Hijacking to use and abuse: the role of host factors during West Nile and Dengue virus replication and membrane mophogenesis
AuthorAktepe, Turgut Esad
MetadataShow full item record
Document TypePhD thesis
Access StatusOpen Access
© 2017 Dr. Turgut Esad Aktepe
Flaviviruses such as West Nile virus and Dengue virus are enveloped, +ssRNA virus from the Flaviviridae family causing infection in North America, Oceania, Africa, Europe, the Middle East and West and central Asia. Due to the limited number of viral proteins encoded by +ssRNA viruses, host factors, such as lipids and proteins, are recruited and required for sufficient viral replication. Host factors are involved in almost every step of +ssRNA virus replication by interacting with viral proteins via a specific motif. It is well established that these viruses manipulate ER homeostasis and distribution to facilitate efficient replication. In this thesis we have investigated the role of the host lipid and protein ceramide and Reticulon 3, respective, alongside the SH3 protein-protein interaction domain. It is well established that +ssRNA viruses manipulate cellular lipid homeostasis and distribution to facilitate efficient replication. Cellular lipid ceramide is redistributed to the West Nile virus strain Kunjin virus (WNVKUN) replication complex (RC) but not to the Dengue virus serotype 2 strain New Guinea C (DENVNGC) RC. Ceramide depletion by prolonged chemical inhibition of serine palmitoyltransferase (SPT) activity with myriocin or inhibition of ceramide synthase with Fumonisin B1 had a significant deleterious effect on WNVKUN replication but enhanced DENVNGC replication. These observations suggest that ceramide production via the de novo and salvage pathway is a requirement for WNVKUN replication but inhibitory for DENVNGC replication. Thus, although these two viruses are from the same genus, they have a differential ceramide requirement for replication. Furthermore, previous studies have shown that a ER membrane-shaping protein Reticulon 3A (RTN3A) plays a crucial role in +ssRNA virus replication by inducing positive-membrane curvature [1, 2]. In this study, we have observed that RTN3A is redistributed and recruited to the RC of WNVKUN and DENVNGC. Subsequent analysis revealed that RTN3A interacts with the viral protein NS4A, known to be responsible for host membrane rearrangements in WNVKUN and DENVNGC infected cells. Via deletion mutagenesis we mapped the interaction between NS4A and RTN3A to the first transmembrane region as removal of the first transmembrane region reduced NS4A – RTN3A interaction by FRET and co-IP experiments. Furthermore, siRNA-mediated knockdown of RTN3A attenuated WNVKUN and DENVNGC viral replication, altered virally induced membrane architecture and severely affected the stability and expression of NS4A. Thus we have demonstrated the importance of RTN3A during Flavivirus replication by interacting with, and stabilizing the NS4A protein. Lastly, we have established that modular interaction domains are crucial during WNV replication. These are generally conserved regions in proteins that are generally 30-200 amino acids. These domains specialize in mediating interactions of proteins with one another, lipids and nucleic acids. Src Homology 3 (SH3) domains are one of the best characterized and most studied modular interaction domains requiring a conserved PxxP binding motif. Previous studies have established that SH3 domains are significant for the replication of numerous viruses. Analysis of the WNV genome allowed us to determine that the viral genome contains a variety of putative SH3 motif. By alignment analysis of different Flavivirus genomes, we have determined that two possible SH3 motifs (within NS1 and NS5) are conserved throughout the Flaviviruses and may play a role in viral replication. Site-directed point mutations and insertion mutations within these motifs suggest that specific mutations have either attenuated or completely abolished WNV replication. This study will determine the interaction between host and viral proteins through these domains and the role of these host proteins during WNV replication.
KeywordsFlavivirus; West Nile virus; Dengue virus; replication; host interaction; membrane; ceramide; Reticulon; SH3 domain
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