PhD

dissertationThe possible role of the cap-binding protein (CBP) in the inhibition of host cell protein synthesis observed following poliovirus infection was examined by comparing the CBP from uninfected and infected HeLa cells. A 26K Mr CBP was detected by chemically crosslinking it to radio-labeled cap structures on mRNA. From both uninfected and infected cells, the CBP was found in the post-ribosomal supernate and the 0-40% ammonium sulfate fractional precipitate of ribosomal salt wash (RSW). The 0-40% ammonium sulfate precipitate from uninfected cells was able to restore the ability of infected cell extracts to translate capped mRNAs. The analogous fraction from infected cells lacked any restoring activity. Approximately half of the 26K Mr CBP in the RSW from uninfected cells sedimented as a large complex, together with the known initiation factor eIF-3. The remaining CBP sedimented more slowly. None of the CBP from infected cells cosedimented with infected cell eIF-3, suggesting that an eIF-3-CBP complex has been disrupted following poliovirus infection. Attempts were made to reconstitute a complex from mixtures of infected cell CBP and uninfected cell eIF-3 and vice versa. Both infected and uninfected cell components appeared to reassociate to the same degree. It was therefore not possible to attribute the disruption of the eIF-3-CBP complex in infected cells to an alteration in either eIF-3 or the CBP. Restoring activity did not cosediment precisely with either the eIF-3-CBP complex or with the peak of slowly sedimenting CBP. The conclusion is that a change in the properties of the 26K Mr CBP occurs following poliovirus infection, reflected by a disruption in the normal eIF-3-CBP association. Attempts to assign this change to either the CBP or eIF-3 were inconclusive. Restoring activity did not correlate solely with the presence of the 26K Mr CBP, indicating that other components are necessary for restoring activity. The change detected in the CBP or the CBP-eIF-3 complex may be related to inhibition of host cell translation following poliovirus infection but involvement of other factors in that inhibition cannot be ruled out