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Identification of Host Cell Factors Associated with Astrovirus Replication in Caco-2 Cells

American Society for Microbiology
Journal of Virology
Authors:
Murillo Andrea at National Autonomous University of Mexico
Murillo Andrea
Rosario Vera-EStrella at National Autonomous University of Mexico
Rosario Vera-EStrella
Bronwyn J Barkla at Southern Cross University
Bronwyn J Barkla
Ernesto Méndez
Ernesto Méndez
Ernesto Méndez
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Abstract

Importance: Astroviruses are common etiological agents of acute gastroenteritis in children and immunocompromised patients. More recently, they have been associated with neurological diseases in mammals, including humans, and are also responsible for different pathologies in birds. In this work we provide evidence that astrovirus RNA replication and virus assembly occurs in contact with cell membranes potentially derived from multiple cell organelles and show that cellular proteins involved in lipid metabolism that associate with these membranes are required for the efficient viral replication. Our findings provide information to the knowledge of astrovirus biology and provide information that might be useful to develop therapeutic interventions to prevent virus replication.
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Identification of Host Cell Factors Associated with Astrovirus
Replication in Caco-2 Cells
Andrea Murillo,
a
Rosario Vera-Estrella,
a
Bronwyn J. Barkla,
b
Ernesto Méndez,
a†
Carlos F. Arias
a
Instituto de Biotecnología, Universidad Nacional Autónoma de México, Colonia Chamilpa, Cuernavaca, Morelos, México
a
; Southern Cross Plant Sciences, Southern Cross
University, Lismore, NSW, Australia
b
ABSTRACT
Astroviruses are small, nonenveloped viruses with a single-stranded positive-sense RNA genome causing acute gastroenteritis in
children and immunocompromised patients. Since positive-sense RNA viruses have frequently been found to replicate in associ-
ation with membranous structures, in this work we characterized the replication of the human astrovirus serotype 8 strain Yuc8
in Caco-2 cells, using density gradient centrifugation and free-flow zonal electrophoresis (FFZE) to fractionate cellular mem-
branes. Structural and nonstructural viral proteins, positive- and negative-sense viral RNA, and infectious virus particles were
found to be associated with a distinct population of membranes separated by FFZE. The cellular proteins associated with this
membrane population in infected and mock-infected cells were identified by tandem mass spectrometry. The results indicated
that membranes derived from multiple cell organelles were present in the population. Gene ontology and protein-protein inter-
action network analysis showed that groups of proteins with roles in fatty acid synthesis and ATP biosynthesis were highly en-
riched in the fractions of this population in infected cells. Based on this information, we investigated by RNA interference the
role that some of the identified proteins might have in the replication cycle of the virus. Silencing of the expression of genes in-
volved in cholesterol (DHCR7,CYP51A1) and fatty acid (FASN) synthesis, phosphatidylinositol (PI4KIII␤) and inositol phos-
phate (ITPR3) metabolism, and RNA helicase activity (DDX23) significantly decreased the amounts of Yuc8 genomic and antige-
nomic RNA, synthesis of the structural protein VP90, and virus yield. These results strongly suggest that astrovirus RNA
replication and particle assembly take place in association with modified membranes potentially derived from multiple cell or-
ganelles.
IMPORTANCE
Astroviruses are common etiological agents of acute gastroenteritis in children and immunocompromised patients. More re-
cently, they have been associated with neurological diseases in mammals, including humans, and are also responsible for differ-
ent pathologies in birds. In this work, we provide evidence that astrovirus RNA replication and virus assembly occur in contact
with cell membranes potentially derived from multiple cell organelles and show that membrane-associated cellular proteins in-
volved in lipid metabolism are required for efficient viral replication. Our findings provide information to enhance our knowl-
edge of astrovirus biology and provide information that might be useful for the development of therapeutic interventions to pre-
vent virus replication.
Astroviruses are common etiological agents of acute gastroen-
teritis in children and immunocompromised patients (1,2).
They have also been associated with neurological diseases in hu-
mans (3) and other mammals, like minks and cattle (4,5). In
birds, astroviruses cause different pathologies, manifesting as hep-
atitis in ducks (6) and poultry enteritis mortality syndrome
(PEMS) in turkeys (7). There is no vaccine or specific antiviral
therapy against astrovirus disease.
Astroviruses are small, nonenveloped viruses with a single-
stranded positive-sense RNA [(⫹)RNA] genome (2). The virus
genome has three open reading frames (ORFs), named ORF1a,
ORF1b, and ORF2. ORF1a is translated into nsp1a, a nonstruc-
tural polyprotein precursor that is subsequently processed by viral
and cellular proteases (2). ORF1b encodes nsp1b, the RNA-de-
pendent RNA polymerase (2,8). ORF2 encodes a precursor struc-
tural polyprotein, named VP90 in the human astrovirus (HAstV)
serotype 8 (HAstV-8) strain Yuc8, which is proteolytically pro-
cessed to produce the final capsid viral proteins VP34, VP27, and
VP25 through a precursor polypeptide named VP70. ORF2 is ex-
pressed from a subgenomic RNA that is coterminal with the
genomic RNA (RNAg) at the 3=end (2,9).
Viruses interact with a large number of cellular proteins during
their replication cycle (10). (⫹)RNA viruses take advantage of
host cells by subverting host protein synthesis and remodeling
membranes, co-opting and modulating protein and ribonucleo-
protein complexes, and altering cellular metabolic pathways dur-
ing infection (11). They utilize intracellular membranes to assem-
Received 12 May 2015 Accepted 28 July 2015
Accepted manuscript posted online 5 August 2015
Citation Murillo A, Vera-Estrella R, Barkla BJ, Méndez E, Arias CF. 2015.
Identification of host cell factors associated with astrovirus replication in Caco-2
cells. J Virol 89:10359 –10370. doi:10.1128/JVI.01225-15.
Editor: K. Kirkegaard
Address correspondence to Carlos F. Arias, arias@ibt.unam.mx.
† Deceased.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/JVI.01225-15.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JVI.01225-15
October 2015 Volume 89 Number 20 jvi.asm.org 10359Journal of Virology
... Astrovirus replication complex dependence on host proteins with NTPase/helicase activity. Notably, proteomic analysis of HAstV8-infected Caco2 cells showed enrichment of membrane-only fractions with the cellular RNA helicase DDX23, detected alongside viral RdRp and protease, established components of RCs [35]. Additionally, siRNA-mediated depletion of DDX23 significantly decreased virus replication [35], indicating the host helicase dependence of astrovirus replication. ...
... Notably, proteomic analysis of HAstV8-infected Caco2 cells showed enrichment of membrane-only fractions with the cellular RNA helicase DDX23, detected alongside viral RdRp and protease, established components of RCs [35]. Additionally, siRNA-mediated depletion of DDX23 significantly decreased virus replication [35], indicating the host helicase dependence of astrovirus replication. In a recent transcriptomic analysis of HAstV1-infected Caco2 cells, cellular helicases HELZ2 and DDX58 transcripts were found to be upregulated in infected cells [16], providing more candidates for the host helicases that can be involved in the RNA replication process. ...
... Viruses employ a range of strategies to ensure the correct association of replication-competent virus-host replication machinery. Co-opting the ER for this purpose has been described for numerous RNA viruses including the Flaviviridae, Coronaviridae, Picornaviridae [36] families, and has also been suggested for astroviruses [16,35,37]. We confirm this localization and identify perinuclear ER membranes as the preferential RNA replication site (Fig 3), similar to several other RNA viruses [38][39][40]. ...
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Article
  • Nov 2013
  • NAT REV MOL CELL BIO
Lipid droplets are intracellular organelles that are found in most cells, where they have fundamental roles in metabolism. They function prominently in storing oil-based reserves of metabolic energy and components of membrane lipids. Lipid droplets are the dispersed phase of an oil-in-water emulsion in the aqueous cytosol of cells, and the importance of basic biophysical principles of emulsions for lipid droplet biology is now being appreciated. Because of their unique architecture, with an interface between the dispersed oil phase and the aqueous cytosol, specific mechanisms underlie their formation, growth and shrinkage. Such mechanisms enable cells to use emulsified oil when the demands for metabolic energy or membrane synthesis change. The regulation of the composition of the phospholipid surfactants at the surface of lipid droplets is crucial for lipid droplet homeostasis and protein targeting to their surfaces.
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