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- EMDB-8733: The C-terminus of the herpes simplex virus pUL25 protein is requi... -

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Entry
Database: EMDB / ID: EMD-8733
TitleThe C-terminus of the herpes simplex virus pUL25 protein is required for release of viral genomes from capsids bound to nuclear pores.
Map dataVirion with deletion of three C-terminal amino acids (578-580) of the UL25 protein
Sampleherpes simplex virus != Human alphaherpesvirus 1

herpes simplex virus

  • Virus: Human alphaherpesvirus 1 (Herpes simplex virus type 1)
Biological speciesHuman alphaherpesvirus 1 (Herpes simplex virus type 1)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.4 Å
AuthorsHuffman JB / Daniel GR / Falck-Pedersen E / Huet A / Smith GA / Conway JF / Homa FL
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI089803 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32 GM08061 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 AIO56346 United States
National Institutes of Health/Office of the DirectorS10 OD019995 United States
CitationJournal: J Virol / Year: 2017
Title: The C Terminus of the Herpes Simplex Virus UL25 Protein Is Required for Release of Viral Genomes from Capsids Bound to Nuclear Pores.
Authors: Jamie B Huffman / Gina R Daniel / Erik Falck-Pedersen / Alexis Huet / Greg A Smith / James F Conway / Fred L Homa /
Abstract: The herpes simplex virus (HSV) capsid is released into the cytoplasm after fusion of viral and host membranes, whereupon dynein-dependent trafficking along microtubules targets it to the nuclear ...The herpes simplex virus (HSV) capsid is released into the cytoplasm after fusion of viral and host membranes, whereupon dynein-dependent trafficking along microtubules targets it to the nuclear envelope. Binding of the capsid to the nuclear pore complex (NPC) is mediated by the capsid protein pUL25 and the capsid-tethered tegument protein pUL36. Temperature-sensitive mutants in both pUL25 and pUL36 dock at the NPC but fail to release DNA. The uncoating reaction has been difficult to study due to the rapid release of the genome once the capsid interacts with the nuclear pore. In this study, we describe the isolation and characterization of a truncation mutant of pUL25. Live-cell imaging and immunofluorescence studies demonstrated that the mutant was not impaired in penetration of the host cell or in trafficking of the capsid to the nuclear membrane. However, expression of viral proteins was absent or significantly delayed in cells infected with the pUL25 mutant virus. Transmission electron microscopy revealed capsids accumulated at nuclear pores that retained the viral genome for at least 4 h postinfection. In addition, cryoelectron microscopy (cryo-EM) reconstructions of virion capsids did not detect any obvious differences in the location or structural organization for the pUL25 or pUL36 proteins on the pUL25 mutant capsids. Further, in contrast to wild-type virus, the antiviral response mediated by the viral DNA-sensing cyclic guanine adenine synthase (cGAS) was severely compromised for the pUL25 mutant. These results demonstrate that the pUL25 capsid protein has a critical role in releasing viral DNA from NPC-bound capsids. Herpes simplex virus 1 (HSV-1) is the causative agent of several pathologies ranging in severity from the common cold sore to life-threatening encephalitic infection. Early steps in infection include release of the capsid into the cytoplasm, docking of the capsid at a nuclear pore, and release of the viral genome into the nucleus. A key knowledge gap is how the capsid engages the NPC and what triggers release of the viral genome into the nucleus. Here we show that the C-terminal region of the HSV-1 pUL25 protein is required for releasing the viral genome from capsids docked at nuclear pores. The significance of our research is in identifying pUL25 as a key viral factor for genome uncoating. pUL25 is found at each of the capsid vertices as part of the capsid vertex-specific component and implicates the importance of this complex for NPC binding and genome release.
History
DepositionMay 15, 2017-
Header (metadata) releaseJun 21, 2017-
Map releaseJun 21, 2017-
UpdateJan 29, 2020-
Current statusJan 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.1
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8733.png

Downloads & links

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Map

FileDownload / File: emd_8733.map.gz / Format: CCP4 / Size: 2.1 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationVirion with deletion of three C-terminal amino acids (578-580) of the UL25 protein
Voxel sizeX=Y=Z: 1.8 Å
Density
Contour LevelBy AUTHOR: 0.1 / Movie #1: 0.1
Minimum - Maximum-0.2220644 - 0.490131
Average (Standard dev.)0.0067447457 (±0.047714412)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions825825825
Spacing825825825
CellA=B=C: 1485.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.81.81.8
M x/y/z825825825
origin x/y/z0.0000.0000.000
length x/y/z1485.0001485.0001485.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-38-19-20
NX/NY/NZ858082
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS825825825
D min/max/mean-0.2220.4900.007

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Supplemental data

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Sample components

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Entire : herpes simplex virus

EntireName: herpes simplex virus
Components
  • Virus: Human alphaherpesvirus 1 (Herpes simplex virus type 1)

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Supramolecule #1: Human alphaherpesvirus 1

SupramoleculeName: Human alphaherpesvirus 1 / type: virus / ID: 1 / Parent: 0 / NCBI-ID: 10298 / Sci species name: Human alphaherpesvirus 1 / Virus type: VIRION / Virus isolate: OTHER / Virus enveloped: Yes / Virus empty: No
Host (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 144 MDa
Virus shellShell ID: 1 / Name: capsid / Diameter: 1200.0 Å / T number (triangulation number): 16

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
10.0 mMTRIS
150.0 mMsodium chlorideNaClSodium chloride
1.0 mMEDTAEthylenediaminetetraacetic acid
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK III / Details: 8 second blot.
DetailsVirion

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 5.0 µm / Calibrated defocus min: 1.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 59000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Digitization - Sampling interval: 14.0 µm / Digitization - Frames/image: 2-6 / Number grids imaged: 1 / Number real images: 4503 / Average exposure time: 1.3 sec. / Average electron dose: 10.0 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 6665
CTF correctionSoftware - Name: CTFFIND3
Initial angle assignmentType: COMMON LINE / Software - Name: Auto3DEM
Final angle assignmentType: COMMON LINE / Software - Name: Auto3DEM
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 6.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Auto3DEM / Number images used: 4666
FSC plot (resolution estimation)

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