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SlyD is a putative folding helper protein from the Escherichia coli cytosol, which consists of an N-terminal prolyl isomerase domain of the FKBP type and a presumably unstructured C-terminal tail. We produced truncated versions without this tail (SlyD) for SlyD from E. coli, as well as for the SlyD orthologues from Yersinia pestis, Treponema pallidum, Pasteurella multocida, and Vibrio cholerae. They are monomeric in solution and unfold reversibly. All SlyD variants catalyze the proline-limited refolding of ribonuclease T1 with very high efficiencies, and the specificity constants (kcat/KM) are equal to approximately 10(6) M(-1) s(-1). These large values originate from the high affinities of the SlyD orthologues for unfolded RCM-T1, which are reflected in low KM values of approximately 1 microM. SlyD also exhibits pronounced chaperone properties. Permanently unfolded proteins bind with high affinity to SlyD and thus inhibit its prolyl isomerase activity. The unfolded protein chains do not need to contain proline residues to be recognized and bound by SlyD. The conservation of prolyl isomerase activity and chaperone properties within the SlyD family suggests that these proteins might act as true folding helpers in the bacterial cytosol. The SlyD proteins are also well suited for biotechnological applications. As fusion partners they facilitate the refolding and increase the solubility of aggregation-prone proteins such as the gp41 ectodomain fragment of HIV-1.

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In order to identify functional regions of the human cytomegalovirus protein pUL97 (i) different 5h fragments of the UL97 open reading frame (ORF) were fused to the coding region of the green fluorescent protein and (ii) recombinant vaccinia viruses (rVV) were generated carrying two full-length and 11 mutated UL97 ORFs. The results indicated the presence of an N-terminal region within pUL97 which changed the intracellular distribution of the fusion proteins. pUL97 was localized in the nucleus, but not in the nucleoli, and was detected in the nuclear matrix fraction. Expression of all pUL97 mutants could be confirmed by Western blot analysis. pUL97-associated ganciclovir (GCV) phos-

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The UL97 protein (pUL97) of human cytomegalovirus (HCMV) is a protein kinase that also phosphorylates ganciclovir (GCV), but its biological function is not yet clear. The M97 protein (pM97) of mouse cytomegalovirus (MCMV) is the homolog of pUL97. First, we studied the consequences of genetic replacement of M97 by UL97. Using the infectious bacterial plasmid clone of the full-length MCMV genome (M. Wagner, S. Jonjic, U. H. Koszinowski, and M. Messerle, J. Virol. 73:7056-7060, 1999), we replaced the M97 gene with the UL97 gene and constructed an MCMV M97 deletion mutant and a revertant virus. In addition, pUL97 and pM97 were expressed by recombinant vaccinia virus to compare both for known functions. Remarkably, pM97 proved not to be the reason for the GCV sensitivity of MCMV. When expressed by the recombinant MCMV, however, pUL97 was phosphorylated and endowed MCMV with the capacity to phosphorylate GCV, thereby rendering MCMV more susceptible to GCV. We found that deletion of pM97, although it is not essential for MCMV replication, severely affected virus growth. This growth deficit was only partially amended by pUL97 expression. When expressed by recombinant vaccinia viruses, both proteins were phosphorylated and supported phosphorylation of GCV, but pUL97 was about 10 times more effective than pM97. One hint of the functional differences between the proteins was provided by the finding that pUL97 accumulates in the nucleus, whereas pM97 is predominantly located in the cytoplasm of infected cells. In vivo testing revealed that the UL97-MCMV recombinant should allow evaluation of novel antiviral drugs targeted to the UL97 protein of HCMV in mice.

Human cytomegalovirus (HCMV) infection appears to be linked to the pathogenesis of atherosclerosis. An association between HCMV infection and an enhanced restenosis rate as well as the induction of vasculopathies after solid organ transplantation has been documented. Knowledge of the cellular and molecular basis of these findings is limited, however. By Northern blot and RT-PCR analysis of human foreskin fibroblasts (HFF) and human coronary artery smooth muscle cells (SMC), we identified extracellular matrix (ECM) genes that were downregulated after HCMV infection, including collagen type I and fibronectin. Quantitative immunoassays showed a significant reduction of soluble collagen type I and fibronectin proteins in supernatants of both cell types. This was shown to be a direct effect of HCMV infection and not due to a response to interferons released from infected cells, since neutralization of alpha and beta interferon activity could not block virus-induced downregulation of matrix proteins. As the amount of ECM depends on both synthesis and degradation, we also assessed the influence of HCMV on the activity of matrix metalloproteinases (MMP). Interestingly, a significant difference in virus-induced matrix degradation could be shown between the two cell types. HCMV upregulated MMP-2 protein and activity in SMC but not in HFF. Thus, HCMV infection of SMC reduces ECM dramatically by inducing two independent mechanisms that influence synthesis as well as degradation of ECM. These may represent molecular mechanisms for HCMV-induced pathogenesis of inflammatory vasculopathies and may facilitate dissemination of HCMV by promoting the detachment of infected cells in vivo.

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