The penetration of various viruses into host cells is accomplished by hijacking the host endocytosis machinery. In the case of severe acute respiratory syndrome coronavirus (SARS-CoV) infection, viral entry is reported to require a low pH in intracytoplasmic vesicles; however, little is known about how SARS-CoV invades such compartments. Here we demonstrate that SARS-CoV mainly utilizes the clathrin-mediated endocytosis pathway for its entry to target cells by using infectious SARS-CoV, as well as a SARS-CoV pseudovirus packaged in the SARS-CoV envelope. The SARS-CoV entered caveolin-1-negative HepG2 cells, and the entry was significantly inhibited by treatment with chlorpromazine, an inhibitor for clathrin-dependent endocytosis, and by small interfering RNAmediated gene silencing for the clathrin heavy chain. Furthermore, the SARS-CoV entered COS7 cells transfected with the mutant of ACE2 with the cytoplasmic tail deleted, SARS-CoV receptor, as well as the wild-type ACE2, and their entries were significantly inhibited by treatment with chlorpromazine. In addition, ACE2 translocated into EEA1-positive early endosomes immediately after the virus attachment to ACE2. These results suggest that when SARS-CoV binds ACE2 it is internalized and penetrates early endosomes in a clathrin-dependent manner and that the cytoplasmic tail of ACE2 is not required for the penetration of SARS-CoV.
These results indicate that PB1 suppresses HCV RNA synthesis, possibly as a HCV RNA polymerase inhibitor. Our results might contribute towards the development of more effective inhibitors for HCV infection from natural plants.
Suppression subtractive hybridization was carried out by using cDNAs of peripheral white blood cells (PWBCs) of banded dogfish (Triakis scyllia) after phorbol 12-myristate 13-acetate (PMA) stimulation. The Trsc-SCYA107, MIP3alpha1 and MIP3alpha2 clones contained an open reading frame encoding 97, 99 and 97 amino acids, respectively. Comparison of the deduced amino acids showed that the banded dogfish MIP3alpha1 and MIP3alpha2 sequences shared 42.3% and 40.0% identity with human SCYA20, respectively, while the Trsc-SCYA107 sequence shared 50.6, 44.2 and 42.0% identity with the catshark (Scyliorhinus canicula) Scca-SCYA107, rainbow trout (Oncorhynchus mykiss) CK4A and CK4B, respectively. The genomic sequences of banded dogfish Trsc-SCYA107, MIP3alpha1 and MIP3alpha2 contain four exons and three introns, and MIP3alpha1 and MIP3alpha2 shared the same intron/exon organization with that of human. The MIP3alpha1 and MIP3alpha2 genes of lipopolysaccharide (LPS)-unstimulated banded dogfish were expressed in gill, kidney and liver, while Trsc-SCYA107 mRNA was detected in various tissues except for brain. However, the constitutive expression of MIP3alpha2 gene was much lower than the Trsc-SCYA107 and MIP3alpha1 genes. RT-PCR analysis of the Trsc-SCYA107 expression in tissues of LPS-stimulated fish showed enhanced expression at 24 h poststimulation in the gill, heart, leydig, spleen and testes, while the expression of MIP3alpha1 and MIP3alpha2 was not influenced by LPS-stimulation in vivo. Furthermore, a relative increase in the expression of the Trsc-SCYA107 and MIP3alpha2 genes in PWBCs was observed at 1-12 h poststimulation with PMA and LPS, with maximal expression observed at 3 h, while MIP3alpha1 expression was observed at 3-12 h poststimulation only with PMA.
The aim of the present study was to determine whether bovine coronavirus (BCV) has the ability to initiate infection in a human colon carcinoma cell line, Caco‐2, that has been established to spontaneously differentiate after confluence. When Caco‐2 cells were infected with BCV, a titer of 5.5 × 106 plaque‐forming units (p.f.u.)/mL was found in the culture supernatant at 5 days postinfection. Two clones, Caco‐2/CA1 and Caco‐2/CA2, were then isolated by monitoring alkaline phosphatase (ALP) and cell proliferation activities. The ALP activity level of CA1 cells was significantly higher than that of CA2 cells, while the level of cell proliferation activity of CA1 was significantly lower than that of CA2. When CA1 and CA2 cells were infected with BCV at confluence, virus hemagglutination (HA) was detected in the culture supernatant at 5 days postinfection for CA1 cells and at 8 days postinfection for CA2 cells. Thus, BCV propagation was substantially delayed in CA2 cells, suggesting that a cellular factor(s) that appears at the differentiation stage may control BCV propagation. BCV‐susceptible CA1 and CA2 cells showing different levels of ALP activity would be useful for further experiments to elucidate the mechanism of BCV propagation.
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