Breakdown of Polysomes

Sellinger, O., Azcurra, J. (1970). The breakdown of polysomes and the stimulation of protein synthesis in cerebral mechanisms of defense against seizures. In A. Lajtha (Ed.), Protein metabolism of the nervous system (pp. 519-532). New York Plenum. 

To eliminate exonucleolytic attack as a cause of mRNA degradation we followed the fate of mRNA stably attached to ribosomes. If an inhibitor of polypeptide chain elongation is added to the incubations to prevent ribosome movement along mRNA, breakdown of polysomes can only result from endonucleolytic cleavage of the mRNA strand connecting ribosomes. Figure 2 shows... 

The early breakdown of polysomes that followed infection with HSV-2 was not prevented by cycloheximide (Fenwick and Walker, 1978) indicating that it did not depend on newly made protein, nor on continuing translation with its associated termination of polypeptide chains and detachment of ribosomes from the messenger. 

Silverstein and Engelhardt (1979) measured the protein-synthesizing activity of polysomes and rates of chain elongation of polypeptides, and found that the translation rate did not alter during the time that the polysomes were declining. They also concluded that a substantial proportion of the larger polysomes that formed after the early breakdown were inactive in protein synthesis and suggested a second block which resulted in inhibition of translation but not breakdown of polysomes and affected specifically cellular and early viral protein synthesis. 

Host protein synthesis was also suppressed in cells infected in the presence of canavanine, an analogue of arginine (Honess and Roizman, 1975). In these circumstances, a-proteins were made but were apparently defective because p-protein synthesis was severely restricted. This implied either that the host-suppressing activity of a-or p-proteins was not eliminated by substituting canavanine for arginine or that the observed suppression, like the breakdown of polysomes, did not depend on proteins synthesized after infection, as if some component of the virus inoculum itself was also involved. 

Figure 2. Polysome breakdown in extracts of interferon-treated and control cells incubated with dsEHA. The cells were treated 1 7 hr with 100 units/ml of interferon. Both control and interferon-treated cells were incubated 1 hr with 1 iig/ml of cycloheximide to increase polysome size according to Fan and Penman (l ) Extracts prepared from these cells were incubated 1 hr at 30 U with the components described in Figure 1, 1 mM ATP, 0.1 mM spars onQTCin and no added dsEHA in A and C or 10 jig/ml of poly(I) poly(C) in B and B. The samples were centrifuged 90 min at 40,000 rpm on 15-40% sucrose gradients and the A260 analyzed with a recording spectrophotometer.

Incubation of an HeLa cell extract with 2 5 oligo(A) resulted in extensive polysome breakdown (Figure 6), whereas no significant changes in polysome pattern could be detected in a control incubation. 

Fig. 1. Schematic representation of the effect of amino acid supply to the liver on protein synthesis by liver polysomes, and on RNA degradation rate and synthesis of purine nucleotides, The diagram indicates interrelationships between those metabolic events which result in reduced RNA breakdown and increased purine biosynthesis when amino acid supply to the liver is increased (Clifford et al., 1972).

Ihara et al. (1983), working with PRV, found that it differs from HSV-1 in that only one immediate-early polypeptide, with an electrophoretic mobility similar to that of ICP4 of HSV, was made after reversing a cycloheximide block. Cellular protein synthesis was inhibited soon after reversal. is a mutant of PRV with a defect in the immediate-early protein which (like tsK of HSV-1 see Section 7.6) is unable to progress from immediate-early to early protein synthesis at the nonpermissive temperature (41°C). This mutant caused significant shut-off of cellular protein and DNA synthesis at 41°C but less than wild-type virus. It was concluded that the immediate-early protein is involved in the shut-off, but either the mutant form is partially defective in this function or some later viral proteins also contribute to the shut-off by wild-type virus. The case for the host-suppressing function of the immediate-early protein would be strengthened if it were confirmed that shut-off occurred after reversal of cycloheximide in the presence of actinomycin, as was reported for polysome breakdown (Ben-Porat et al., 1971). 

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