A Putative Lipid-Associating Motif in the West Nile Virus NS4A Protein Is Required for Efficient Virus Replication

Flavivirus replication is intimately associated with re-organized cellular membranes. These virus-induced changes in membrane architecture form three distinct membranous “organelles” that have specific functions during the flavivirus life cycle. One of these structures is the replication complex in which the flaviviral RNA is replicated to produce progeny genomes. We have previously observed that this process is strictly dependent on cellular cholesterol. In this study we have identified a putative cholesterol recognition/interaction amino acid consensus (CRAC) motif within the West Nile virus strain Kunjin virus (WNVKUN) NS4A protein. Site-directed mutagenesis of this motif within a WNVKUN infectious clone severely attenuated virus replication and the capacity of the mutant viruses to form the replication complex. Replication of the mutant viruses also displayed reduced co-localization with cellular markers recruited to replication sites during wild-type virus replication. In addition, we observed that the mutant viruses were significantly impaired in their ability to remodel cytoplasmic membranes. However, after extensive analysis we are unable to conclusively reveal a role for the CRAC motif in direct cholesterol binding to NS4A, suggesting additional complex lipid-protein and protein-protein interactions. We believe this study highlights the crucial role for this region within NS4A protein in recruitment of cellular and viral proteins to specialized subdomains on membrane platforms to promote efficient virus replication.

[1]  C. Simmons,et al.  The Host Protein Reticulon 3.1A Is Utilized by Flaviviruses to Facilitate Membrane Remodelling. , 2017, Cell reports.

[2]  J. Mackenzie,et al.  Conserved amino acids within the N-terminus of the West Nile virus NS4A protein contribute to virus replication, protein stability and membrane proliferation. , 2015, Virology.

[3]  J. Saiz,et al.  The Composition of West Nile Virus Lipid Envelope Unveils a Role of Sphingolipid Metabolism in Flavivirus Biogenesis , 2014, Journal of Virology.

[4]  C. Rice,et al.  Flavivirus Replication Complex Assembly Revealed by DNAJC14 Functional Mapping , 2012, Journal of Virology.

[5]  Ronald J. Moore,et al.  Dengue Virus Infection Perturbs Lipid Homeostasis in Infected Mosquito Cells , 2012, PLoS pathogens.

[6]  M. Veit,et al.  Intrinsic membrane association of the cytoplasmic tail of influenza virus M2 protein and lateral membrane sorting regulated by cholesterol binding and palmitoylation. , 2011, The Biochemical journal.

[7]  N. Heaton,et al.  Dengue virus-induced autophagy regulates lipid metabolism. , 2010, Cell host & microbe.

[8]  N. Heaton,et al.  Dengue virus nonstructural protein 3 redistributes fatty acid synthase to sites of viral replication and increases cellular fatty acid synthesis , 2010, Proceedings of the National Academy of Sciences.

[9]  Y. Maehara,et al.  Expression profile of lipid metabolism‐associated genes in hepatitis C virus‐infected human liver , 2010, Hepatology research : the official journal of the Japan Society of Hepatology.

[10]  J. Mackenzie,et al.  The Endoplasmic Reticulum Provides the Membrane Platform for Biogenesis of the Flavivirus Replication Complex , 2010, Journal of Virology.

[11]  M. Labow,et al.  Cholesterol biosynthesis modulation regulates dengue viral replication. , 2009, Virology.

[12]  K. Nave,et al.  Cholesterol Regulates the Endoplasmic Reticulum Exit of the Major Membrane Protein P0 Required for Peripheral Myelin Compaction , 2009, The Journal of Neuroscience.

[13]  J. Krijnse-Locker,et al.  Modification of intracellular membrane structures for virus replication , 2008, Nature Reviews Microbiology.

[14]  Annick Thomas,et al.  Hydrophobic substitutions in the first residue of the CRAC segment of the gp41 protein of HIV. , 2008, Biochemistry.

[15]  R. Parton,et al.  Cholesterol manipulation by West Nile virus perturbs the cellular immune response. , 2007, Cell host & microbe.

[16]  Clemens Vonrhein,et al.  Crystal Structure of the RNA Polymerase Domain of the West Nile Virus Non-structural Protein 5* , 2007, Journal of Biological Chemistry.

[17]  J. Mackenzie,et al.  West Nile virus strain Kunjin NS5 polymerase is a phosphoprotein localized at the cytoplasmic site of viral RNA synthesis. , 2007, The Journal of general virology.

[18]  Ralf Bartenschlager,et al.  The Non-structural Protein 4A of Dengue Virus Is an Integral Membrane Protein Inducing Membrane Alterations in a 2K-regulated Manner* , 2007, Journal of Biological Chemistry.

[19]  S. Robbins,et al.  Erlin-1 and erlin-2 are novel members of the prohibitin family of proteins that define lipid-raft-like domains of the ER , 2006, Journal of Cell Science.

[20]  J. Mackenzie,et al.  Regulated Cleavages at the West Nile Virus NS4A-2K-NS4B Junctions Play a Major Role in Rearranging Cytoplasmic Membranes and Golgi Trafficking of the NS4A Protein , 2006, Journal of Virology.

[21]  J. Hancock,et al.  Biogenesis of caveolae: a structural model for caveolin-induced domain formation , 2006, Journal of Cell Science.

[22]  J. Mackenzie.  Wrapping Things up about Virus RNA Replication , 2005, Traffic.

[23]  E. Möncke-Buchner,et al.  The influenza virus ion channel and maturation cofactor M2 is a cholesterol-binding protein , 2005, European Biophysics Journal.

[24]  R. Epand,et al.  Caveolin scaffolding region and cholesterol-rich domains in membranes. , 2005, Journal of molecular biology.

[25]  M. Gale,et al.  Disruption of hepatitis C virus RNA replication through inhibition of host protein geranylgeranylation , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[26]  L. Wodicka,et al.  Genomic analysis of the host response to hepatitis C virus infection , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[27]  J. Mackenzie,et al.  Markers for trans-Golgi Membranes and the Intermediate Compartment Localize to Induced Membranes with Distinct Replication Functions in Flavivirus-Infected Cells , 1999, Journal of Virology.

[28]  J. Mackenzie,et al.  Nascent flavivirus RNA colocalized in situ with double-stranded RNA in stable replication complexes. , 1999, Virology.

[29]  V. Papadopoulos,et al.  Printed in U.S.A. Copyright © 1998 by The Endocrine Society Peripheral-Type Benzodiazepine Receptor Function in Cholesterol Transport. Identification of a Putative Cholesterol Recognition/Interaction Amino Acid Sequence and Consensus Pattern* , 2022 .

[30]  E. G. Westaway,et al.  trans-Complementation of Flavivirus RNA Polymerase Gene NS5 by Using Kunjin Virus Replicon-Expressing BHK Cells , 1998, Journal of Virology.

[31]  J. Mackenzie,et al.  Ultrastructure of Kunjin virus-infected cells: colocalization of NS1 and NS3 with double-stranded RNA, and of NS2B with NS3, in virus-induced membrane structures , 1997, Journal of virology.

[32]  J. Mackenzie,et al.  Proteins C and NS4B of the flavivirus Kunjin translocate independently into the nucleus. , 1997, Virology.

[33]  E. G. Westaway,et al.  Subgenomic replicons of the flavivirus Kunjin: construction and applications , 1997, Journal of virology.

[34]  P. Young,et al.  Immunolocalization of the dengue virus nonstructural glycoprotein NS1 suggests a role in viral RNA replication. , 1996, Virology.

[35]  E. Koonin.  Computer-assisted identification of a putative methyltransferase domain in NS5 protein of flaviviruses and lambda 2 protein of reovirus. , 1993, The Journal of general virology.

[36]  J. H. Strauss,et al.  Nucleotide sequence of yellow fever virus: implications for flavivirus gene expression and evolution. , 1985, Science.

[37]  H. Lei,et al.  The dengue virus-induced autophagy. , 2008 .

[38]  J. Mackenzie,et al.  Replication and gene function in Kunjin virus. , 2002, Current topics in microbiology and immunology.

[39]  V. Papadopoulos,et al.  Peripheral-Type Benzodiazepine Receptor , 1999 .

[40]  P. Young,et al.  Improved membrane preservation of flavivirus-infected cells with cryosectioning. , 1996, Journal of virological methods.

[41]  E. Berger,et al.  Double immunofluorescent staining of alpha 2,6 sialyltransferase and beta 1,4 galactosyltransferase in monensin-treated cells: evidence for different Golgi compartments? , 1993, Journal of cellular biochemistry.

[42]  David J. Brakenridge,et al.  On Influenza , 1890, Transactions. Medico-Chirurgical Society of Edinburgh.