Novel Mutations That Control the Sphingolipid and Cholesterol Dependence of the Semliki Forest Virus Fusion Protein

Flavivirus membrane fusion is triggered by acidic pH and mediated by the major envelope protein E. A structurally very similar fusion protein is found in alphaviruses, and these molecules are designated class II viral fusion proteins. In contrast to that of flaviviruses, however, alphavirus fusion has been shown to be absolutely dependent on the presence of cholesterol and sphingomyelin in the target membrane, suggesting significant differences in the fusion protein-membrane interactions that lead to fusion. With the flavivirus tick-borne encephalitis virus (TBEV), we have therefore conducted a study on the lipid requirements of viral fusion with liposomes and on the processes preceding fusion, specifically, the membrane-binding step and the fusion-associated oligomeric switch from E protein dimers to trimers. As with alphaviruses, cholesterol had a strong promoting effect on membrane binding and trimerization of the fusion protein, and-as shown by the use of cholesterol analogs-the underlying interactions involve the 3␤-hydroxyl group at C-3 in both viral systems. In contrast to alphaviruses, however, these effects are much less pronounced with respect to the overall fusion of TBEV and can only be demonstrated when fusion is slowed down by lowering the temperature. The data presented thus suggest the existence of structurally related interactions of the flavivirus and alphavirus fusion proteins with cholesterol in the molecular processes required for fusion but, at the same time, point to significant differences between the class II fusion machineries of these viruses.Entry of enveloped viruses into cells involves binding to specific receptors and fusion of the viral membrane with a cellular membrane. Both processes are controlled by viral surface glycoproteins. The proteins mediating fusion possess structural elements that interact with lipids in the target membrane, and in some cases, a specific lipid requirement has been demonstrated for these processes (reviewed in reference 16). The nature and specificity of these protein-lipid interactions, however, are still much less well understood than those of protein-mediated receptor binding.Most of the viral fusion proteins characterized so far can be divided into two classes (20). Class I comprises homotrimeric spike proteins that are synthesized as precursors and require proteolytic cleavage for their activation. They have aminoterminal or amino-proximal fusion peptides and form a coiledcoil postfusion structure. Such proteins are present in orthomyxoviruses, paramyxoviruses, retroviruses, and filoviruses (reviewed in references 34 and 43). In contrast, the class II viral fusion proteins of flaviviruses (E protein) and alphaviruses (E1 protein) are not spiky projections but are, instead, oriented parallel to the viral membrane and form an icosahedral network in the viral envelope. They do not appear to have the propensity to form alpha-helical coiled coils, they are not proteolytically cleaved but require the cleavage of a second, accessory protein for activatio...

Section: Discussionmentioning

confidence: 82%

Section: Discussionmentioning

confidence: 99%

Involvement of Lipids in Different Steps of the Flavivirus Fusion Mechanism

Stiasny

Koessl

Heinz

2003

“…2, bottom panel). Although cell surface expression of some alphavirus envelope protein mutants can be rescued by incubation of infected cells at 28°C (32,33), these conditions did not rescue E1 transport of the Y47/48A mutant (data not shown). Immunofluorescent staining confirmed that the double mutant did not cause secondary infection of cocultured nonelectroporated BHK cells (data not shown), in keeping with the E1 transport defect.…”

Section: Analysis Of Interactions At the Chikv E3-e2 Interfacementioning

confidence: 99%

The Role of E3 in pH Protection during Alphavirus Assembly and Exit

Uchime

Fields

Kielian

2013

Self Cite

Alphaviruses are small enveloped viruses whose surface is covered by spikes composed of trimers of E2/E1 glycoprotein heterodimers. During virus entry, the E2/E1 dimer dissociates within the acidic endosomal environment, freeing the E1 protein to mediate fusion of the viral and endosome membranes. E2 is synthesized as a precursor, p62, which is cleaved by furin in the late secretory pathway to produce mature E2 and a small peripheral glycoprotein, E3. The immature p62/E1 dimer is acid resistant, but since p62 is cleaved before exit from the acidic secretory pathway, low pH-dependent binding of E3 to the spike complex is believed to prevent premature fusion. Based on analysis of the structure of the Chikungunya virus E3/E2/E1 complex, we hypothesized that interactions of E3 residues Y47 and Y48 with E2 are important in this binding. We then directly tested the in vivo role of E3 in pH protection by alanine substitutions of E3 Y47 and Y48 (Y47/48A) in Semliki Forest virus. The mutant was nonviable and was blocked in E1 transport to the plasma membrane and virus production. Although the Y47/48A mutant initially formed the p62/E1 heterodimer, the dimer dissociated during transport through the secretory pathway. Neutralization of the pH in the secretory pathway successfully rescued dimer association, E1 transport, and infectious particle production. Further mutagenesis identified the critical contact as the cation-interaction of E3 Y47 with E2. Thus, E3 mediates pH protection of E1 during virus biogenesis via interactions strongly dependent on Y47 at the E3-E2 interface. E nveloped viruses use membrane fusion to deliver their genomes into host cells (reviewed in references 1, 2, and 3). These viral fusion reactions can be triggered by various combinations of receptor/coreceptor binding and/or by the low-pH environment of the endocytic pathway. Fusion is mediated by specialized viral fusion proteins, which are highly regulated for deployment at the correct time and place during entry. Suppression of the fusion reaction during virus biogenesis is as crucial as its correct triggering during virus entry. Viruses with low-pH-triggered fusion reactions have evolved several mechanisms to protect their membrane fusion proteins from the low pH of the exocytic pathway. Influenza virus (4) and hepatitis C virus (5) express small membrane proteins, termed viroporins, that act as ion channels and can neutralize the exocytic pathway as a protection mechanism. The alphavirus and flavivirus fusion proteins are synthesized with "companion" proteins whose dimeric interactions protect the fusion protein from low pH (reviewed in reference 6). Maturation of the companion protein by furin cleavage primes the virus to respond to low pH during entry (7,8).Alphaviruses such as Semliki Forest virus (SFV) are plusstrand RNA viruses enveloped in a lipid bilayer containing a lattice of trimeric spikes of E1 and E2 glycoprotein heterodimers (9). The companion protein E2 binds receptors on the cell surface, leading to clathrin-mediated endocytosis...

“…Selection for growth on cholesterol-depleted insect cells was used to isolate three SFV mutants, termed srf (sterol requirement in function) mutants (4,5,27). Each mutant contains a single amino acid substitution in domain II of E1.…”

mentioning

confidence: 99%

Second-Site Revertants of a Semliki Forest Virus Fusion-Block Mutation Reveal the Dynamics of a Class II Membrane Fusion Protein

Chanel-Vos

Kielian

2006

Self Cite

The alphavirus Semliki Forest virus (SFV) infects cells through low-pH-induced membrane fusion mediated by the E1 protein, a class II virus membrane fusion protein. During fusion, E1 inserts into target membranes via its hydrophobic fusion loop and refolds to form a stable E1 homotrimer. Mutation of a highly conserved histidine (the H230A mutation) within a loop adjacent to the fusion loop was previously shown to block SFV fusion and infection, although the mutant E1 protein still inserts into target membranes and forms a homotrimer. Here we report on second-site mutations in E1 that rescue the H230A mutant. These mutations were located in a cluster within the hinge region, at the membrane-interacting tip, and within the groove where the E1 stem is believed to pack. Together the revertants reveal specific and interconnected aspects of the fusion protein refolding reaction.The entry of enveloped viruses into host cells requires the fusion of the viral membrane with the host cell membrane, a reaction that can occur either at the plasma membrane or in the low-pH environment of the endocytic pathway. The fusion reaction is carried out by viral membrane fusion proteins. To date, two classes of these proteins have been defined based on structural features (17). The E1 proteins of alphaviruses such as Semliki Forest virus (SFV) and the E proteins of flaviviruses such as dengue virus (DEN) and tick-borne encephalitis virus (TBE) are the current members of the class II membrane fusion proteins (reviewed in references 11, 12, 15, and 22).The SFV fusion reaction is triggered by low pH and promoted by the presence of cholesterol in the target membrane (reviewed in references 12 and 15). During this process, the E1 fusion protein inserts into the target membrane and reorganizes into homotrimers (E1HT) that are resistant to trypsin digestion and to dissociation by sodium dodecyl sulfate (SDS) at 30°C (28,29). The structure of the E1 ectodomain, here referred to as E1*, reveals that the prefusion form of E1 contains three domains composed almost entirely of ␤-sheets (17, 24). Domain I is a central globular domain that contains the E1 N terminus. Domain II is composed of two elongations that emanate from domain I. Each elongation contains a loop at its tip, the fusion peptide loop (termed the cd loop) at the tip of the first elongation and the ij loop at the tip of the second. A flexible region termed the hinge is located approximately at the boundary between domains I and II. Domain III is linked to domain I at the other side of the molecule from domain II and has a typical immunoglobulin-like fold. The stem region, a portion of which is missing in the E1* molecule, connects domain III to the transmembrane (TM) domain. In the neutral-pH conformation, the fusion peptide loop and the TM domain are present at opposite ends of the E1 molecule, and E1 is arranged tangentially to the virus membrane to form an icosahedral shell (17, 24, 31). The prefusion structure of the flavivirus E protein ectodomain shows an overall fold striking...