown in this study (Fig.…”

The Interaction of Alphavirus E1 Protein with Exogenous Domain III Defines Stages in Virus-Membrane Fusion

“…The gradients were centrifuged in a TL-55 rotor at 54,000 rpm for 3 h at 4°C and collected in 7 fractions. The percentage of total virus radioactivity associated with the liposomes in the top three fractions was determined as previously described (38).…”