Autographa californica nucleopolyhedrovirus infection results in Sf9 cell cycle arrest at G2/M phase.
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 envelope proteins, ODV-E66 and ODV-EC27, were not detected by Western blot analyses. Since the cells were sustained at the boundary of G1 and S phases for the duration of this experiment, the decreased amount of enveloped ODV in the nucleus could be due to several factors, including decreased levels of proteins expressed from late genes, aberrant microvesicles, or the necessity of G2/M phasing of the infected cell for efficient production and maturation of intranuclear microvesicles. These data indicate that AcMNPV infection results in cell cycle arrest in G2/M phase and this arrest may be due to a viral-encoded protein(s) that has cdc2-associated kinase activity. (ABSTRACT TRUNCATED)
Braunagel
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SC
Department of Entomology, Texas A&M University, and Texas Agriculture Experimental Station, College Station 77843, USA.
Parr
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R
Belyavskyi
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M
Summers
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MD
D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002453:Cell Cycle; D002455:Cell Division; D002460:Cell Line; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis; D005434:Flow Cytometry; D005468:Fluorodeoxyuridylate / Q000494:pharmacology; D016195:G2 Phase; D006131:Growth Inhibitors / Q000494:pharmacology; D008898:Mimosine / Q000494:pharmacology; D008938:Mitosis; D009036:Moths / Q000821:virology; D015739:Nocodazole / Q000494:pharmacology; D017924:Nucleopolyhedroviruses / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D011494:Protein Kinases / Q000378:metabolism; D018411:Spodoptera; D014779:Virus Replication
D004279:DNA, Viral; D006131:Growth Inhibitors; D005468:Fluorodeoxyuridylate; D008898:Mimosine; D011494:Protein Kinases; C102333:histone H1 kinase; D016203:CDC2 Protein Kinase; D015739:Nocodazole