Many viruses require the cleavage-activation of membrane fusion proteins by host proteases in the course of infection. This knowledge is based on historical studies of Sendai virus in the 1970s. From the 1970s to the 1990s, avian influenza virus and Newcastle disease virus were studied, showing a clear link between virulence and the cleavage-activation of viral membrane fusion proteins (hemagglutinin and fusion proteins) by host proteases. In these viruses, cleavage of viral membrane fusion proteins by furin is the basis for their high virulence. Subsequently, from the 2000s to the 2010s, the importance of TMPRSS2 in activating the membrane fusion proteins of various respiratory viruses, including seasonal influenza viruses, was demonstrated. In late 2019, severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) emerged and caused a pandemic. The virus continues to mutate, producing variants that have caused global pandemics. The spike protein of SARS-CoV-2 is characterized by two cleavage sites, each of which is cleaved by furin and TMPRSS2 to achieve membrane fusion. SARS-CoV-2 variants exhibit altered sensitivity to these proteases. Thus, studying the cleavage-activation of membrane fusion proteins by host proteases is critical for understanding the ongoing pandemic and developing countermeasures against it.

Helicobacter pylori uses a cluster of polar, sheathed flagella for swimming motility. A search for homologs of H. pylori proteins that were conserved in Helicobacter species that possess flagellar sheaths but were underrepresented in Helicobacter species with unsheathed flagella identified several candidate proteins. Four of the identified proteins are predicted to form part of a tripartite efflux system that includes two transmembrane domains of an ABC transporter (HP1487 and HP1486), a periplasmic membrane fusion protein (HP1488), and a TolC-like outer membrane efflux protein (HP1489). Deleting hp1486/hp1487 and hp1489 homologs in H. pylori B128 resulted in reductions in motility and the number of flagella per cell. Cryo-electron tomography studies of intact motors of the Δhp1489 and Δhp1486/hp1487 mutants revealed many of the cells contained a potential flagellum disassembly product consisting of decorated L and P rings, which has been reported in other bacteria. Aberrant motors lacking specific components, including a cage-like structure that surrounds the motor, were also observed in the Δhp1489 mutant. These findings suggest a role for the H. pylori HP1486-HP1489 tripartite efflux system in flagellum stability. Three independent variants of the Δhp1486/hp1487 mutant with enhanced motility were isolated. All three motile variants had the same frameshift mutation in fliL, suggesting a role for FliL in flagellum disassembly.

The membrane surface of enveloped viruses contains dedicated proteins enabling the fusion of the viral with the host cell membrane. Working with these proteins is almost always challenging because they are membrane-embedded and naturally metastable. Fortunately, based on a range of different examples, researchers now have several possibilities to tame membrane fusion proteins, making them amenable for structure determination and immunogen generation. This review describes the structural and functional similarities of the different membrane fusion proteins and ways to exploit these features to stabilise them by targeted mutational approaches. The recent determination of two herpesvirus membrane fusion proteins in prefusion conformation holds the potential to apply similar methods to this group of viral fusogens. In addition to a better understanding of the herpesviral fusion mechanism, the structural insights gained will help to find ways to further stabilise these proteins using the methods described to obtain stable immunogens that will form the basis for the development of the next generation of vaccines and antiviral drugs.

Bombyx mori nucleopolyhedrovirus (BmNPV) is the primary pathogen of silkworms that causes severe economic losses in sericulture. GP64 is the key membrane fusion protein that mediates budded virus (BV) fusion with the host cell membrane. Previously, we found that the n-region of the GP64 signal peptide (SP) is required for protein secretion and viral pathogenicity; however, our understanding of BmNPV GP64 remains limited. Here, we first reported that BmNPV GP64 retained its SP in the mature protein and virion in only host cells but did not retain in nonhost cells. Uncleaved SP mediates protein targeting to the cytomembrane or secretion in Bombyx mori cells. The exitance of the n-region extended the transmembrane helix length, which resulted in the cleavage site to be located in the helix structure and thus blocked cleavage from signal peptidase (SPase). Without the n-region, the protein fails to be transported to the cytomembrane, but this failure can be rescued by the cleavage site mutation of SP. Helix-breaking mutations in SP abolished protein targeting to the cytomembrane and secretion. Our results revealed a previously unrecognized mechanism by which SP of membrane fusion not only determines protein localization but also determines viral pathogenicity, which highlights the escape mechanism of SP from the cleavage by SPase. IMPORTANCE BmNPV is the primary pathogen of silkworms, which causes severe economic losses in sericulture. BmNPV and Autographa californica multiple nucleopolyhedrovirus (AcMNPV) are closely related group I alphabaculoviruses, but they exhibit nonoverlapping host specificity. Recent studies suppose that GP64 is a determinant of host range, while knowledge remains limited. In this study, we revealed that BmNPV GP64 retained its SP in host cells but not in nonhost cells, and the SP retention is required for GP64 secretion across the cytomembrane. This is the first report that a type I membrane fusion protein retained its SP in mature proteins and virions. Our results unveil the mechanism by which SP GP64 escapes cleavage and the role of SP in protein targeting. This study will help elucidate an important mechanistic understanding of BmNPV infection and host range specificity.

Couples increasingly experience infertility and seek help from assisted reproductive techniques to become pregnant. However, 5%-15% of the couples that are selected for in vitro fertilisation (IVF) experience a total fertilisation failure (TFF), where no zygotes develop despite oocytes and semen parameters appear to be normal. We hypothesise that TFF during IVF could be related to improper membrane fusion of gametes.
To investigate the membrane integrity and fusion proteins in spermatozoa from men in couples experiencing TFF.
A total of 33 infertile couples, 17 of which experienced TFF during IVF and 16 matched control couples with normal IVF fertilisation rates, were selected and the men re-called to deliver an additional semen sample. Proteins involved in gamete membrane fusion on spermatozoa (IZUMO1, SPESP1 and Syncytin-1) as well as O-glycosylation patterns (Tn and GALNT3), were investigated by immunofluorescence. The DNA fragmentation index, acrosomal integrity and viability of spermatozoa were determined by flow and image cytometry.
No significant changes in the expression of GALNT3, Tn and Syncytin-1 were observed between the TFF and control groups. The fraction of spermatozoa expressing SPESP1, the median IZUMO1 staining intensity, and the percentage of viable acrosome-intact spermatozoa were significantly lower in the TFF group compared to controls. Furthermore, following progesterone-induced acrosomal exocytosis, a significant difference in the fraction of spermatozoa expressing SPESP1 and the median IZUMO1 staining intensity were observed between the control and TFF group.
Our results indicate that acrosomal exocytosis, IZUMO1 and SPESP1 expression in spermatozoa could play a crucial role in achieving fertilisation during IVF. However, the size of our cohort was quite small, and our results need to be validated with quantitative methods in larger cohorts.

The signal peptide (SP) of integrated membrane proteins is removed cotranslationally or posttranslationally in the endoplasmic reticulum, while GP64, a membrane fusion protein of Bombyx mori nucleopolyhedrovirus (BmNPV), retains its SP in the mature protein and virion. In this study, we revealed that uncleaved SP is a key determinant with additional functions in infection. First, uncleaved SP endows BmNPV with strong virulence; second, SP retention-induced BmNPV infection depends on cholesterol recognition amino acid consensus domain 1 (CRAC1) and CRAC2. In contrast, the recombinant virus with SP-cleaved GP64 has reduced infectivity, and only CRAC2 is required for BmNPV infection. Furthermore, we showed that cholesterol in the plasma membrane is an important fusion receptor that interacts with CRAC2 of GP64. Our study suggested that BmNPV GP64 is a key cholesterol-binding protein and uncleaved SP determines GP64\'s unique dependence on the CRAC domains. IMPORTANCE BmNPV is a severe pathogen that mainly infects silkworms. GP64 is the key membrane fusion protein that mediates BmNPV infection, and some studies have indicated that cholesterol and lipids are involved in BmNPV infection. A remarkable difference from other membrane fusion proteins is that BmNPV GP64 retains its SP in the mature protein, but the cause is still unclear. In this study, we investigated the reason why BmNPV retains this SP, and its effects on protein targeting, virulence, and CRAC dependence were revealed by comparison of recombinant viruses harboring SP-cleaved or uncleaved GP64. Our study provides a basis for understanding the dependence of BmNPV infection on cholesterol/lipids and host specificity.

Some antimicrobial peptides (AMPs) and membrane fusion-catalyzing peptides (FPs) stabilize bicontinuous inverted cubic (QII) phases. Previous authors proposed a topological rationale: since AMP-induced pores, fusion intermediates, and QII phases all have negative Gaussian curvature (NGC), peptides which produce NGC in one structure also do it in another. This assumes that peptides change the curvature energy of the lipid membranes. Here I test this with a Helfrich curvature energy model. First, experimentally, I show that lipid systems often used to study peptide NGC have NGC without peptides at higher temperatures. To determine the net effect of an AMP on NGC, the equilibrium phase behavior of the host lipids must be determined. Second, the model shows that AMPs must make large changes in the curvature energy to stabilize AMP-induced pores. Peptide-induced changes in elastic constants affect pores and QII phase differently. Changes in spontaneous curvature affect them in opposite ways. The observed correlation between QII phase stabilization and AMP activity doesn\'t show that AMPs act by lowering pore curvature energy. A different rationale is proposed. In theory, AMPs could simultaneously stabilize QII phase and pores by drastically changing two particular elastic constants. This could be tested by measuring AMP effects on the individual constants. I propose experiments to do that. Unlike AMPs, FPs must make only small changes in the curvature energy to catalyze fusion. It they act in this way, their fusion activity should correlate with their ability to stabilize QII phases.

Fusion-associated small transmembrane (FAST) proteins are a diverse family of nonstructural viral proteins. Once expressed on the plasma membrane of infected cells, they drive fusion with neighboring cells, increasing viral spread and pathogenicity. Unlike viral fusogens with tall ectodomains that pull two membranes together through conformational changes, FAST proteins have short fusogenic ectodomains that cannot bridge the intermembrane gap between neighboring cells. One orthoreovirus FAST protein, p14, has been shown to hijack the actin cytoskeleton to drive cell-cell fusion, but the actin adaptor-binding motif identified in p14 is not found in any other FAST protein. Here, we report that an evolutionarily divergent FAST protein, p22 from aquareovirus, also hijacks the actin cytoskeleton but does so through different adaptor proteins, Intersectin-1 and Cdc42, that trigger N-WASP-mediated branched actin assembly. We show that despite using different pathways, the cytoplasmic tail of p22 can replace that of p14 to create a potent chimeric fusogen, suggesting they are modular and play similar functional roles. When we directly couple p22 with the parallel filament nucleator formin instead of the branched actin nucleation promoting factor N-WASP, its ability to drive fusion is maintained, suggesting that localized mechanical pressure on the plasma membrane coupled to a membrane-disruptive ectodomain is sufficient to drive cell-cell fusion. This work points to a common biophysical strategy used by FAST proteins to push rather than pull membranes together to drive fusion, one that may be harnessed by other short fusogens responsible for physiological cell-cell fusion.

The highly conserved extracellular domain of the transmembrane protein M2 (M2e) of the influenza A virus is a promising target for the development of broad-spectrum vaccines. However, M2e is a poor immunogen by itself and must be linked to an appropriate carrier to induce an efficient immune response. In this study, we obtained recombinant mosaic proteins containing tandem copies of M2e fused to a lipopeptide from Neisseria meningitidis surface lipoprotein Ag473 and alpha-helical linkers and analyzed their immunogenicity. Six fusion proteins, comprising four or eight tandem copies of M2e flanked by alpha-helical linkers, lipopeptides, or a combination of both of these elements, were produced in Escherichia coli. The proteins, containing both alpha-helical linkers and lipopeptides at each side of M2e repeats, formed nanosized particles, but no particulate structures were observed in the absence of lipopeptides. Animal study results showed that proteins with lipopeptides induced strong M2e-specific antibody responses in the absence of external adjuvants compared to similar proteins without lipopeptides. Thus, the recombinant M2e-based proteins containing alpha-helical linkers and N. meningitidis lipopeptide sequences at the N- and C-termini of four or eight tandem copies of M2e peptide are promising vaccine candidates.

Allogeneic immune rejection is a major barrier for the application of human pluripotent stem cells (hPSCs) in regenerative medicine. A broad spectrum of immune cells, including T cells, natural killer (NK) cells, and antigen-presenting cells, which either cause direct cell killing or constitute an immunogenic environment, are involved in allograft immune rejection. A strategy to protect donor cells from cytotoxicity while decreasing the secretion of inflammatory cytokines of lymphocytes is still lacking. Here, we engineered hPSCs with no surface expression of classical human leukocyte antigen (HLA) class I proteins via beta-2 microglobulin (B2M) knockout or biallelic knockin of HLA-G1 within the frame of endogenous B2M loci. Elimination of the surface expression of HLA class I proteins protected the engineered hPSCs from cytotoxicity mediated by T cells. However, this lack of surface expression also resulted in missing-self response and NK cell activation, which were largely compromised by expression of β2m-HLA-G1 fusion proteins. We also proved that the engineered β2m-HLA-G5 fusion proteins were soluble, secretable, and capable of safeguarding low immunogenic environments by lowering inflammatory cytokines secretion in allografts. Our current study reveals a novel strategy that may offer unique advantages to construct hypoimmunogenic hPSCs via the expression of membrane-bound and secreted β2m-HLA-G fusion proteins. These engineered hPSCs are expected to serve as an unlimited cell source for generating universally compatible \"off-the-shelf\" cell grafts in the future.