1.G.3 The Viral Pore-forming Membrane Fusion Protein-3 (VMFP3) Family

The tick-borne encephalitis virus (TBEV) is a Class II fusion protein, consisting primarily of β-sheet structure with internal fusion peptides forming loops at the tips of β-strands. These proteins are in the virion, parallel to the viral membrane. There they exist as dimers that become β-stranded trimers upon membrane insertion and fusion (White et al. 2008). Fusion function depends on proteolytic processing. Other Class II VFPs include SFV E1/E2.

The flavivirus dengue virus (DV) infects cells through a low-pH-triggered membrane fusion reaction mediated by the viral envelope protein E. E is an elongated transmembrane protein with three C-terminal TMSs and is organized as a homodimer on the mature virus particle. During fusion, the E protein homodimer dissociates, inserts the hydrophobic fusion loop into target membranes, and refolds into a trimeric hairpin in which domain III (DIII) packs against the central trimer. It is clear that E refolding drives membrane fusion. Liao et al. (2010) used truncated forms of the DV E protein to reconstitute trimerization in vitro. Protein constructs containing domains I and II (DI/II) were monomeric and interacted with membranes to form core trimers. DI/II-membrane interaction and trimerization occurred efficiently at both neutral and low pH. The DI/II core trimer was relatively unstable and could be stabilized by binding exogenous DIII or by the formation of mixed trimers containing DI/II plus E protein with all three TMSs. The mixed trimer has unoccupied DIII interaction sites that specifically bind exogenous DIII at either low or neutral pH. Protein constructs containing domains I and II (DI/II) were monomeric and interacted with membranes to form core trimers. DI/II-membrane interaction and trimerization occurred efficiently at both neutral and low pH. The DI/II core trimer was relatively unstable and could be stabilized by binding exogenous DIII or by the formation of mixed trimers containing DI/II plus E protein with all three TMSs. The mixed trimer has unoccupied DIII interaction sites that specifically bind exogenous DIII at either low or neutral pH. Truncated DV E proteins thus reconstitute hairpin formation and define properties of key domain interactions during DV fusion.


 

References:

Apellaniz B., Huarte N., Largo E. and Nieva JL. (2014). The three lives of viral fusion peptides. Chem Phys Lipids. 181:40-55.

Leung, J.Y., G.P. Pijlman, N. Kondratieva, J. Hyde, J.M. Mackenzie, and A.A. Khromykh. (2008). Role of nonstructural protein NS2A in flavivirus assembly. J. Virol. 82: 4731-4741.

Liao, M., C. Sánchez-San Martín, A. Zheng, and M. Kielian. (2010). In vitro reconstitution reveals key intermediate states of trimer formation by the dengue virus membrane fusion protein. J. Virol. 84: 5730-5740.

Shrivastava, G., G. Visoso-Carvajal, J. Garcia-Cordero, M. Leon-Juarez, B. Chavez-Munguia, T. Lopez, P. Nava, N. Villegas-Sepulveda, and L. Cedillo-Barron. (2020). Dengue Virus Serotype 2 and Its Non-Structural Proteins 2A and 2B Activate NLRP3 Inflammasome. Front Immunol 11: 352.

Shrivastava, G., J. García-Cordero, M. León-Juárez, G. Oza, J. Tapia-Ramírez, N. Villegas-Sepulveda, and L. Cedillo-Barrón. (2017). NS2A comprises a putative viroporin of Dengue virus 2. Virulence 8: 1450-1456.

Tomar, P.P.S., M. Krugliak, A. Singh, and I.T. Arkin. (2022). Zika M-A Potential Viroporin: Mutational Study and Drug Repurposing. Biomedicines 10:.

White, J.M., S.E. Delos, M. Brecher, and K. Schornberg. (2008). Structures and mechanisms of viral membrane fusion proteins: multiple variations on a common theme. Crit. Rev. Biochem. Mol. Biol. 43: 189-219.

Zhang, X., R. Jia, H. Shen, M. Wang, Z. Yin, and A. Cheng. (2017). Structures and Functions of the Envelope Glycoprotein in Flavivirus Infections. Viruses 9:.

Examples:

TC#NameOrganismal TypeExample
1.G.3.1.1

Tick-borne encephalitis virus (TBEV) (Class II) polyprotein of 3414 aas.  Residues 281 - 776 include the envelop protein that includes the viral fusion protein (Zhang et al. 2017).

Virus

Tick-borne encephalitis virus polyprotein (P14336)

 
1.G.3.1.10

Yellow Fever Virus polyprotein fragment of 220 aas and 2 TMSs.  This fragment should include the E (envelope) protein, the M (membrane) protein and the pr protein Their involvement in membrane fusion and flavivirus maturation is discussed by Crampon et al. 2023.

Polyprotein fragment of Yellow Fever Virus

 
1.G.3.1.2

Polyprotein (3391aas) (includes the membrane fusion protein, envelope protein E (495aas; 38% identical to residues 282-774 in 1.G.3.1.1) (Liao et al., 2010)).  The fusion peptides are residues 98 - 113 in V7SFC4 and residues 378 - 393 in P14340 (Apellániz et al. 2014).

Virus

Polyprotein of Dengue virus (P14340)
Envelope protein E (B7SFC4)

 
1.G.3.1.3

Polyprotein of 3432 aas of the Flavi-glycoprotein family.  The type II fusion peptide is residues 392 - 407 (Apellániz et al. 2014).

Viruses (Flaviviridae)

Polyprotein of Japanese encephalitis virus

 
1.G.3.1.4

Non-structural protein 2B of 131 aas and 2 TMSs.

NS2B of Usutu virus

 
1.G.3.1.5

NS2B of 131 aas and 2 TMSs.

NS2B of Murray Valley encephalitis virus

 
1.G.3.1.6

NS2A viroporin of 218 aas and 4 - 8 TMSs (Shrivastava et al. 2017).

NS2A of Dengue Virus

 
1.G.3.1.7

Polyprotein of 3433 aas and about 20 - 24 TMSs.  It includes the N2Sa protein which has been shown to be a viroporin (Leung et al. 2008).

N2Sa of Kunjin Virus

 
1.G.3.1.8

Non-structural protein, NSP 2B, of 122 aas and 2 TMSs. It forms a viroporin (Shrivastava et al. 2020).

NSP2B of Dengue virus

 
1.G.3.1.9

Zika viroporin, ZikV-M (the M protein) of 75 aas and 1 C-terminal TMS. Flaviviruses contain several important human pathogens. Among these, the Zika virus is an emerging etiological agent. One of its structural proteins, prM, plays an essential role in viral maturation and assembly, making it an attractive drug and vaccine development target. Tomar et al. 2022 have characterized ZikV-M as a potential viroporin candidate using three different bacterium-based assays which were used to identify potential ZikV-M blockers. Mutational analyses of conserved amino acids in the transmembrane domain of other flaviviruses, including West Nile and Dengue viruses, were performed to study their role in ion channel activity. Thus, ZikV-M is a potential ion channel that can be used as a drug target for high throughput screening and drug repurposing (Tomar et al. 2022). P2X7R is expressed in cancer and immune system cell surfaces. ATP plays a key role in numerous metabolic processes due to its abundance in the tumour microenvironment. P2X7R plays an important role in cancer by interacting with ATP. The unusual property of P2X7R is that stimulation with low doses of ATP causes the opening of a permeable channel for sodium, potassium, and calcium ions, whereas sustained stimulation with high doses of ATP favours the formation of a non-selective pore. The latter effect induces a change in intracellular homeostasis that leads to cell death.

ZikV-M viroporin of Zika Virus