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Cell signalling in response to Coxsackievirus B3 infection White, Michaela


Coxsackievirus B3 is a member of the Picornaviridae, genus Enterovirus. The virus is non-enveloped with a positive sense RNA genome of approximately 7.4kb. CVB3 is the most common etiologic agent associated with acute viral myocarditis and chronic dilated cardiomyopathy, and this importance of CVB3 as a human pathogen makes its mode of infection an area of intense interest for study in the development of antivirals. Recent studies have demonstrated an important role for the Src family kinase (SFK) Lck in CVB3 infection in mice and Jurkat T cells. In Lck-/- mice CVB3 infection is severely attenuated and replication in JCaM cells, a Jurkat cell line that lacks Lck, occurs at levels several log-fold lower than in Lck-expressing Jurkat cells. This study aimed to identify whether HeLa and Mouse Embryonic Fibroblasts (MEF) cells, which express little or no Lck, also require SFK for efficient CVB3 replication and to elucidate which of the SFK are required. HeLa cells expressing dominant negative (DN) or wild-type (wt) Fyn or Src were infected with CVB3 and viral yields were measured. The results found that CVB3 replication was not inhibited in the presence of DN Fyn or Src relative to wt cDNA. Treatment of HeLa and MEF cells with the SFK inhibitor PP2 also did not inhibit CVB3 infection, however, replication was inhibited in Jurkat cells by PP2, as previously shown. To elucidate whether SFK were involved in infection of MEF cells, CVB3 replication was measured in knockout MEF cell lines with triple mutations in Src, Yes and Fyn (SYF). Viral titers measured at 0-24 hours post infection in MEF and SYF cells displayed no reduction in titer in the knockout cell lines, indicating that the SFK Fyn, Src and Yes are dispensable during CVB3 infection in these cell lines. The results have demonstrated that SFK are not required in HeLa or MEF cells during CVB3 infection. This study also sought to establish whether PI3K was required for CVB3 infection. Treatment of HeLa cells with PI3K inhibitors wortmannin or LY294002 did not cause any decrease in viral titer, indicating that PI3K function is not required for CVB3 infection. Treatment of HeLa cells with cytochalasin D, or with RhoGTPase inhibitor Clostridium difficile toxin A had no effect on CVB3 replication. These results show that the actin network plays no function in CVB3 replication. Our conclusions are that CVB3 uptake into HeLa and MEF cells differs from the pathways shown for either Adenovirus (which like CVB3 also uses CAR as its primary receptor) or echo virus (which utilizes DAF, the CVB3 secondary receptor), where actin rearrangements are required.

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