Using correlative cryo-SXT and cryo-SIM imaging to study the morphology of membranous rearrangements of Hepatitis C Virus & West Nile Virus-infected cells

Supervisor Organisation PhD Awarding Entity: phd location
ALBA & CNB-CSIC
Universitat Autònoma de Barcelona
ALBA Synchrotron, Barcelona, Spain

Research Focus

The project will focus on locating specific viral proteins within infected cells by Hepatitis C virus (HCV) and West Nile virus using a cryo correlative imaging approach, building on previous studies performed at the Alba synchrotron to investigate by cryo Soft X-ray Tomograghy (cryo-SXT) the cell ultrastructural changes caused by HCV infection.

By establishing protocols and correlative workflows using the cryo 3D structured illumination microscope currently in development (cryo-3D-SIM), specific viral proteins conveniently tagged will be imaged. Afterwards, cryo Soft X-ray Tomograghy will allow revealing the ultrastructure of the infected cells in close-to-native conditions, therefore putting in context the protein location within the cellular environment.

The resolution of cryo-3D-SIM is 2X better than conventional visible light fluorescence microscopy, thus narrowing the resolution gap and allowing more accurate correlation between the two imaging techniques.

Methodology

Cryo-soft X-ray tomography (cryo-SXT) is the only imaging technology that can provide nanoscale 3D information from cryo-preserved, unstained whole cells. This technique provides unique capabilities to capture high resolution (30nm) images of the cellular architecture maintaining the native state of the samples, enabling investigating the viral replication in unprecedented detail.

Cryo-3D-SIM allows fast 3D imaging of volumetric specimens at resolutions beyond those achievable with the confocal microscope, making it an excellent complement to SXT at cryogenic temperatures, matching its penetration depth and detecting fluorescent molecules with wavelength-dependent resolutions of up to 150nm, therefore allowing correlation with the imaged 3D ultrastructure.

This Correlative Light X-ray Tomography approach (CLXT) enables new insights into viral infection by providing complementary morphological, specific localization of proteins and chemical information beyond what is achievable through the use of any individual technique alone. CLXT can provide state-of-art information for viral infection research.

Aim 1

Cryo-SXT to study the morphology of membranous rearrangements during the infection of West Nile virus (WNV)
Evaluate reversion of morphological changes after treatment with antiviral drugs by cryo-SXT and deep UV fluorescence microspectroscopy (secondment at SOLEIL)

Aim 2

Characterization and optimization of the cryo-3D- SIM to establish workflows to locate HCV ns5a in the native cellular environment by correlative cryo-3D-SIM and cryo-SXT on the same infected cells.

Report describing the results of testing cryo-cell preparation on micropatterned grids (project deliverable).

Aim 3

Locate ns5a-cherry and sigma-GFP in HCV replicated cells by cryo-3D-SIM/SXT to understand their role in the formation of the viral replication sites, virion morphogenesis and viral egress

Pictures Attached

PhD Researcher

Name: Kevin Mamprin

University: Autonomous University of Barcelona

Supervisor’s Name: Dr. Ana Joaquina Pérez Berná / Dr. Pablo Gastaminza

Kevin Mamprin completed his Bachelor Degree in Chemistry at the University of Trieste. During his Bachelor’s thesis he worked in the lab of Dr. Marchesan on supramolecular nanostructures which had given him the opportunity to work with EMs and XRM both of them deeply fascinated him. Later He obtained a Master’s Degree in Clinical and Forensic Chemistry at the university of Turin. His Master’s project was carried out in collaboration between the University of Padua Hospital and the University of Padova in Dr. Castagliuolo’s lab, here he developed the passion for host-pathogen interactions. Kevin has then begun his PhD at the ALBA Synchrotron within the CLEXM Consortium where he will examine the host-viruses interactions using correlative Cryo-SXT and Cryo-SIM imaging.

His research interest lies in the application of different microscopy technics to study and characterized the host-pathogen interaction. This comprehended the optimization of the sampled preparation in order to obtain the most native state of cells. But also explore different technics that could be used together to obtain a more complete understanding of this phenomenon.

CLICK HERE TO GO TO THE LAB PAGE