Porcine reproductive and respiratory syndrome virus (PRRSV) replicates in differentiated macrophages. In macrophages, heparan sulphate
glycosaminoglycans mediate the initial PRRSV attachment and the receptor
sialoadhesin mediates both PRRSV attachment and internalization into endosomes. Upon a pH drop, PRRSV is uncoated and its genome is released from the endosomes into the cytoplasm, which allows virus replication. However, expression of heparan sulphate and
sialoadhesin in non-susceptible cells only allows virus internalization, but no virus uncoating and
infection, indicating that other factors are involved. In the present study, it is shown that treatment of macrophages with serum (mainly the
alpha-globulin fraction) inhibited PRRSV
infection without affecting attachment and internalization. Because
alpha-globulins contain several
protease inhibitors, macrophages were treated with different
protease inhibitors to investigate the involvement of
proteases in PRRSV uncoating. Treatment of macrophages with broadly active inhibitors of
serine or aspartic
proteases, but not
cysteine- or metallo-
proteases, inhibited PRRSV uncoating and
infection. Further investigation using specific inhibitors indicated that the aspartic
protease cathepsin E is involved during PRRSV uncoating, but did not allow identification of the
serine protease involved. The involvement of
cathepsin E during PRRSV uncoating was confirmed by partial co-localization of internalized PRRSV with
cathepsin E. Furthermore,
cathepsin E expression increased with macrophage cultivation, which was positively correlated with an increased susceptibility to PRRSV
infection. Together, these data show that, in macrophages, both the aspartic
protease cathepsin E and an unidentified
trypsin-like serine protease are involved in uncoating of internalized PRRSV and subsequent
infection.