No abstract

SummaryBaculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), has the ability to transduce mammalian cell lines without replication. The general objective of this study was to detect the transcription and expression of viral immediate-early genes in human cells and to examine the interactions between viral components and subnuclear structures. Viral capsids were seen in large, discrete foci in nuclei of both dividing and non-dividing human cells. Concurrently, the transcription of viral immediate-early transregulator genes (ie-1, ie-2) and translation of IE-2 protein were detected. Quantitative microscopy imaging and analysis showed that virus transduction altered the size of promyelocytic leukaemia nuclear bodies, which are suggested to be involved in replication and transcription of various viruses. Furthermore, altered distribution of the chromatin marker Draq5™ and histone core protein (H2B) in transduced cells indicated that the virus was able to induce remodelling of the host cell chromatin. To conclude, this study shows that the non-replicative insect virus, baculovirus and its proteins can induce multiple changes in the cellular machinery of human cells.

H ؉ -pyrophosphatase (H ؉ -PPase) catalyzes pyrophosphate-driven proton transport against the electrochemical potential gradient in various biological membranes. All 50 of the known H ؉ -PPase amino acid sequences contain four invariant glutamate residues. In this study, we use site-directed mutagenesis in conjunction with functional studies to determine the roles of the glutamate residues Glu 197 , Glu 202 , Glu 550 , and Glu 649 in the H ؉ -PPase of Rhodospirillum rubrum (R-PPase). All residues were replaced with Asp and Ala. The resulting eight variant R-PPases were expressed in Escherichia coli and isolated as inner membrane vesicles. All substitutions, except E202A, generated enzymes capable of PP i hydrolysis and PP i -energized proton translocation, indicating that the negative charge of Glu 202 is essential for R-PPase function. The hydrolytic activities of all other PPase variants were impaired at low Mg 2؉ concentrations but were only slightly affected at high Mg 2؉ concentrations, signifying that catalysis proceeds through a three-metal pathway in contrast to wild-type R-PPase, which employs both two-and three-metal pathways. Substitution of Glu 197 , Glu 202 , and Glu 649 resulted in decreased binding affinity for the substrate analogues aminomethylenediphosphonate and methylenediphosphonate, indicating that these residues are involved in substrate binding as ligands for bridging metal ions. Following the substitutions of Glu 550 and Glu 649 , R-PPase was more susceptible to inactivation by the sulfhydryl reagent mersalyl, highlighting a role of these residues in maintaining enzyme tertiary structure. None of the substitutions affected the coupling of PP i hydrolysis to proton transport.