Autographa californica Nucleopolyhedrovirusac75is Required for the Nuclear Egress of Nucleocapsids and Intranuclear Microvesicle Formation

Autographa californica nucleopolyhedrovirus (AcMNPV)orf75(ac75) is a highly conserved gene of unknown function. In this study, we constructed anac75knockout AcMNPV bacmid and investigated the role ofac75in the baculovirus life cycle. The expression and distribution of the Ac75 protein were characterized, and its interaction with another viral protein was analyzed to further understand its function. Our data indicated thatac75was required for the nuclear egress of nucleocapsids, intranuclear microvesicle formation, and subsequent budded virion (BV) formation, as well as occlusion-derived virion (ODV) envelopment and embedding of ODVs into polyhedra. Western blot analyses showed that two forms of 18 and 15 kDa of FLAG-tagged Ac75 protein were detected. Ac75 was associated with both nucleocapsid and envelope fractions of BV, but only the nucleocapsid fraction of ODV; the 18-kDa form was associated with only BVs, whereas the 15-kDa form was associated with both types of virion. Ac75 was predominantly localized in the intranuclear ring zone during infection and exhibited a nuclear rim distribution during the early phase of infection. A phase-separation assay suggested that Ac75 was not an integral membrane protein. A coimmunoprecipitation assay revealed an interaction between Ac75 and the integral membrane protein Ac76, and bimolecular fluorescence complementation assays identified the sites of the interaction within the cytoplasm, at the nuclear membrane and ring zone in AcMNPV-infected cells. Our results have identifiedac75as a second gene that is required for both the nuclear egress of nucleocapsids and the formation of intranuclear microvesicles.IMPORTANCEDuring the baculovirus life cycle, the morphogenesis of both budded virions (BVs) and occlusion-derived virions (ODVs) is proposed to involve a budding process at the nuclear membrane, which occurs while nucleocapsids egress from the nucleus or when intranuclear microvesicles are produced. However, the exact mechanism of virion morphogenesis remains unknown. In this study, we identifiedac75as a second gene, in addition toac93, that is essential for the nuclear egress of nucleocapsids, intranuclear microvesicle formation, and subsequent BV formation, as well as ODV envelopment and embedding of ODVs into polyhedra. Ac75 is not an integral membrane protein. However, it interacts with an integral membrane protein (Ac76) and is associated with the nuclear membrane. These data enhance our understanding of the commonalities between nuclear egress of nucleocapsids and intranuclear microvesicle formation and may help to reveal insights into the mechanism of baculovirus virion morphogenesis.

The Study of Residence Time and Flow Rate of Lubricating Oil Through the Top Ring Zone of a Gasoline Engine

With the ever increasing demand on lubricant manufacturers to improve fuel economy, fuel economy retention and increase the drain intervals of their lubricants there is an increasing need to understand which factors effect the degradation of the lubricant in the sump. The harsh conditions in the ring pack have been shown to be responsible for the majority of the lubricant degradation in the engine. Therefore in this investigation the flow rate of lubricant through, and residence time of lubricant in, the top ring zone of a gasoline engine with respect to load and speed has been experimentally measured. An initial effect of the lubricant viscosity on these parameters has also been investigated. The theory and experimental methods are described before the experimental results are presented and discussed. The work has shown that the overall effect of engine speed on sump lubricant degradation is nominal, and that the level of sump degradation increases with increasing engine load.