CRISPR/Cas9-Mediated Knockout of DNAJC14 Verifies This Chaperone as a Pivotal Host Factor for RNA Replication of Pestiviruses.

Pestiviruses like bovine viral diarrhea virus (BVDV) are a threat to livestock. For pestiviruses, cytopathogenic (cp) and noncytopathogenic (noncp) strains are distinguished in cell culture. The noncp biotype of BVDV is capable of establishing persistent infections, which is a major problem in disease control. The noncp biotype rests on temporal control of viral RNA replication, mediated by regulated cleavage of nonstructural protein 2-3 (NS2-3). This cleavage is catalyzed by the autoprotease in NS2, the activity of which depends on its cellular cofactor, DNAJC14. Since this chaperone is available in small amounts and binds tightly to NS2, NS2-3 translated later in infection is no longer cleaved. As NS3 is an essential constituent of the viral replicase, this shift in polyprotein processing correlates with downregulation of RNA replication. In contrast, cp BVDV strains arising mostly by RNA recombination show highly variable genome structures and display unrestricted NS3 release. The functional importance of DNAJC14 for noncp pestiviruses has been established so far only for BVDV-1. It was therefore enigmatic whether replication of other noncp pestiviruses is also DNAJC14 dependent. By generating bovine and porcine DNAJC14 knockout cells, we could show that (i) replication of 6 distinct noncp pestivirus species (A to D, F, and G) depends on DNAJC14, (ii) the pestiviral replicase NS3-5B can assemble into functional complexes in the absence of DNAJC14, and (iii) all cp pestiviruses replicate their RNA and generate infectious progeny independent of host DNAJC14. Together, these findings confirm DNAJC14 as a pivotal cellular cofactor for the replication and maintenance of the noncp biotype of pestiviruses.IMPORTANCE Only noncp pestivirus strains are capable of establishing life-long persistent infections to generate the virus reservoir in the field. The molecular basis for this biotype is only partially understood and only investigated in depth for BVDV-1 strains. Temporal control of viral RNA replication correlates with the noncp biotype and is mediated by limiting amounts of cellular DNAJC14 that activate the viral NS2 protease to catalyze the release of the essential replicase component NS3. Here, we demonstrate that several species of noncp pestiviruses depend on DNAJC14 for their RNA replication. Moreover, all cp pestiviruses, in sharp contrast to their noncp counterparts, replicate independently of DNAJC14. The generation of a cp BVDV in the persistently infected animal is causative for onset of mucosal disease. Therefore, the observed strict biotype-specific difference in DNAJC14 dependency should be further examined for its role in cell type/tissue tropism and the pathogenesis of this lethal disease.

Phase space structure and chaotic scattering in near-integrable systems.

We investigate the bifurcation phenomena and the change in phase space structure connected with the transition from regular to chaotic scattering in classical systems with unbounded dynamics. The regular systems discussed in this paper are integrable ones in the sense of Liouville, possessing a degenerated unstable periodic orbit at infinity. By means of a McGehee transformation the degeneracy can be removed and the usual Melnikov method is applied to predict homoclinic crossings of stable and unstable manifolds for the perturbed system. The chosen examples are the perturbed radial Kepler problem and two kinetically coupled Morse oscillators with different potential parameters which model the stretching dynamics in ABC molecules. The calculated subharmonic and homoclinic Melnikov functions can be used to prove the existence of chaotic scattering and of elliptic and hyperbolic periodic orbits, to calculate the width of the main stochastic layer and of the resonances, and to predict the range of initial conditions where singularities in the scattering function are found. In the second example the value of the perturbation parameter at which channel transitions set in is calculated. The theoretical results are supplemented by numerical experiments.

Regional factors affecting proliferation in the large intestine of the rat.

Colonic neoplasia is more frequent in the distal colon than in the proximal colon in spontaneous human disease and in carcinogen-induced tumors in rodents. The possibility that this may reflect regional differences in morphology and in proliferative responses to fasting and refeeding was explored in this study in rats. Scanning electron microscopy revealed that the density of colonic crypts was 36% higher in the distal than in the proximal colon, while light microscopy revealed that distal crypts had 70% more colonocytes than proximal crypts. Thus, the number of colonocytes per unit area in the distal colon is approximately twice that in the proximal colon. Proliferation was assessed by the uptake of bromodeoxyuridine in vivo and showed that regions of the distal colon had greater suppression of proliferation during fasting than the cecum, and greater enhancement of proliferation during refeeding than that observed in the cecum or the proximal colon. Changes in proliferation associated with fasting and refeeding were accompanied by changes in the concentrations of short chain fatty acids, but the data did not support the hypothesis of a direct relationship between increasing concentrations of short chain fatty acids and enhanced proliferation. Regional differences in morphology and proliferation could be relevant to the greater susceptibility of the distal colon to neoplasia.