# Influence of CCL2-mediated modulation of ALIX in the budding and replication of viruses from multiple families

## TL;DR

This study shows that CCL2 signaling affects ALIX, which is important for the release of several viruses, including HIV-1, SIV, and EIAV.

## Contribution

A novel CRISPR-based system was developed to study ALIX's role in virus budding without altering viral genes or ESCRT components.

## Key findings

- CCL2 knockout reduces HIV-1 production by 10-fold due to ALIX sequestration in F-actin.
- CCL2 addition rescues virus production in CCL2 knockout cells by mobilizing ALIX.
- SIV and EIAV budding is inhibited in CCL2 knockout cells but rescued by CCL2, while HSV-1 and DENV replication is unaffected.

## Abstract

Signaling by C-C motif ligand 2 (CCL2), a β-chemokine, modulates HIV-1 budding and release by mobilizing ALG-2-interacting protein X (ALIX) from the F-actin cytoskeleton to the cytosol. Immunodepleting CCL2 in the medium sequesters ALIX to F-actin. We developed a novel tool to study HIV budding and release without mutating viral late domains or silencing ESCRT genes, but by blocking CCL2 signaling using CRISPR-Cas9 knockout (KO) of the CCL2 or CCR2 genes. We knocked out CCL2 (CCL2KO) and CCR2 (CCR2KO) singly or together (double knockout) in HeLa cells and confirmed that knockout was associated with the absence of CCL2 or CCR2 expression. In KO cells, ALIX was associated with the F-actin cytoskeleton, while in control cells, it was associated with the cytosolic soluble fraction. In KO cells, HIV-1 production was profoundly reduced (10-fold). Strikingly, for CCL2KO cells, the addition of CCL2 mobilized ALIX to the soluble fraction, and virus production was stimulated to levels higher than those of untreated HeLa cells. We utilized these cells to test the involvement of ALIX in the budding and/or replication of several viruses, including Simian Immunodeficiency Virus (SIV), Equine Infectious Anemia Virus (EIAV), Herpes Simplex Virus type 1 (HSV-1), Dengue virus (DENV), and Hazara virus (HAZV). Budding and release of SIV and EIAV were both inhibited in CCL2KO cells and rescued by CCL2 addition. Replication of HSV-1 and DENV was unaffected in CCL2KO cells, confirming that ALIX is not involved in their replication. Finally, HAZV replication was affected by CCL2 signaling. Our studies indicate that CCL2 signaling and ALIX mobilization are important for several viral families.

C-C motif ligand 2 (CCL2) plays a regulatory role in the budding and release of HIV-1 in macrophages and HeLa cells. CCL2 signaling mobilizes ALG-2-interacting protein X (ALIX) from the F-actin cytoskeleton to the soluble cytosol, where it is accessible for recruitment by the HIV-1 Gag polyprotein in the assembling virions at the plasma membrane. In previous studies, CCL2 immunodepletion, which blocks CCL2 signaling, resulted in ALIX sequestration to the F-actin cytoskeleton and inhibited virus production. Here, we developed a HeLa CCL2 gene knockout cell line and found that abrogation of CCL2 signaling can be restored by CCL2 addition, as evidenced by the restoration of ALIX to the cytosolic fraction and rescue of HIV-1 release. Employing such a system, we tested Simian Immunodeficiency Virus, Equine Infectious Anemia Virus, Herpes Simplex Virus type 1, Dengue, and Hazara virus for their dependence on ALIX for virus replication. The results indicate that CCL2 signaling and ALIX release from F-actin may play a role in the replication of several viruses.