Degradation of the encephalomyocarditis virus and hepatitis A virus 3C proteases by the ubiquitin/26S proteasome system in vivo.

We have isolated stably transfected mouse embryonic fibroblast cell lines that inducibly express either the mature encephalomyocarditis virus (EMCV) or hepatitis A virus (HAV) 3C protease and have used these cells to demonstrate that both proteins are subject to degradation in vivo by the ubiquitin/26S proteasome system. The detection of 3C protease expression in these cells requires inducing conditions and the presence of one of several proteasome inhibitors. Both 3C proteases are incorporated into conjugates with ubiquitin in vivo. HAV 3C protease expression has deleterious effects on cell viability, as determined by observation and counting of cells cultured in the absence or presence of inducing conditions. The EMCV 3C protease was found to be preferentially localized to the nucleus of induced cells, while the HAV 3C protease remains in the cytoplasm. The absence of polyubiquitinated EMCV 3C protease conjugates in nuclear fraction preparations suggests that localization to the nucleus can protect this protein from ubiquitination.

Signals in hepatitis A virus P3 region proteins recognized by the ubiquitin-mediated proteolytic system.

The hepatitis A virus 3C protease and 3D RNA polymerase are present in low concentrations in infected cells. The 3C protease was previously shown to be rapidly degraded by the ubiquitin/26S proteasome system and we present evidence here that the 3D polymerase is also subject to ubiquitination-mediated proteolysis. Our results show that the sequence (32)LGVKDDWLLV(41) in the 3C protease serves as a protein destruction signal recognized by the ubiquitin-protein ligase E3alpha and that the destruction signal for the RNA polymerase does not require the carboxyl-terminal 137 amino acids. Both the viral 3ABCD polyprotein and the 3CD diprotein were also found to be substrates for ubiquitin-mediated proteolysis. Attempts to determine if the 3C protease or the 3D polymerase destruction signals trigger the ubiquitination and degradation of these precursors yielded evidence suggesting, but not unequivocally proving, that the recognition of the 3D polymerase by the ubiquitin system is responsible.

Kinetic studies and structural models of the association of E. coli sigma(70) RNA polymerase with the lambdaP(R) promoter: large scale conformational changes in forming the kinetically significant intermediates.

The kinetics of interaction of Esigma(70) RNA polymerase (R) with the lambdaP(R) promoter (P) were investigated by filter binding over a broad range of temperatures (7.3-42 degrees C) and concentrations of RNA polymerase (1-123 nM) in large excess over promoter DNA. Under all conditions examined, the kinetics of formation of competitor-resistant complexes (I(2), RP(o)) are single-exponential with first order rate constant beta(CR). Interpretation of the polymerase concentration dependence of beta(CR) in terms of the three step mechanism of open complex formation yields the equilibrium constant K(1) for formation of the first kinetically significant intermediate (I(1)) and the forward rate constant (k(2)) for the conformational change converting I(1) to the second kinetically significant intermediate I(2): R + P-->(K(1))<--I(1)(k(2))-->I(2). Use of rapid quench mixing allows K(1) and k(2) to be individually determined over the entire temperature range investigated, previously not possible at this promoter using manual mixing. Given the large (>60 bp) interface formed in I(1), its relatively small binding constant K(1) at 37 degrees C at this [salt] (approximately 6 x 10(6) M(-1)) strongly argues that binding free energy is used to drive large-scale structural changes in polymerase and/or promoter DNA or other coupled processes. Evidence for coupling of protein folding is provided by the large and negative activation heat capacity of k(a)[DeltaC(o,++)(a)= -1.5(+/-0.2)kcal K(-1)], now shown to originate directly from formation of I(1) [DeltaC(o)(1)= -1.4(+/-0.3)kcal K(-1)] rather than from the formation of I(2) as previously proposed. The isomerization I(1)-->I(2) exhibits relatively slow kinetics and has a very large temperature-independent Arrhenius activation energy [E(act)(2)= 34(+/-2)kcal]. This kinetic signature suggests that formation of the transition state (I(1)-I(2)++ involves large conformational changes dominated by changes in the exposure of polar and/or charged surface to water. Structural and biochemical data lead to the following hypotheses to interpret these results. We propose that formation of I(1) involves coupled folding of unstructured regions of polymerase (beta, beta' and sigma(70)) and bending of promoter DNA (in the -10 region). We propose that interactions with region 2 of sigma(70) and possibly domain 1 of beta induce a kink at the -11/-12 base pairs of the lambdaP(R) promoter which places the downstream DNA (-5 to +20) in the jaws of the beta and beta' subunits of polymerase in I(1). These early interactions of beta and beta' with the DNA downstream of position -5 trigger jaw closing (with coupled folding) and subsequent steps of DNA opening.