Mouse hepatitis virus type 2 enters cells through a clathrin-mediated endocytic pathway independent of Eps15.
It has recently been shown that cell entry of mouse hepatitis virus type 2 (MHV-2) is mediated through endocytosis (Z. Qiu et al., J. Virol. 80:5768-5776, 2006). However, the molecular mechanism underlying MHV-2 entry is not known. Here we employed multiple chemical and molecular approaches to determine the molecular pathways for MHV-2 entry. Our results showed that MHV-2 gene expression and infectivity were significantly inhibited when cells were treated with chemical and physiologic blockers of the clathrin-mediated pathway, such as chlorpromazine and hypertonic sucrose medium. Furthermore, viral gene expression was significantly inhibited when cells were transfected with a small interfering RNA specific to the clathrin heavy chain. However, these treatments did not affect the infectivity and gene expression of MHV-A59, demonstrating the specificity of the inhibitions. In addition, overexpression of a dominant-negative mutant of caveolin 1 did not have any effect on MHV-2 infection, while it significantly blocked the caveolin-dependent uptake of cholera toxin subunit B. These results demonstrate that MHV-2 utilizes the clathrin- but not caveolin-mediated endocytic pathway for entry. Interestingly, when the cells transiently overexpressed a dominant-negative form (DIII) of Eps15, which is thought to be an essential component of the clathrin pathway, viral gene expression and infectivity were unaffected, although DIII expression blocked transferrin uptake and vesicular stomatitis virus infection, which are dependent on clathrin-mediated endocytosis. Thus, MHV-2 entry is mediated through clathrin-dependent but Eps15-independent endocytosis.
Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205-7199, USA.
Yinghui
Y
Pu
Xuming
X
Zhang
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D002772:Cholera Toxin / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism*; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D005799:Genes, Dominant; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D006517:Murine hepatitis virus / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; D002966:Clathrin; C082729:Eps15 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D002772:Cholera Toxin