Palmitoylated Cysteines in Chikungunya Virus nsP1 Are Critical for Targeting to Cholesterol-Rich Plasma Membrane Microdomains with Functional Consequences for Viral Genome Replication

Abstract

In mammalian cells, alphavirus replication complexes are anchored to the plasma membrane. This interaction with lipid bilayers is mediated through the viral methyl/guanylyltransferase nsP1 and reinforced by palmitoylation of cysteine residue(s) in the C-terminal region of this protein. Lipid content of membranes supporting nsP1 anchoring remains poorly studied. Here, we explore the membrane binding capacity of nsP1 with regard to cholesterol. Using the medically important chikungunya virus (CHIKV) as a model, we report that nsP1 cosegregates with cholesterol-rich detergent-resistant membrane microdomains (DRMs), also called lipid rafts. In search for the critical factor for cholesterol partitioning, we identify nsP1 palmitoylated cysteines as major players in this process. In cells infected with CHIKV or transfected with CHIKV trans-replicase plasmids, nsP1, together with the other nonstructural proteins, are detected in DRMs. While the functional importance of CHIKV nsP1 preference for cholesterol-rich membrane domains remains to be determined, we observed that U18666A- and imipramine-induced sequestration of cholesterol in late endosomes redirected nsP1 to these compartments and simultaneously dramatically decreased CHIKV genome replication. A parallel study of Sindbis virus (SINV) revealed that nsP1 from this divergent alphavirus displays a low affinity for cholesterol and only moderately segregates with DRMs. Behaviors of CHIKV and SINV with regard to cholesterol, therefore, match with the previously reported differences in the requirement for nsP1 palmitoylation, which is dispensable for SINV but strictly required for CHIKV replication. Altogether, this study highlights the functional importance of nsP1 segregation with DRMs and provides new insight into the functional role of nsP1 palmitoylated cysteines during alphavirus replication. IMPORTANCE Functional alphavirus replication complexes are anchored to the host cell membranes through the interaction of nsP1 with the lipid bilayers. In this work, we investigate the importance of cholesterol for such an association. We show that nsP1 has affinity for cholesterol-rich membrane microdomains formed at the plasma membrane and identify conserved palmitoylated cysteine(s) in nsP1 as the key determinant for cholesterol affinity. We demonstrate that drug-induced cholesterol sequestration in late endosomes not only redirects nsP1 to this compartment but also dramatically decreases genome replication, suggesting the functional importance of nsP1 targeting to cholesterol-rich plasma membrane microdomains. Finally, we show evidence that nsP1 from chikungunya and Sindbis viruses displays different sensitivity to cholesterol sequestering agents that parallel with their difference in the requirement for nsP1 palmitoylation for replication. This research, therefore, gives new insight into the functional role of palmitoylated cysteines in nsP1 for the assembly of functional alphavirus replication complexes in their mammalian host.

New Insights into Chikungunya Virus Infection and Pathogenesis

“…CHIKV nsP3 was proposed to be involved in spherule assembly by recruiting BIN1/amphiphysin 2, an F-BAR protein involved in membrane curvature, but there is still a lack of clear evidence (92) (Figure 3). Furthermore, the contribution of defined membrane lipid species should be considered with a special attention to fatty acid molecular species that determine the fluidity or curvature of the lipid bilayer, depending on the length and saturation of their fatty acyl chains, and to negatively charged phospholipids and cholesterol that regulate nsP1 capping activity and membrane affinity, respectively (74,93), and could alternatively promote host cofactor coalescence to the replication site to assist spherule assembly.…”

Role of Host-Mediated Post-Translational Modifications (PTMs) in RNA Virus Pathogenesis

“…HIV-1 glycoprotein palmitoylation is required for gp160 incorporation and infectivity [ 148 ]. Alphavirus TF (transframe) protein is palmitoylated and is involved in its localization to the plasma membrane and incorporation into a virus [ 149 ], and palmitoylated nsP1 is involved in anchoring replication complex to the plasma membrane [ 154 ]. The examples mentioned above show that the palmitoylation of viral proteins is crucial for viral replication.…”

Small-Molecule Inhibitors of Chikungunya Virus: Mechanisms of Action and Antiviral Drug Resistance

“…The middle part of nsP1, spanning amino acid residues 245 to 264, contains an amphipathic helix responsible for association of the alphavirus replication complex (RC) with membranes ( 49 ). Specifically, the presence of amphipathic helix and palmitoylated cysteines 417 to 419 ( 50 ) allows nsP1 and nsP1-containing replication complexes to anchor to cholesterol-enriched membrane microdomains ( 51 ). Importantly, site-directed mutagenesis of conserved residues in alphavirus nsP1 indicated that abrogation of nsP1 enzymatic activities is detrimental for virus replication ( 52 ).…”