Inhibition of host cell protein synthesis by poliovirus.

The mechanism of poliovirus inhibition of host cell protein synthesis was investigated. In the first study, the involvement of input virion capsid proteins in shutoff was investigated by infecting HeLa cells with poliovirus which had been previously irradiated with ultraviolet light. The loss of the host cell shutoff function followed one-hit inactivation kinetics and possessed a D37 of 306 ergs/mm2. When compared to energy levels required to inactivate other poliovirus functions as well as other RNA and protein functions reported elsewhere, it was determined that the loss of this function was due to damage to the virion RNA and not the input virion proteins. The data confirm the hypothesis that a viral RNA or protein product of the input virion genome is responsible for host cell shutoff and demonstrate that complete inhibition of host cell protein synthesis can be accomplished by one infectious viral genome per cell. In the second study, control of protein synthesis in vitro translation systems prepared from poliovirus-infected cells was examined. Cell-free extracts utilizing endogenous mRNA were prepared from uninfected HeLa cells, early-infected cells, and late-infected cells. The systems were then fractionated into a post-ribosomal supernatant, polysomal pellet, and ribosomal salt wash and the initiation and elongation steps of translation were assayed by incorporation of formyl [35S] methionine and [3H] leucine in vitro. It was found that while the post-ribosomal supernatants from uninfected and infected cells exhibited little preference for either cell or viral mRNA translation, the ribosomal salt washes from infected cells possessed mRNA specificity. Uninfected cell salt wash functioned equally well with either cell or viral mRNA translation. Ribosomal salt washes from early-infected an late-infected cells functioned reasonably well with mRNA translation but had little activity for stimulating initiation of cell mRNA translation. This disparity was evident at all concentrations of infected ribosomal salt wash and mixing of infected an uninfected salt washes also demonstrated an inhibitory activity present in the infected preparation only when tested with cell mRNA but not viral mRNA translation. Ternary complex formation of eIF2, GTP, and f[35S] met-tRNA/f/met was assayed in infected cells and the eIF2 activity was found to be at near normal levels. The results indicate that infected cell ribosomal salt washes have been altered such that they no longer function with cell mRNA translation but retain activity for viral mRNA translation, a property which alone explains many aspects of host cell shutoff and later preferential viral translation. In addition the data indicate that the block in initiation is subsequent to ternary complex formation. In the third study the ribosomal salt washes from uninfected and late-infected cells were fractionated to characterize the mRNA-specific component. Partial (NH4)2SO4 fractionation was used to separate the initiation factors into two groups, a 0-40% (NH4)2SO4 cut or A fraction which contained eIF3 and eIF4B and a 40-70% (NH4)2SO4 cut or B fraction which contains the other activities. Evaluations in the HeLA translation system demonstrated that the mRNA-specific characteristic was confirmed to the A fraction. Examination of the initiation factor requirements of the HeLa system by the use of purified initiation factors from reticulocytes showed that it was dependent only on the addition of eIF3 and eIF2 and had no requirement for the other known factors. Evaluation of the crude ribosomal salt washes and fractions with globin mRNA translation in the heterologous translation system which is dependent upon the entire known factor showed that both infected crude ribosomal salt wash and A fraction were also defective in this system. Separation of the infected cell A fraction on a Sepharose 4B-CL and subsequent evaluation with globin mRNA translation demonstrated that a functional eIF4B activity was present in infected cells but not a functional eIF3. While much of the data would suggest that an altered eIF3 was responsible for the mRNA specific effect, other conflicting data does not rule out the possible involvement of a hitherto, underscribed component which co-purifies eIF3.