- Volume 82, Issue 2, 2001

The persistence-associated transcript 1 (PAT1) is actively expressed during persistent infection with Hz-1 virus, while transcription of the rest of the viral genes is shut down. Previously, results of a series deletion of the PAT1 promoter suggested that the regions from nucleotides −312 to −212 and nucleotides −158 to −90 negatively regulate the promoter activity. Here, the negative regulatory effect of the −312/−90 fragment was confirmed using a heterologous IE0 promoter of Autographa californica multiple nucleopolyhedrovirus. Further, the negative regulation of the −312 to −212 region was orientation-independent. The results of electrophoresis mobility shift assays showed that cellular protein(s) bind specifically to DNA fragments −312/−212 and −158/−90. In each of these fragments, a GATA element was identified by computer-assisted analysis. Mutating both GATA elements in the −312/−90 fragment completely eliminated its negative effect on IE0 promoter activity, while mutating only one of these elements had little or no effect. Together, these results suggest that the GATA element has a negative regulatory role on the IE0 and PAT1 promoters.

Complementation of movement-deficient potato virus X (PVX) coat protein (CP) mutants, namely PVX.CP-Xho lacking the 18 C-terminal amino acid residues and PVX.ΔCP lacking the entire CP gene, was studied by transient co-expression with heterologous proteins. These data demonstrated that the potyvirus CPs and both the major and minor CPs of beet yellows closterovirus could complement cell-to-cell movement of PVX.CP-Xho but not PVX.ΔCP. These data also indicated that the C-terminally truncated PVX CP lacked a movement function which could be provided in trans by the CPs of other filamentous viruses, whereas another movement determinant specified by some region outside the most C-terminal part of the PVX CP could not be complemented either by potyvirus or closterovirus CPs. Surprisingly, the CP of spherical cocksfoot mottle sobemovirus rescued all of the PVX CP movement functions, complementing the spread of PVX.CP-Xho and, to a lesser extent, PVX.ΔCP. Both these mutants were also rescued by the tobacco mosaic virus (TMV) movement protein (MP). To shed light on the movement function of PVX CP, attempts were made to complement PVX.CP-Xho by a series of TMV MP mutants. An internal deletion abolished complementation, suggesting that the internal region of TMV MP, which includes a number of overlapping functional domains important for cell-to-cell transport, provides an activity complementing movement determinant(s) specified by the C-terminal region of PVX CP.

Prion rods, i.e. insoluble infectious aggregates of the N-terminally truncated form of the prion protein, PrP 27–30, and the corresponding recombinant protein, rPrP(90–231), were autoclaved in water, bovine lipid or lipid–water mixtures for 20 min at temperatures from 100 to 170 °C. A protocol was developed for the quantitative precipitation of small amounts of protein from large excesses of lipid. PrP remaining undegraded after autoclaving was quantified by Western blot and degradation factors were calculated. The Arrhenius plot of the rate of degradation vs temperature yielded linear relationships for prion rods in water or lipid–water as well as for rPrP(90–231) in lipid–water. The presence of lipids increased the heat stability of prion rods, especially at lower temperatures. Prion rods had a much higher thermal stability compared to rPrP. Autoclaving of prion rods in pure lipid gave different results – not simple degradation but bands indicative of covalently linked dimers, tetramers and higher aggregates. The heat stability of prion rods in pure lipid exceeded that in lipid–water mixtures. Degradation factors larger than 104 were reached at 170 °C in the presence of lipids and at 150 °C in the absence of lipids. The linear correlation of the data allows cautious extrapolation to conditions not tested, i.e. temperatures higher than 170 °C. A factual basis for assessing the biological safety of industrial processes utilizing potentially BSE-or scrapie-contaminated animal fat is provided.