Host Poly Research Articles

Replication of alfalfa mosaic virus RNA: Evidence for a soluble replicase in healthy and infected tobacco leaves

Soluble RNA-dependent RNA polymerases from healthy and alfalfa mosaic virus (AMV)-infected leaves were purified more than 400-fold from the 100,000 g supernatant of leaf homogenates, using ammonium sulfate precipitation, Sephadex G-100 filtration, and chromatography on DEAE Sephadex A25 and P11-phosphocellulose. The DEAE-chromatography step eliminated host poly(U)polymerase-like activity, cellular RNases, and endogenous RNA, yielding a typical RNA-dependent RNA polymerase (“DEAE-enzyme”) which was dependent on exogenous RNA. On nondenaturing 4–30% gradient slab gels, the activity of the enzyme was recovered in two peaks containing proteins of 340K and 160K. RNA products were synthesized in the presence of the “DEAE-enzyme” from healthy and infected leaves: When an excess of AMV RNA was introduced into the reaction mixture, only synthesis of minus strand RNA was detected. However, when limiting amounts of AMV RNA were used as template some small plus strands were detected, suggesting that synthesized minus strands may in turn serve as template. In both cases, the products were partially double stranded, and of very heterogenous size. Their size depended on the length of the RNA template which in turn varied according to the degree of RNase contaimination of the enzyme fractions. These results are the first demonstration that both healthy and AMV-infected tobacco leaves contain a soluble replicase, although its possible in vivo role in viral replication is yet to be demonstrated.

Poly (A) polymerase activity in L cells following encephalomyocarditis virus infection.

Poly (A) polymerase activity has been measured in crude cytoplasmic extracts of mouse L cells infected with encephalomyocarditis (EMC) virus. After infection there is first a decrease in enzyme activity followed by an increase which itself precedes detectable virus RNA and protein synthesis. The activity of the enzyme then declines before the release of mature virions and cell death take place. The early inhibition of poly (A) polymerase activity is correlated with the virus-induced shut-off of cellular protein synthesis but it is not due to inhibition of the synthesis of cellular enzyme and occurs in the absence of virus replication. The poly (A) polymerase is not synthesized after infection and modification of its activity can be reversed late in the virus cycle. These results indicate that host poly (A) polymerase activity can be regulated by the virus and further show that there is a correlation between the modification of poly (A) polymerase activity and the biosynthesis of poly (A).

Localization of host poly(A)+ mRNA in the ribosome profile of mengovirus-infected Ehrlich ascites tumor cells

The distribution of poly(A)+ mRNA among polysomes, monosomes, and ribosome-free supernatant fractions after mengovirus infection of Ehrlich ascites tumor (EAT) cells was investigated employing sucrose gradient centrifugation of their corresponding postnuclear supernatants. Poly(A)+ mRNA was isolated from sucrose gradient fractions and quantitated in a cell-free protein synthesizing system from uninfected EAT cells. It was also localized by annealing [3H]-poly(U) to the poly(A)-tracts of mRNA present in the sucrose gradient fractions. Both experiments revealed a gradual shift of host poly(A)+ mRNA from large to small polysomes and monosomes, respectively, with the time postinfection. The greatest part of host template RNA appears to remain ribosome-bound and only a fraction seems to be detached from the ribosomes in the course of mengovirus infection. At the end of the infectious cycle, 8 h postinfection, approximately 70% of the poly(A)+ mRNA detected in uninfected cells is still biologically active, but not translated in vivo, in agreement with data from the [3H]poly(U)hybridization experiment.

Inhibition of host protein synthesis by vaccinia virus: fate of cell mRNA and synthesis of small poly (A)-rich polyribonucleotides in the presence of actinomycin D.

Purified vaccinia virus rapidly inhibited HeLa cell protein synthesis in the presence of actinomycin D. Under these conditions host polyribosomes were extensively degraded but the mRNA was stable as indicated by a greater than 90% recovery of prelabeled polyadenylylated RNA. Although actinomycin D prevented the synthesis of host mRNA and poly(A) in uninfected cells, incorporation of adenosine into poly(A) was inhibited by less than 50% in infected cells. Further analysis indicated that there was little or no normal size viral mRNA but that a unique class of small poly(A)-rich RNA was made in the presence of actinomycin D. From measurements of the RNase resistance and base composition of the RNA, approximately 40% of the nucleotide sequence was estimated to be poly(A). The poly(A)-rich RNA was found associated with small polyribosomes and monoribosomes that were inactive in protein synthesis. It was suggested that the poly(A) segment of the RNA is formed by the poly(A) polymerase previously found in vaccinia virus cores and that the inactive RNA, by competing with host mRNA, may contribute to the virus-mediated inhibition of host protein synthesis observed in the presence of actinomycin D.