| Summary: | 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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