abinScience Helps Decode How African Swine Fever Virus I10L Dismantles Host Autophagy Defenses

I10L: A Key Autophagy-Suppressing Protein Encoded in the ASFV Right Variable Region

The researchers carried out a systematic screen of ten proteins encoded in the right variable region of the ASFV genome (L11L, I10L, I9R, I8L, and others), overexpressing each in HeLa cells and measuring changes in the autophagy markers LC3-II and SQSTM1/p62. I10L stood out, significantly increasing levels of both LC3-II and SQSTM1/p62 in a time- and dose-dependent manner. GFP-LC3 puncta assays further confirmed that I10L expression markedly increased the number of autophagosomes within cells.

Figure 1. ASFV I10L expression induces accumulation of autophagy markers

I10L Blocks the Fusion Step of Autophagic Flux, Not Autophagy Initiation

To distinguish between “enhanced autophagy initiation” and “blocked autophagic flux,” the researchers used chloroquine (CQ) and bafilomycin A1 (Baf-A1) to inhibit autophagosome–lysosome fusion, and EBSS starvation or INK128 treatment to activate autophagy initiation. When autophagic flux was already blocked by CQ or Baf-A1, additional I10L expression did not further raise LC3-II or SQSTM1/p62 levels, suggesting I10L acts at the same node as these inhibitors. The same lack of additive effect was observed under ATG5 knockdown. Using the RFP-GFP-LC3 dual fluorescent reporter to track autophagic flux, cells expressing I10L showed a marked accumulation of yellow puncta — representing immature autophagosomes — directly confirming that I10L blocks fusion between autophagosomes and lysosomes.

Figure 2. I10L expression suppresses autophagic flux

I10L-Mediated Autophagy Blockade Requires Cooperation Between Its Transmembrane Domain and C-Terminal Region

The full-length I10L protein is 170 amino acids long, comprising a short N-terminal segment, a transmembrane domain (TM), and a C-terminal functional region. Immunofluorescence localization showed that both full-length I10L and a truncated construct containing only the TM domain (I10L-NTM) localized to LAMP1-positive lysosomal compartments, whereas the TM-deleted truncation showed no such localization. Functional assays demonstrated that deleting either the TM domain or the C-terminal region alone completely abolished the autophagy-inhibitory effect. The TM domain anchors I10L to the lysosome, while the C-terminus carries out the effector function — both are indispensable.

Figure 3. The TM domain and C-terminus are both essential for I10L-mediated autophagy blockade

I10L Specifically Binds RAB7, a Marker of Late Endosomes/Lysosomes

RAB7 is a core regulatory GTPase on late endosomes/lysosomes that serves as an “anchoring bridge” during autophagosome–lysosome fusion. The researchers found that I10L colocalizes extensively with RAB7-marked late endosomes/lysosomes, but not with RAB5 (early endosomes) or RAB11 (recycling endosomes). Co-IP experiments confirmed that I10L binds specifically to RAB7, with no interaction with RAB5, RAB11, or other SNARE proteins. In ASFV-infected primary porcine alveolar macrophages (PAMs), endogenous I10L also interacted and colocalized with RAB7. Overexpressing RAB7 alleviated I10L-induced accumulation of autophagy markers, while RAB7 knockdown prevented I10L from further enhancing the autophagy blockade — demonstrating that I10L exerts its inhibitory effect by hijacking RAB7. Split-fluorescence assays further mapped the interaction interface precisely to amino acids 67–148 in the C-terminus of I10L.

Figure 4. I10L interacts with RAB7

I10L Competitively Occupies RAB7, Blocking Its Binding to the HOPS Subunit VPS39

The HOPS complex binds RAB7 through its VPS39 subunit to drive membrane fusion at late endosomes/lysosomes. Co-IP results showed that I10L expression significantly weakened the interaction between RAB7 and VPS39 in a dose-dependent manner. Immunofluorescence further confirmed that full-length I10L markedly reduced colocalization between VPS39 and endogenous RAB7, an effect not seen with either the TM or C-terminal truncations alone. Notably, I10L did not affect the interaction between the HOPS subunit VPS41 and STX17, indicating that I10L specifically targets the RAB7–VPS39 interface rather than disrupting the entire HOPS complex. Infection with ASFV-ΔI10L restored the RAB7–VPS39 interaction, further supporting this finding.

Figure 5. I10L prevents RAB7-VPS39 association

I10L Selectively Disrupts Assembly of the STX17–SNAP29–VAMP8 SNARE Complex

Disruption of the RAB7–HOPS axis ultimately prevents proper assembly of the downstream SNARE complex. Co-IP experiments showed that I10L expression significantly reduced the interactions between STX17 and SNAP29, and between STX17 and VAMP8, while leaving the direct interaction between VAMP8 and SNAP29 unaffected — a pattern consistent with the immunofluorescence colocalization data. In ASFV infection models, the STX17–SNAP29 interaction was markedly reduced in cells infected with ASFV-WT, but this effect was absent with ASFV-ΔI10L. I10L did not interfere with the YKT6–SNAP29–STX7 complex, highlighting its high selectivity for the STX17 pathway.

Figure 6. I10L inhibits formation of the STX17-SNAP29-VAMP8 complex

I10L Deletion Significantly Impairs ASFV Replication in Primary Macrophages

When ASFV-ΔI10L and the parental ASFV-WT virus were used to infect PAMs and pBMDMs, qPCR and Western blot both showed that ASFV-ΔI10L replicated significantly less efficiently, with lower viral genome copy numbers and p72 protein levels than the wild-type virus. LC3-II and SQSTM1/p62 levels declined in cells infected with ASFV-ΔI10L, indicating that autophagic flux was partially restored once I10L was removed. Based on these findings, the study proposes an overall mechanistic model: early in infection, ASFV expresses I10L, which competitively binds RAB7, blocks HOPS assembly, and consequently disrupts SNARE complex formation — causing autophagosomes to accumulate without being degraded and thereby promoting ASFV replication.

Figure 7. Loss of ASFV I10L restores virus-mediated autophagy