The species-specific effects of guinea pig adaptive mutations in Marburg virus VP40 and L on the protein’s functions and viral fitness

Marburg virus (MARV) is a highly pathogenic virus that causes severe, often lethal diseases specifically in humans and non-human primates. In rodents MARV is non-pathogenic. However, sequential passaging of MARV in rodents results in selection of a rodent-lethal virus. Lofts et al. established a gui...

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Bibliographic Details
Main Author: Köhler, Alexander
Contributors: Becker, Stephan (Prof. Dr.) (Thesis advisor)
Format: Dissertation
Language:English
Published: Philipps-Universität Marburg 2017
Medizin
Subjects:
L
Online Access:PDF Full Text
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Summary:Marburg virus (MARV) is a highly pathogenic virus that causes severe, often lethal diseases specifically in humans and non-human primates. In rodents MARV is non-pathogenic. However, sequential passaging of MARV in rodents results in selection of a rodent-lethal virus. Lofts et al. established a guinea pig-lethal MARV containing only four non-silent amino acid changes in viral genome (Lofts et al., 2007). One amino acid substitution (D184N) was detected in the viral matrix protein VP40 and three amino acid substitutions were detected in the RNA-dependent RNA polymerase L (S741C, D758A and A759D). We analyzed the effects of the guinea pig-adaptive mutations on the functions of VP40 and L in a comparative study including human and guinea pig cells. Functional analyses were performed with ectopically expressed VP40D184N and L mutants by using different assays. The influence of the D184N mutation in VP40 on the replicative capacities of MARV in guinea pig cells was analyzed by infection of cells with recombinant MARV containing the D184N mutation in VP40. The first part of the study demonstrated that a recombinant rMARV containing only the D184N amino acid substitution in VP40 displayed a higher level of viral fitness specifically in guinea pig, but not in human cells. The mutant virus showed higher replicative capacities, enlarged inclusion bodies and enhanced infectivity only in guinea pig cells. Detailed comparative analysis of VP40 functions, in human and guinea pig cells, indicated that the membrane binding capabilities and the interferon antagonistic function were not altered by the D184N amino acid substitution. However, presence of the D184N mutation in VP40 enhanced the production of VP40-induced virus-like particles (VLPs) specifically in guinea pig cells. In addition, the amount of NP in infectious virus-like particles (iVLPs) and virus preparations was enhanced in presence of the D184N mutation in VP40 specifically in guinea pig cells. These data might partially explain the higher infectivity of VP40D184N containing iVLPs compared to wildtype VP40 containing iVLPs. Most importantly, the inhibitory capacity of VP40 on replication and transcription was species-specifically lowered by the D184N mutation in VP40, allowing significantly higher levels of replication and transcription in guinea pig cells. The second part of the study focused on the importance of the mutations in the L protein observed in the guinea pig-lethal MARV. Interestingly, only the S714C substitution increased replication in both species while the other mutations, D758A and A759D severely impaired the polymerase function. All L mutant proteins displayed proper expression and were able to localize into inclusion bodies which represent the sites of viral transcription/replication. Finally, co-expression of plasmids encoding L with the S741C amino acid substitution and VP40D184N in guinea pig cells resulted in eight fold higher levels of replication and transcription in comparison to human cells. This suggested that the D184N mutation in VP40 and the S741C mutation in L together significantly improve replication of MARV in guinea pig cells. Altogether these data suggests that the D184N substitution in VP40 and the S714C substitution in L can contribute to the increased pathogenicity of guinea pig-lethal MARV.
DOI:https://doi.org/10.17192/z2017.0337