ACERT Highlights: Recent Highlights

National Institute of General Medical Sciences
	National Biomedical Resource for Advanced ESR Spectroscopy

Recent Highlights

Highly Basic Clusters in the Herpes Simplex Virus 1 Nuclear Egress Complex Drive Membrane Budding by Inducing Lipid Ordering

During replication of herpesviruses, capsids escape from the nucleus into the cytoplasm by budding at the inner nuclear membrane. This unusual process is mediated by the viral nuclear egress complex (NEC) that deforms the membrane around the capsid by oligomerizing into a hexagonal, membrane-bound scaffold. Here, we found that highly basic membrane-proximal regions (MPRs) of the NEC alter lipid order by inserting into the lipid headgroups and promote negative Gaussian curvature. We also find that the electrostatic interactions between the MPRs and the membranes are essential for membrane deformation. One of the MPRs is phosphorylated by a viral kinase during infection, and the corresponding phosphomimicking mutations block capsid nuclear egress. We show that the same phosphomimicking mutations disrupt the NEC-membrane interactions and inhibit NEC-mediated budding in vitro, providing a biophysical explanation for the in vivo phenomenon. Our data suggest that the NEC generates negative membrane curvature by both lipid ordering (ESR) and protein scaffolding and that phosphorylation acts as an off switch that inhibits the membrane-budding activity of the NEC to prevent capsid-less budding.

Publication: mBio 12, (4), e01548-21 (2021), PMC8406295.

Figure: UL31 and UL34 MPR peptides interact in the presence of membranes. (A) Schematic depicting peptide location within the NEC sequence. (B, C, and D) Representative experimental DEER data (left) and reconstructed interpeptide distance distributions (right) of at least two individual experiments. The "C" indicates the location of spin-labeled Cys. (B) UL31^(C1-50)/UL34^(174-194) and UL31^(1-C51)/UL34^(174-194). The blue line in the left panel is the denoised curve. Schematic depicts interpeptide distance in Ångstroms and associated standard error of the mean (68% confidence interval of the mean) for each tested probe location. (C) UL31^(C40-50)/UL34^(174-194), UL31^(41-C51)/UL34^(174-194), (D) UL31^(C21-42)/UL34^(174-194), or UL31^(22-C43)/UL34^(174-194). The peptides were mixed with POPC/POPS/POPA 3:1:1 SUVs in 1:200 P/L ratio. Each combination was repeated two times. (E) Model of HSV-1 UL31 and UL34 MPRs attached to greyscale HSV-1 NEC crystal structure based on DEER measurements. Native amino acid sequences were substituted for cysteine probe locations and are shown as filled in circles. UL34 MPR is alpha helical in homologous structures and depicted as such, while UL31 is shown as unstructured. (F) Bottom of homology-modeled HSV-1 NEC onto PRV NEC. Inset shows distance measurement between Q51_UL31 and C182_UL34 shown as spheres. A174_UL34 and I189_UL34 are shown as spheres. Images taken in PyMoL.

Michael K. Thorsen (Department of Molecular Biology and Microbiology, Graduate Program in Cellular, Molecular and Developmental Biology, Tufts University School of Medicine, Boston, MA)
Alex Lai (ACERT)
Michelle W. Lee (Department of Bioengineering, Department of Chemistry and Biochemistry, California NanoSystems Institute, University of California, Los Angeles, Los Angeles,CA)
David P. Hoogerheide (Center for Neutron Research, National Institute of Standards and Technology, Gaithersburg, MD)
Gerard C. L. Wong (Department of Bioengineering, Department of Chemistry and Biochemistry, California NanoSystems Institute, University of California, Los Angeles, Los Angeles,CA)
Jack H. Freed (ACERT)
Ekaterina E. Heldwein (Department of Molecular Biology and Microbiology, Graduate Program in Cellular, Molecular and Developmental Biology, Tufts University School of Medicine, Boston, MA)

Site Map

Home

About ACERT
ACERT News
Personnel

Research
   Available Resources
   Technologies
   Research Highlights
   Collaborations

Outreach
   Dissemination
   Training/Workshops
   Publications
   Useful Links

ACERT is supported by grant 1R24GM146107 from the National Institute of General Medical Sciences (NIGMS), part of the National Institutes of Health.

National Biomedical Resource for Advanced ESR Spectroscopy

Baker Laboratory of Chemistry
259 East Ave.
Ithaca, NY 14853

National Institute of
General Medical Sciences