L12

Amino Acid Sequence

3D Structure (/structures/AF-0000000211971556-model_v1.cif)

Function

Catalyzes the removal of terminal sialic acid residues from viral and cellular glycoconjugates. Cleaves off the terminal sialic acids on the glycosylated HA during virus budding to facilitate virus release. Additionally helps virus spread through the circulation by further removing sialic acids from the cell surface. These cleavages prevent self-aggregation and ensure the efficient spread of the progeny virus from cell to cell. Otherwise, infection would be limited to one round of replication. Described as a receptor-destroying enzyme because it cleaves a terminal sialic acid from the cellular receptors. May facilitate viral invasion of the upper airways by cleaving the sialic acid moieties on the mucin of the airway epithelial cells. Likely to plays a role in the budding process through its association with lipid rafts during intracellular transport. May additionally display a raft-association independent effect on budding. Plays a role in the determination of host range restriction on replication and virulence. Sialidase activity in late endosome/lysosome traffic seems to enhance virus replication.

Mechanism

The ubiquitin-proteasome pathway (UPP) constitutes the principal pathway for protein degradation in eukaryotic cells, mediating the breakdown of target proteins through ubiquitin tagging and subsequent proteasomal degradation. In an ATP-dependent manner, the ubiquitin-activating enzyme (E1) activates ubiquitin (Ub) and conjugates it to a ubiquitin-conjugating enzyme (E2). E3 ligase subsequently catalyzes the transfer of Ub to the target protein, leading to the formation of a polyubiquitin chain that completes the ubiquitination mark. The 19S regulatory particle of the 26S proteasome recognizes this ubiquitination signal and, after deubiquitination, mediates the ATP-dependent delivery of the substrate into the 20S proteasome for degradation.

Description

First, co-treatment of L12 with the protein synthesis inhibitor cycloheximide (CHX) led to near-complete degradation of NA protein within 10 hours (Fig. 5A). To determine whether L12 induces NA degradation via the ubiquitin-proteasome pathway, we conducted inhibition experiments using three specific inhibitors: the proteasome inhibitor MG-132, the lysosomal inhibitor chloroquine (CQ), and the autophagy inhibitor bafilomycin A1 (Fig. 5B). The results showed that MG-132 effectively blocked L12-induced NA degradation, while chloroquine and bafilomycin A1 had no significant inhibitory effects. Furthermore, we co-transfected 293T cells with hemagglutinin (HA)-tagged ubiquitin and NA-Flag plasmids, followed by treatment with L12 and MG-132. This resulted in a noticeable increase in NA protein ubiquitination levels, suggesting that L12 may induce NA protein degradation through the ubiquitin-proteasome system (UPS) (Fig. 5C). Additionally, NA-Flag-transfected 293T cells treated with increasing concentrations of L12 showed no significant changes in NA mRNA levels compared to control groups at 48 hours post-RNA lysis buffer administration (Fig. 5D). Collectively, these findings confirm that L12 reduces NA protein levels through proteasome mediated degradation rather than transcriptional regulation.

Description

The H274Y mutation significantly reduces oseltamivir efficacy, posing challenges to clinical management. As a result, the antiviral activities of compounds L1–L19 against the oseltamivir resistant H1N1-H274Y strain in MDCK cells were performed by using oseltamivir (OSP) as a reference control. Among these, compound L12, with a nine-carbon linker, exhibited an EC50 of 0.68 mM—157 times more potent than oseltamivir—highlighting its excellent antiviral effects.