Phenotypic Consequences In vivo and In vitro of Rearranging the P Gene of RABV HEP-Flury

Abstract

Phosphoprotein (P) of the Rabies virus (RABV) is critically required for viral replication and pathogenicity. Here we manipulated infectious cDNA clones of the RABV HEP-Flury to translocate the P gene from its wild-type position 2 to 1, 3, or 4 in gene order, using an approach which left the viral nucleotide sequence unaltered. The recovered viruses were evaluated for the levels of gene expression, growth kinetics in cell culture, lethality in suckling mice and protection of mice. The results showed that viral replication was affected by the absolute value of N protein which was regulated by P protein. Viral lethality in suckling mice was consistent with the ratio of P mRNA in one complete transcription. The protection of mice induced by viruses was related to the antibody titer 5 weeks post-infection which might be regulated by G protein. However, the ability to induce cell apoptosis and viral spread were not only related to the viral replication but also to the ratio of related gene which affected by the gene position. These findings might not only improve the understanding of phenotype of RABV and P gene rearrangement, but also help rabies vaccine candidate construction.

Phosphoprotein Gene of Wild-Type Rabies Virus Plays a Role in Limiting Viral Pathogenicity and Lowering the Enhancement of BBB Permeability

“…Our results demonstrated that rHEP-SH-P showed significant lower pathogenicity in mice compared to GD-SH-01, which was consistent with previous studies that show that G protein mainly contributes to the pathogenicity of RABV, rather than P protein (Dietzschold et al, 1983;Ito et al, 2001;Pulmanausahakul et al, 2008), whereas the P gene facilitated viral pathogenesis, as previously reported that P gene-deficient RABV strain was apathogenic to host (Morimoto et al, 2005). Our previous study demonstrated that the lower expression of P gene resulted in weaker pathogenic outcomes (Mei et al, 2017). In the present study, despite that the G gene expression of HEP-Flury and rHEP-SH-P was not significantly different, rHEP-SH-P bearing suppressed P gene was less pathogenic than HEP-Flury.…”

“…The RNP is a key role in the viral assembly and replication; RABV P protein serves as a component of RNP affecting viral replication indirectly. Actually, the formation of RNP was influenced by the optimal ratio of N:P:L (Pattnaik and Wertz, 1990;Mei et al, 2017), which was reflected by the suppressed N and L expression in the current study and suggested that the low level of P gene expression limited the viral replication by obstructing the formation of RNP. A previous study suggested the poor proliferative capacity of rHEP-SH-P in vitro (Tian et al, 2017b); in the current study, we observed relatively lower viral growth of rHEP-SH-P in vivo, which was due to the suppressed P gene.…”

“…Phosphoprotein (P) gene of RABV plays a major role in the neuro-invasiveness by mediating infection of peripheral nerves (Yamaoka et al, 2013) and serves as an interferon antagonist resulting in immune escape (Ito et al, 2010). In our previous study, we rearranged the P gene of RABV and found that low level of P gene expression suppressed N gene and result in pathogenicity attenuation (Mei et al, 2017), which suggested that the viral replication and pathogenicity were consistent with P gene expression indirectly. However, the role of P protein in the course of RABV invasion in vivo was unclear so far.…”

Phosphoprotein Gene Contributes to the Enhanced Apoptosis Induced by Wild-Type Rabies Virus GD-SH-01 In Vitro

“…All virus stocks were prepared in NA cells and stored at -80°C. Viruses were titrated in NA cells using fluorescein isothiocyanate-conjugated antibodies directed against N of RABV (FujiRebio Diagnostic Inc., PA, United States), as described previously ( Mei et al, 2017 ).…”

The Deoptimization of Rabies Virus Matrix Protein Impacts Viral Transcription and Replication

“…Virus spread assay was conducted using NA cells on 6-well plate as described previously [19,20]. Briefly, NA cells were infected with RABV at an MOI of 0.01, and incubated for 2 h at 37 • C. The inoculum was removed and cells were washed with PBS.…”