HIV-1 Nef interacts with the cyclin K/CDK13 complex to antagonize SERINC5 for optimal viral infectivity

Abstract

SUMMARY HIV-1-negative factor (Nef) protein antagonizes serine incorporator 5 (SERINC5) by redirecting this potent restriction factor to the endosomes and lysosomes for degradation. However, the precise mechanism remains unclear. Using affinity purification/mass spectrometry, we identify cyclin K (CycK) and cyclin-dependent kinase 13 (CDK13) as a Nef-associated kinase complex. CycK/CDK13 phosphorylates the serine at position 360 (S360) in SERINC5, which is required for Nef downregulation of SERINC5 from the cell surface and its counteractivity of the SERINC5 antiviral activity. To understand the role of S360 phosphorylation, we generate chimeric proteins between CD8 and SERINC5 to study their response to Nef. Nef not only downregulates but, importantly, also binds to this chimera in an S360-dependent manner. Thus, S360 phosphorylation increases interactions between Nef and SERINC5 and initiates the destruction of SERINC5 by the endocytic machinery.

Cul3-KLHL20 E3 ubiquitin ligase plays a key role in the arms race between HIV-1 Nef and host SERINC5 restriction

“…To understand how SERINC5 is polyubiquitinated, we interrogated the presence of E3 ubiquitin ligases in SERINC5 protein complexes by mass spectrometry. FLAG-tagged SERINC5 was purified from HEK293T cells by an anti-FLAG affinity column and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS), as we reported 18 . Four independent experiments were conducted from which a total of 25 ubiquitin E3 ligase-associated proteins were identified, which consisted of Cul1, Cul3, and Cul4B (Fig.…”

“…In particular, Nef interacts with SERINC5 via its largest intracellular loop 4 (ICL4) 17 . In addition, Nef directs Cyclin K/Cyclin-dependent kinase 13 (CDK13) complex to phosphorylate a serine residue (S360) at SERINC5 in ICL4, which bridges SERINC5 with the adapter protein 2 (AP2) complex via Nef for endocytosis and degradation 18 .…”

SERINC5: One antiviral factor to bind them all

“…Studies on the effect of Nef on SERINC5 found that Nef N-terminal region (residues 32 to 39) is crucial for its interaction with SERINC5, while the C-terminal endocytic sorting motif (E 160 xxxLL 165 ) mediates binding with AP2 [ 9 , 28 ]. Furthermore, it has been shown that Cyclin K/Cyclin-dependent kinase 13 (CDK13) complex-mediated phosphorylation of a serine residue (S360) within the intracellular loop 4 (ICL4) of SERINC5 enhances Nef-AP2-SERINC5 interaction [ 29 ]. However, S360 residue is not conserved among SERINCs [ 8 ].…”

The Emerging Role of the Serine Incorporator Protein Family in Regulating Viral Infection

“…Chai et al used affinity purification/mass spectrometry to identify a complex of cyclin K (CycK) and cyclindependent kinase 13 (CDK13) that interacts with Nef to antagonize SERINC5 for optimal viral infectivity (Figure 3②). Mechanistically, the CycK-CDK13 complex phosphorylates the serine at position 360 in SERINC5, resulting in downregulation of SERINC5 from the cell surface (Chai et al, 2021) (Figure 3③).…”