The pathway of cell senescence: WI-38 cells arrest in late G1 and are unable to traverse the cell cycle from a true G0 state.

Senescent human diploid fibroblasts have an undefined arrest state partially characterized by the differential expression of cell cycle-regulated genes and a failure to complete the mitogen-stimulated cascade of signalling events that lead to DNA synthesis. We present evidence that this arrest state precludes the entry of senescent fibroblasts into a normally reversible G0 or quiescent state. Both nuclear association kinetics and quinacrine dihydrochloride nuclear fluorescence show chromatin condensation patterns consistent with arrest in late G1 and exclusion of senescent cells from the G0 phase of the cell cycle. Steady-state thymidine kinase mRNA levels indicate that some of the signalling cascades initiated from a functional G0 state may be intact in senescent cells, at least qualitatively, and that this expression may represent an abortive attempt to complete pathways required for DNA replication. Taken together, the evidence suggests that growth arrest in senescent cells likely occurs in a physiologic state fundamentally distinct from that of the G0, quiescent state that is achieved by nonproliferating young cells. A full response to serum or growth factor addition, leading from quiescence to DNA synthesis, may require cells to initiate this traverse from a true G0 state. If so, senescent cells would be excluded from this pathway.

Rotavirus RNA polymerase requires the core shell protein to synthesize the double-stranded RNA genome.

Rotavirus cores contain the double-stranded RNA (dsRNA) genome, RNA polymerase VP1, and guanylyltransferase VP3 and are enclosed within a lattice formed by the RNA-binding protein VP2. Analysis of baculovirus-expressed core-like particles (CLPs) has shown that VP1 and VP2 assemble into the simplest core-like structures with replicase activity and that VP1, but not VP3, is essential for replicase activity. To further define the role of VP1 and VP2 in the synthesis of dsRNA from viral mRNA, recombinant baculoviruses containing gene 1 (rBVg1) and gene 2 (rBVg2) of SA11 rotavirus were generated and used to express recombinant VP1 (rVP1) and rVP2, respectively. After purification, the proteins were assayed individually and together for the ability to catalyze the synthesis of dsRNA in a cell-free replication system. The results showed that dsRNA was synthesized only in assays containing rVP1 and rVP2, thus establishing that both proteins are essential for replicase activity. Even in assays containing a primer-linked mRNA template, neither rVP1 nor rVP2 alone directed RNA synthesis. Characterization of the cis-acting replication signals in mRNA recognized by the replicase of rVP1 and rVP2 showed that they were the same as those recognized by the replicase of virion-derived cores, thus excluding a role for VP3 in recognition of the mRNA template by the replicase. Analysis of RNA-protein interactions indicated that the mRNA template binds strongly to VP2 in replicase assays but that the majority of the dsRNA product neither is packaged nor stably associates with VP2. The results of replicase assays performed with mutant VP2 containing a deletion in its RNA-binding domain suggests that the essential role for VP2 in replication is linked to the protein's ability to bind the mRNA template for minus-strand synthesis.

Nucleotide and amino acid sequence analysis of the rotavirus nonstructural RNA-binding protein NS35.

NS35, a basic protein encoded by gene 8 of SA11 rotavirus, possesses RNA-binding activity and is essential for genome replication. To identify conserved regions in the NS35 gene and its protein product, we determined the nucleotide sequences of the NS35 gene for the mammalian and avian rotaviruses Wa, DS1, SA11 (Patton and Ramig strains), NCDV, and Ty-1 and compared them and their deduced amino acid sequences to those reported for SA11 (Both strain), OSU, and UK. The results indicated that the NS35 genes of the mammalian rotaviruses are 1058-1059 bases in length and encode proteins of 317 amino acids that exhibit high levels of sequence conservation (> or = 83%). The NS35 gene of the turkey rotavirus Ty-1 differed from those of the mammalian rotaviruses with respect to size of the predicted protein (315 amino acids) and of the gene (1042 bases). NS35 of Ty-1 exhibited a relatively low degree of amino acid homology (52-57%) with NS35 of the mammalian viruses. Phylogenetic analysis of the NS35 gene indicated that avian (TY-1) and mammalian rotaviruses are distantly related. Comparison of the predicted sequences of NS35 showed that all possessed a conserved basic domain of 37 amino acids at residues 205-241 that may serve as the RNA-binding domain. Electrophoretic examination showed that NS35 contains a disulfide bond probably located in the amino-terminal half of the protein. Comparison of NS35 genes at the nucleotide level revealed two regions of extensive conservation, (i) a 75-base (b) sequence that includes the 35-base 5'-noncoding region and the first 30 bases of the open reading frame for NS35, and (ii) a 28-b sequence in the 3'-noncoding region of the gene. Secondary structure predictions for the NS35 mRNA suggest that the 75-base sequence can fold to produce a stem double-loop structure. Such a structure may serve as a packaging signal for the assortment of NS35 mRNA into replicase particles.