• 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2020-03
  • 2020-07
  • 2020-08
  • 2021-03
  • br Material and methods br Results br Discussion


    Material and methods
    Discussion Our previous deep sequence analysis in MA104 cells infected with wild-type RV ZTR-68 revealed about 40 small RNAs that showed differential expression. After comparison with multiple databases, RT-PCR verification, filtering by differences in reads, and expression analysis by qRT-PCR, we selected the small RNA named RV-vsRNA1755 for further functional analysis. In our present study, we performed BLAST analysis of the RV-vsRNA1755 sequence in NCBI, and confirmed that it was derived from the rotavirus NSP4 gene, and not from host cells. NSP4, encoded by gene segment 10, is a 175 amino AT13387 multifunctional protein and essential for rotavirus replication, transcription, and morphogenesis. NSP4 is synthesized as an ER transmembrane glycoprotein and consists of three hydrophobic domains (H1−H3) and extended cytoplasmic domain containing a coiled-coil region and a flexible C-terminus. The coiled-coil domain of NSP4 has some multifunctional binding sites including integrin I domains, caveolin and VP4. Our results reveal that RV-vsRNA 1755 expression is another regulation feature of rotavirus NSP4 coiled-coil region. Further qRT-PCR analyses revealed that RV-vsRNA1755 expression was upregulated following infection with different RV genotypes, including G1, G2, G3 and G4. Moreover, RV-vsRNA1755 expression was upregulated following RV infection in different host cells (MA104, HT-29, and Caco2 cells), and the RV-vsRNA1755 expression level increased with increased duration virus infection and with increased MOI. These results indicated that RV-vsRNA1755 is a small RNA molecule encoded by multiple genotypes of RV in infected host cells, which may be a common regulatory mechanism in RV infection. To further characterize RV-vsRNA1755, we used Northern blot hybridization to determine its expression levels in control and RV-infected MA104 cells. At 24hpi, we detected a 23-nt small RNA (3′ probe) that matched the size of RV-vsRNA1755 determined by deep sequencing. Our present results showed significantly lower RV-vsRNA1755 levels in Dicer and AGO2-silenced cells, suggesting that Dicer and AGO2 may play an important role in RV-vsRNA1755 biogenesis. Immunoprecipitation (IP) analysis showed that the mature vsRNA1755 sequence was enriched in Dicer and AGO2 IP but not AGO1. Dicer and AGO2-mediated biogenesis of the mature vsRNA1755. These results support the hypothesis that RV-vsRNA1755 is a viral small RNA encoded by RV. Our computational prediction and dual-luciferase reporter assay results revealed that RV-vsRNA1755 targets the host cell insulin growth factor 1 receptor (IGF1R), which is involved in the PI3K/Akt pathway. Autophagy can be induced by RV infection in host cells [37], [38], and here we showed that RV-vsRNA1755 was related to this induction. We found that using RV-vsRNA1755 mimics in MA104 cells led to activating of the autophagy process, similar to the results of RV infection. Moreover, our analysis of the expression levels of IGF1R and key genes of the PI3K/Akt pathway revealed that RV-vsRNA1755 mimics regulated the key proteins in the autophagy pathway. The RV yield was increased in both MA104 cells transfected with RV-vsRNA1755 mimics and MA104 cells transfected with IGF1R siRNA. In the current study, we characterized a new small RNA encoded by RV, and provided insight into a complementary mechanism, other than calcium/calmodulin - dependent kinase kinase-β (CaMKK-β) signaling, in the process of RV-initiated autophagy. To summarize our present understanding of the mechanisms, in the early stage of viral infection, RV encodes RV-vsRNA1755, which activates autophagy by inhibiting activation of the PI3K/Akt/mTOR pathway. RV further utilizes the autophagic membrane transport pathway to promote self-replication. Protein VP7 is transported from the endoplasmic reticulum to the viroplasm, and the DLP virus particles are assembled with VP7 to form infectious VLP virus particles (Fig. 7). The biological characteristics and the biological functions in autophagy initiation of RV-vsRNA1755 during RV infection was investigated. However, RV releases the inhibition of PI3K/Akt pathway in the later stage of infection. Other function and regulatory mechanisms of RV-vsRNA1755 may exist during RV infection and need to be uncovered, in the future. A secreted form of NSP4, which contains the integrin I domain binding site, is involved in diarrhea induction [39]. NSP4 modulates membrane trafficking by orchestrating the activation of autophagy to benefit rotavirus replication [40]. Perhaps one day, RV-vsRNA1755 will become bio-marker and treatment target of Rotaviruses-induced viral diarrhea.