Zika virus disrupts the cellular machinery regulating cell division, and alters the expression of hundreds of genes guiding the formation and development of neurons and astrocytes, according to new research.
Existing evidence indicates that zika virus (ZIKV) infection is associated with microcephaly — a condition in which baby’s head is abnormally small, often because the brain has not developed properly — and other fetal brain defects. Despite the association, cellular alterations caused by the virus are largely unknown.
Elucidating the underpinnings of ZIKV infection is very important to develop tools to combat it, says Stevens Rehen, head of the study and scientist at both D’ Or Institute for Research and Education (IDOR) and Institute of Biomedical Sciences at Federal University of Rio de Janeiro (UFRJ).
Infected Neural Stem Cells
A previous study published by the same Brazilian scientists noted that the pool of infected neural stem cells was completely depleted after one week. In the present study, they decided to explore how neural cells react to ZIKV infection before dying.
To do so, human neural cells were exposed to a strain of ZIKV collected from a Brazilian patient and coaxed to become neurospheres, organized aggregates of neural cells resembling fetal brain tissue.
Combining proteomics and mRNA transcriptional profiling, the researchers evaluated the interactome map – a whole set of molecular interactions – of the infected neural cells in order to understand the ZIKV-related impairment on cellular metabolism.
The analysis revealed more than 500 genes/proteins altered in the infected developing brain cells.
A few of them associated with DNA damage and chromosomal instability, such as aneuploidy. Others, normally active during cellular division, were silenced in infected cells, thwarting their ability to multiply.
In addition, genes driving cell specialization were repressed in ZIKV infected cells. As a result, neural cells lacked the guidance to differentiate into specialized brain cells.
Also, a common strategy employed by viruses when infecting cells was observed: factors involved in viral replication were upregulated.
Shotgun Proteomics and Data Analyses
Qualitative and quantitative proteomic analyses were performed in a two dimensional nano UPLC (2D-RP/RP) in a Acquity UPLC M-Class System, which is connected in line to a Synapt G2-Si mass spectrometer. The mass spectrometer executed Data-Independent Acquisitions (DIA), employing more specifically Ultra-definition Data-independent Mass Spectrometry (UDMSE) method.
Peptide loads were carried to separation in a nanoACQUITY UPLC HSS T3 Column. Peptide elution was achieved using an acetonitrile gradient from 7% to 40% (v/v) for 95 min at a flow rate of 0.4 μL/min directly into a Synapt G2-Si. For every measurement, the mass spectrometer was operated in resolution mode with an m/z resolving power of about 40 000 FWHM, using ion mobility with a cross-section resolving power at least 40 Ω/ΔΩ. MS/MS analyses were performed by nano-electrospray ionization in positive ion mode nanoESI (+) and a NanoLock Spray ionization source.
The lock mass channel was sampled every 30 sec. The mass spectrometer was calibrated with an MS/MS spectrum of [Glu1]-Fibrinopeptide B human (Glu- Fib) solution that was delivered through the reference sprayer of the NanoLock Spray source.
Proteins were identified and quantitative data were processed by using dedicated algorithms and cross-matched with the Uniprot human proteome database, version 2015/11 (70,225 entries), with the default parameters for ion accounting and quantitation45. The databases used were reversed on-the-fly during the database queries and appended to the original database to assess the false- positive identification rate.
For proper spectra processing and database searching conditions, we used Progenesis QI for proteomics software package with Apex3D, peptide 3D, and ion accounting informatics (Waters). This software starts with LC- MS data loading, then performs alignment and peak detection, which creates a list of interesting peptide ions (peptides) that are explored within Peptide Ion Stats by multivariate statistical methods; the final step is protein identity.
Funding for the work was provided by the Brazilian Development Bank; Funding Authority for Studies and Projects; National Council of Scientific and Technological Development; Foundation for Research Support in the State of Rio de Janeiro; and the São Paulo Research Foundation.
Image: Human neural stem cells are the main target of Zika virus infection. Immunofluorescence of neural stem cells, nuclei are in yellow, cytoplasm in pink. Credit: D’Or Institute for Research and Education (IDOR)