Advances in Viroporin Function and Structure: A Comparative Analysis of Alphavirus 6K with Well-Characterized Viroporins

Abstract

Viruses encode ion channel proteins called viroporins to assist in infection and immune evasion. The alphavirus 6K protein is classified as a member of the viroporin family of proteins. Several studies have characterized the role of 6K in alphavirus budding and infection since its discovery in the late 1970s. In this review, we summarize 6K research and discuss some unanswered questions regarding 6K biology. We highlight the similarities and differences between 6K and viroporins of clinically relevant viruses-influenza A virus, HIV-1, hepatitis C virus, and SARS-CoV-2-and address their importance as therapeutic targets. The sensitivity of these viroporins to common inhibitors and their ability to functionally complement each other underscore their potential as targets for broad-spectrum antiviral therapies.

Keywords: HCV p7; HIV-1 Vpu; IAV M2; SARS-CoV-2 envelope; alphavirus 6K; alphavirus TF; antivirals; viroporins.

Figure 4

Monomeric membrane topology and oligomeric channel structures of viroporins. (A) Membrane topologies of monomeric IAV M2, HIV-1 Vpu, HCV p7 (Gt 1b), SARS-CoV-2 E, and SINV 6K (figure created with BioRender.com, https://www.biorender.com/, accessed on 1 June 2025). (B–E) Channel structures of (B) hexameric IAV M2 with the fourth chain hidden (PDB accession 3LBW) [197], (C) pentameric HIV-1 Vpu (PDB accession 1PI7) [198], (D) hexameric HCV p7 (Gt 5a) (PDB accession 2M6X) [199], and (E) pentameric SARS-CoV-2 E (PDB accession 7K3G) [200] proteins, showing important residues involved in channel function. Abbreviations: Gt, genotype; PDB, protein data bank. Structures were generated using UCSF ChimeraX software [201].

Figure 1

Schematic representation of the alphavirus genome and the translation of the structural polyprotein. The alphavirus genome is made up of two ORFs. At the 5′ end of the positive-sense viral RNA is the non-structural ORF, which encodes the non-structural proteins: nsP1, nsP2, nsP3, and nsP4. The structural ORF at the 3′ end encodes the structural proteins: capsid, envelope glycoproteins (pE2, E1), 6K, or TF. The structural ORF is transcribed from an internal subgenomic protomer in the minus-strand antigenome template. Occasionally, −1PRF occurs due to the presence of a conserved slippery codon (UUUUUUA motif) in the sequence of 6K, resulting in the translation of TF in place of 6K and E1. TF has the same N-terminal sequence as 6K with a unique C-terminal sequence. Abbreviations: UTR, untranslated region; nsP, non-structural protein; ORF, open reading frame; CP, capsid protein; E, envelope glycoprotein; 6K, 6kDa protein; TF, TransFrame protein; PRF, programmed ribosomal frameshifting (figure created with BioRender.com, https://www.biorender.com/, accessed on 1 June 2025).

Figure 2

Sequences and topological models of SINV 6K and TF proteins. SINV 6K and TF represent the sequence features and topological models of alphavirus viroporins. (A) Predicted domains and important residues of SINV 6K are shown. Cleavage sites at the start and end of its sequence are recognized by the signalase enzyme in the ER lumen during polyprotein processing. 6K has two interfacial domains (red boxes) near the N-terminus of the predicted ion channel domain [63]. The first is in the pre-transmembrane region, while the second may be a part of the ion channel TMD occupying the amphipathic zone of cellular membranes. Aromatic residues outside these interfacial domains, which may be involved in membrane interaction, are underlined in black. Cysteine residues are emboldened in green, and basic residues are highlighted in yellow. Cysteines can undergo post-translational modifications or form disulfide bonds. The conserved basic residue R34 is important for channel activity [35]. The conserved slip site where −1 PRF occurs is indicated with a black arrow. The E1 translocation signal at the 6K C-terminus is shown in orange. (B) The two-TMD model of SINV 6K is shown. 6K N- and C-termini are in the ER lumen and accessible by signalase enzyme (black arrows) for cleavage from pE2 and E1. 6K has two transmembrane helices: the first is involved in the formation of an oligomeric ion channel [63,64], while the second is needed for E1 translocation [60]. The two helices are separated by a short cytoplasmic loop containing some or all of the four cysteine residues—C35, 36, 38, and 39—which are not palmitoylated [65]. If these residues are present in the ER membrane, they are no longer accessible for palmitoylation. This scenario may place the R34 residue in the hydrophobic region of the ER membrane, leading to membrane destabilization. The furin cleavage site in pE2 that yields E3 and E2 is shown with a white arrowhead. (C) The one-TMD model of SINV 6K is depicted [66]. 6K has a single TMD with the N-terminus in the cytosol and the C-terminus in the ER lumen. This occurs as E2 TM2 does not span the ER membrane but is bound at the cytosolic interface. Cysteine residues in 6K are inaccessible to the cytosolic palmitoylation machinery. 6K is cleaved from pE2 by an unknown protease (red arrow). (D) Predicted domains and important residues in the sequence of SINV TF viroporin are shown. TF has a unique C-terminal sequence (blue) containing five additional cysteines (emboldened in green) and basic residues (highlighted in yellow). (E) TF has a single TMD with the N-terminus in the ER lumen and the C-terminus in the cytosol. In approximately 20% of translation events, E2 TM2 undergoes membrane integration and stimulates −1 PRF to produce TF. Cysteine residues in TF at the C-terminus of the TMD are accessible for palmitoylation in the cytosol. For sequence alignment of 6K and TF proteins from different alphaviruses, refer to the review by Ramsey and Mukhopadhyay (2017) [65]. Abbreviations: SINV, Sindbis virus; 6K, 6kDa protein; E, envelope glycoprotein; TMD, transmembrane domain; TF, TransFrame protein; ER, endoplasmic reticulum; C, cysteine; PRF, programmed ribosomal frameshifting (figure created with BioRender.com https://www.biorender.com/, accessed on 1 June 2025).

Figure 3

Known and potential roles of 6K and TF proteins during the alphavirus life cycle. 6K and TF are expressed in the internal membranes of the ER and the Golgi apparatus. TF localizes to the plasma membrane and gets incorporated into the budding virion. Based on the existing literature, the known (green boxes) and potential (red boxes) roles of these proteins are highlighted alongside their intracellular locations. Abbreviations: 6K, 6kDa protein; TF, TransFrame protein; CPVII, cytopathic vacuole II; ER, endoplasmic reticulum; nsP, non-structural protein (figure created with BioRender.com, https://biorender.com (accessed on 1 June 2025).