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

Abstract

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...

Single-Site Glycoprotein Mutants Inhibit a Late Event in Sindbis Virus Assembly

“…Isoleucine shares few of histidine's properties, so this suggests that the importance of histidine specifically at this location is limited; however, histidine may still have some importance due to the fact that the WT titer is not fully restored by the compensatory mutation. Histidine's limited importance in the assembly of infectious SV particles is also suggested by the previously reported data that show that many different second-site revertants can rescue the H230A mutant in SFV (7). Further experiments, such as replacing histidine with a panel of different amino acids, could provide confirmation of this hypothesis and could lead to more information regarding the biochemical cause of this phenotype.…”

“…The investigators found that when this histidine was replaced with alanine, SFV particles either were not fully assembled when grown at 37°C or were assembled but were noninfectious when grown at 28°C (6). The investigators later identified several second-site revertant mutations that rescued the H230A mutant but found no revertants to the wildtype sequence (7).…”

Functions of the Stem Region of the Semliki Forest Virus Fusion Protein during Virus Fusion and Assembly

“…BHK-21 cells were incubated with serial dilutions of the culture medium at 37°C for 90 to 120 min. Twenty millimolar NH 4 Cl was then added to prevent secondary infection, and the cells were incubated at 28°C overnight. The infected cells (termed infectious centers) were then quantitated by immunofluorescence, using Rab, a polyclonal antibody to the SFV envelope proteins.…”

“…BHK-21 cells infected by RNA electroporation were diluted 1:10 with nonelectroporated cells and incubated at 37°C for 3 h to allow cell attachment to coverslips. The cells were cultured for ϳ16 h at 28°C in the presence of 20 mM NH 4 Cl to prevent secondary infection and then treated with medium at the indicated pH for 1 min at 37°C to trigger cell-cell fusion. After an additional culture at 28°C for 3 h, the cells were fixed in 3% formaldehyde at room temperature for 20 min.…”

“…BHK-21 cells were electroporated with wt or mutant RNAs and cultured on 22-mm square coverslips in a six-well plate at 28°C for ϳ16 h in the presence of 20 mM NH 4 Cl to prevent secondary infection. To prepare labeled target cells, CHO cells were split onto a 60-mm plate 1 day before the experiment and had just achieved confluence at the time of labeling.…”