Bovine ephemeral fever virus uses a clathrin-mediated and dynamin 2-dependent endocytosis pathway that requires Rab5 and Rab7 as well as microtubules.

The specific cell pathways involved in bovine ephemeral fever virus (BEFV) cell entry have not been determined. In this work, colocalization of the M protein of BEFV with clathrin or dynamin 2 was observed under a fluorescence microscope. To better understand BEFV entry, we carried out internalization studies with a fluorescently labeled BEFV by using a lipophilic dye, 3,30-dilinoleyloxacarbocyanine perchlorate (DiO), further suggesting that BEFV uses a clathrin-mediated endocytosis pathway. Our results suggest that clathrin-mediated and dynamin 2-dependent endocytosis is an important avenue of BEFV entry. Suppression of Rab5 or Rab7a through the use of a Rab5 dominant negative mutant and Rab7a short hairpin RNA (shRNA) demonstrated that BEFV requires both early and late endosomes for endocytosis and subsequent infection in MDBK and Vero cells. Treatment of BEFV-infected cells with nocodazole significantly decreased the M protein synthesis and viral yield, indicating that microtubules play an important role in BEFV productive infection, likely by mediating trafficking of BEFV-containing endosomes. Furthermore, BEFV infection was strongly blocked by different inhibitors of endosomal acidification, suggesting that virus enters host cells by clathrin-mediated and dynamin 2-dependent endocytosis in a pH-dependent manner.

Intracellular cleavage of sigmaA protein of avian reovirus.

By Western blot analyzes of expression of avian reovirus proteins, one unknown fragment was detected by an anti-sigmaA monoclonal antibody in virus-infected cells lysate. It was interesting to note that RNA interference against sigmaA resulted in the suppression of the unknown fragment. Using various lengths of sigmaA constructs conjugated with different tags, we present evidences to demonstrate that the fragment comes from the cleavage of sigmaA and is the larger carboxyl-terminus, termed sigmaAC. Cleavage of sigmaA simultaneously produces a smaller amino-terminus, named sigmaAN. sigmaAC could be seen early in viral infection and accumulated with time and dose of infection, indicating that the derived products are not just transient intermediates of protein degradation. The same type of cleaved products were also observed in different genotypes and serotypes of ARV as well as in different cell lines, suggesting that this intracellular modification of sigmaA is common to all ARVs. Similar localization of sigmaAC in both cytosol and nucleus with sigmaA suggested that further modification of sigmaA may be important for its function. Our evidences suggest that besides the outer capsid protein muB, sigmaA may also have post-translational cleavage which has never been reported before even in related mammalian reovirus.