Abstract
Most filoviruses cause severe disease in humans. For example, Ebola virus (EBOV) is responsible for the two most extensive outbreaks of filovirus disease to date, with case fatality rates of 66% and 40%, respectively. In contrast, Reston virus (RESTV) is apparently apathogenic in humans, and while transmission of RESTV from domestic pigs to people results in seroconversion, no signs of disease have been reported in such cases. The determinants leading to these differences in pathogenicity are not well understood, but such information is needed in order to better evaluate the risks posed by the repeated spillover of RESTV into the human population and to perform risk assessments for newly emerging filoviruses with unknown pathogenic potential. Interestingly, RESTV and EBOV already show marked differences in their growth in vitro, with RESTV growing slower and reaching lower end titers. In order to understand the basis for this in vitro attenuation of RESTV, we used various life cycle modeling systems mimicking different aspects of the virus life cycle. Our results showed that viral RNA synthesis was markedly slower when using the ribonucleoprotein (RNP) components from RESTV, rather than those for EBOV. In contrast, the kinetics of budding and entry were indistinguishable between these two viruses. These data contribute to our understanding of the molecular basis for filovirus pathogenicity by showing that it is primarily differences in the robustness of RNA synthesis by the viral RNP complex that are responsible for the impaired growth of RESTV in tissue culture.
Highlights
Ebolaviruses comprise a genus in the family of filoviruses
Tetracistronic minigenome assays were performed as previously described [4], with the indicated modifications to assess individual steps of the virus life cycle. 293T cells were transfected with plasmids encoding a codon-optimized T7-polymerase (125 ng), a tetracistronic minigenome (125 ng), and expression plasmids for the Ebola virus (EBOV) ribonucleoprotein complex (RNP) proteins NP (62.5 ng), VP35 (62.5 ng), VP30 (37.5 ng), and L (500 ng). transcription and replication-competent virus-like particles (trVLPs) in the supernatant were harvested at the time points indicated below for the different experiments and used to infect 293T cells pre-transfected with expression plasmids for T-cell immunoglobulin and mucin domain 1 (TIM-1) and EBOV or Reston virus (RESTV) RNP proteins
The efficiency of morphogenesis and budding was compared using EBOV tetracistronic minigenomes encoding either EBOV VP40, RESTV VP40, or no VP40. trVLPs were harvested at time points indicated in the figures and used to infect target cells pre-transfected with expression plasmids for TIM-1 and EBOV RNP proteins, as described above
Summary
Ebolaviruses comprise a genus in the family of filoviruses (order Mononegavirales). They contain a negative-sense, single-strand RNA genome encoding seven structural proteins. They allow a reductionist approach analyzing individual aspects of the viral life cycle and provide a system in which different virus components can be exchanged These systems are based on minigenomes, i.e., miniature versions of the viral genome in which viral genes have been removed and replaced with a reporter gene, while the non-coding terminal regions (called leader and trailer) that carry sequence signals important for recognition by the viral RNP proteins remain unchanged. Expression of such monocistronic minigenomes in mammalian cells together with the RNP proteins results in minigenome replication and transcription (i.e., viral RNA synthesis), and subsequent translation of minigenome-encoded reporter mRNAs leads to reporter activity, reflecting these processes. Microorganisms 2020, 8, 1215 efficiency of the RESTV RNP proteins in mediating viral RNA synthesis, rather than differences in entry or budding, that is responsible for the impaired growth of RESTV in vitro, and contributes to the differences in pathogenicity between these viruses
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