Autographa californicaNucleopolyhedrovirus Infection Results in Sf9 Cell Cycle Arrest at G2/M Phase

Abstract

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 ...

Temporal expression profiling of novel Spodoptera litura nucleopolyhedrovirus-encoded microRNAs upon infection of Sf21 cells

“…There have been various studies reporting perturbations in cell cycle and apoptotic mechanisms after baculovirus infection. In one study, AcMNPV was observed to arrest the Sf9 cell cycle at the G 2 /M stage (Braunagel et al, 1998) whilst in another study, synchronized Sf9 cells when infected at the G 1 or S phase were arrested in the S phase, whilst those infected at the G 2 /M stage were blocked at the G 2 /M stage (Ikeda & Kobayashi, 1999). In yet another study, Helicoverpa armigera nucleopolyhedrovirus was found to arrest cell growth at the G 2 /M stage (Zhou et al, 2004).…”

Analysis of baculovirus IE1 in living cells: dynamics and spatial relationships to viral structural proteins

“…of 10. Our employment of an asynchronous culture for this experiment may have been the cause of the observed variation (Braunagel et al, 1998;Ikeda & Kobayashi, 1999). Typically, a stationary (preexpansion) phase of the bright IE1-associated structures required 2-3 h, after which the IE1-associated structures began to expand.…”

Cell-cycle-dependent resistance toBacillus thuringiensisCry1C toxin in Sf9 cells

“…A gradual release of the arrested cells from the 'nocodazole barrier' occurred, allowing cells to complete mitosis, recover and grow similarly to untreated cells. The recovery of nocodazole-treated cells lasted several hours (Braunagel et al, 1998) with the appearance of the first cell divisions only 6 hours after washing off the nocodazole (data not shown).…”

“…Nocodazole (10 µg/ml) (Braunagel et al, 1998) was added to the cells 24 hours after culture dilution. The response to Cry1C was evaluated 24 to 48 hours after nocodazole addition.…”