Baculovirus infection results in the induction of membrane structures within the nucleoplasm of the host cells. The source of these membranes is unclear; however, using the normal dynamics of cellular membranes and the nuclear envelope as a model, it is possible that the cell cycle might play a role in the regulation of formation of these intranuclear membranes. Therefore, one goal of this study was to investigate the effect of baculovirus infection on the cell cycle of Sf9 host cells. Since few data are available on the cell cycle of insect cells, the first task was to define Sf9 cell cycle kinetics. The cell cycle phase distribution of Sf9 cells grown in suspension culture was determined to be evenly distributed (29% of the cells in G1, 33% in S, and 36% in G2/M phase), with the duration of G1 and S phases both being about 6 h and the combined duration of G2/M phase being about 8 h. When Sf9 cells were infected with AcMNPV (Autographa californica nuclear polyhedrosis virus), approximately 84% of the cells were arrested in G2/M phase by 18-24 h p.i. Concomitant with the viral-induced arrest in G2/M phase, high levels of both cdc2-associated histone H1 kinase activity and cyclin B protein were detected. By 24 h p.i. cyclin B was no longer detected; however, cdc2-associated histone H1 kinase activity remained throughout the infection. These data suggested that early in infection, cyclin B/cdc2 complex may be used to regulate the transition from G2 to M phase, but prolonged arrest may be due to a protein(s) encoded by AcMNPV. DNA hybridization analysis showed that the maximal rate of viral DNA replication occurred before G2/M arrest. We noted that viral DNA replication still occurred late in infection, when the majority of the cells were arrested in G2/M phase. Since cellular DNA replication normally does not occur during G2 or M phase, experiments were designed to determine if viral DNA replication could occur even when host cell DNA replication was arrested. Sf9 cells were arrested and "frozen" at the boundary of G1/S phase using 5-fluoro-2'deoxyuridine (FdUrd) treatment and then infected with AcMNPV In the blocked, infected cells, viral DNA replication was detected; however, cellular DNA remained at steady-state levels. These results suggested that cellular DNA replication was not necessary for viral DNA replication and show that viral DNA replication was not significantly inhibited by FdUrd treatment. It was a surprise to detect viral DNA replication when the host cells were "frozen" at G1/S phase. We wanted to determine if the viral infection was progressing to the stage of progeny virus production. Our data showed that progeny budded virus (BV) and virus-induced intranuclear microvesicles were produced in the frozen, infected cells; however, the intranuclear microvesicles had an unusual structure. They were irregular in shape and thickened compared to those observed in a normal infection. Very few enveloped nucleocapsids were visible in the nucleus of the frozen, infected cells and the occluded-derived virus ...

Rotavirus nonstructural protein 4 (NSP4) is known to function as an intracellular receptor at the endoplasmic reticulum (ER) critical to viral morphogenesis and is the first characterized viral enterotoxin.Exogenously added NSP4 induces diarrhea in rodent pups and stimulates secretory chloride currents across intestinal segments as measured in Ussing chambers. Circular dichroism studies further reveal that intact NSP4 and the enterotoxic peptide (NSP4 114-135 ) that is located within the extended, C-terminal amphipathic helix preferentially interact with caveola-like model membranes. We now show colocalization of NSP4 and caveolin-1 in NSP4-transfected and rotavirus-infected mammalian cells in reticular structures surrounding the nucleus (likely ER), in the cytosol, and at the cell periphery by laser scanning confocal microscopy. A direct interaction between NSP4 residues 112 to 140 and caveolin-1 was determined by the Pro-Quest yeast twohybrid system with full-length NSP4 and seven overlapping deletion mutants as bait, caveolin-1 as prey, and vice versa. Coimmunoprecipitation of NSP4-caveolin-1 complexes from rotavirus-infected mammalian cells demonstrated that the interaction occurs during viral infection. Finally, binding of caveolin-1 from mammalian cell lysates to Sepharose-bound, NSP4-specific synthetic peptides confirmed the yeast two-hybrid data and further delineated the binding domain to amino acids 114 to 135. We propose that the association of NSP4 and caveolin-1 contributes to NSP4 intracellular trafficking from the ER to the cell surface and speculate that exogenously added NSP4 stimulates signaling molecules located in caveola microdomains.Rotaviruses (RV) cause severe, life-threatening gastroenteritis in children and animals worldwide and in immunocompromised and elderly adults (46). The RV genome is composed of 11 segments of double-stranded RNA that encodes five nonstructural and six structural proteins (17). Nonstructural protein 4 (NSP4), encoded by RV gene 10, initially was identified as an endoplasmic reticulum (ER) transmembrane glycoprotein essential to RV morphogenesis by serving as an intracellular receptor to double-layered particles (DLPs) (5,44,67,66). NSP4 residues 161 to 175 bind the outer coat protein (VP6) of the DLPs, which facilitates translocation into the ER and the addition of two viral proteins, VP7 and VP4, and a transient ER membrane (40,66,67); NSP4 is sufficient for the budding of DLPs into the ER lumen (33). The ER transient viral envelope is eventually removed by an unknown mechanism prior to virus release (40,44). Because the NSP4 sequence lacks classical ER retention signals and does not appear to be retrieved by retrograde transport and the two N-linked, high-mannose glycosylation sites remain sensitive to endoglycosidase H (endo H) digestion, the current tenet is that NSP4 does not enter or traffic through the Golgi (5, 16).In addition to facilitating RV maturation at the ER, NSP4 functions as the first described viral enterotoxin that induces diarrhea in neonatal...

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Sterol carrier protein-2 (SCP-2) was independently discovered as a soluble protein that binds and transfers cholesterol as well as phospholipids (nonspecific lipid transfer protein, nsLTP) in vitro. Physiological functions of this protein are only now beginning to be resolved. The gene encoding SCP-2 also encodes sterol carrier protein-x (SCP-x) arising from an alternate transcription site. In vitro and in vivo SCP-x serves as a peroxisomal 3-ketoacyl-CoA thiolase in oxidation of branchedchain lipids (cholesterol to form bile acids; branched-chain fatty acid for detoxification). While peroxisomal SCP-2 facilitates branched-chain lipid oxidation, the role(s) of extraperoxisomal (up to 50% of total) are less clear. Studies using transfected fibroblasts overexpressing SCP-2 and hepatocytes from SCP-2/SCP-x gene-ablated mice reveal that SCP-2 selectively remodels the lipid composition, structure, and function of lipid rafts/caveolae. Studies of purified SCP-2 and in cells show that SCP-2 has high affinity for and selectively transfers many lipid species involved in intracellular signaling: fatty acids, fatty acyl CoAs, lysophosphatidic acid, phosphatidylinositols, and sphingolipids (sphingomyelin, ceramide, mono-di-and multi-hexosylceramides, gangliosides). SCP-2 selectively redistributes these signaling lipids between lipid rafts/caveolae and intracellular sites. These findings suggest SCP-2 serves not only in cholesterol and phospholipid transfer, but also in regulating multiple lipid signaling pathways in lipid raft/caveolae microdomains of the plasma membrane.

HDL-mediated reverse-cholesterol transport as well as phosphoinositide signaling are mediated through plasma membrane microdomains termed caveolae/lipid rafts. However, relatively little is known regarding mechanism(s) whereby these lipids traffic to or are targeted to caveolae/lipid rafts. Since sterol carrier protein-2 (SCP-2) binds both cholesterol and phosphatidylinositol, the possibility that SCP-2 might interact with caveolin-1 and caveolae was examined. Double immunolabeling and laser scanning fluorescence microscopy showed that a small but significant portion of SCP-2 colocalized with caveolin-1 primarily at the plasma membrane of L-cells and more so within intracellular punctuate structures in hepatoma cells. In SCP-2 overexpressing L-cells, SCP-2 was detected in close proximity to caveolin, 48 +/- 4 A, as determined by fluorescence resonance energy transfer (FRET) and immunogold electron microscopy. Cell fractionation of SCP-2 overexpressing L-cells and Western blotting detected SCP-2 in purified plasma membranes, especially in caveolae/ lipid rafts as compared to the nonraft fraction. SCP-2 and caveolin-1 were coimmunoprecipitated from cell lysates by anti-caveolin-1 and anti-SCP-2. Finally, a yeast two-hybrid assay demonstrated that SCP-2 directly interacts with caveolin-1 in vivo. These interactions of SCP-2 with caveolin-1 were specific since a functionally related protein, phosphatidyinositol transfer protein (PITP), colocalized much less well with caveolin-1, was not in close proximity to caveolin-1 (i.e., >120 A), and was not coimmunoprecipitated by anti-caveolin-1 from cell lysates. In summary, it was shown for the first time that SCP-2 (but not PITP) selectively interacted with caveolin-1, both within the cytoplasm and at the plasma membrane. These data contribute significantly to our understanding of the role of SCP-2 in cholesterol and phosphatidylinositol targeted from intracellular sites of synthesis in the endoplasmic reticulum to caveolae/lipid rafts at the cell surface plasma membrane.