Kinetic Analysis of the Steps of the Polyomavirus Lytic Cycle

Abstract

Kinetic studies of the accumulation of early and late transcripts, early and late proteins, genomes, and live virus, during the lytic cycle of murine polyomavirus wild-type A2, were carried out in synchronized NIH 3T3 cells released from G 0 by the addition of serum after infection. This first-time simultaneous analysis of all parameters of the virus life cycle led to new insights concerning the transcriptional control at the early-to-late transition. During the early phase, early transcripts were synthesized at very low levels, detectable only by reverse transcription-PCR, from 6 h postinfection (hpi). Large T protein could be detected by 8 hpi (while infected cells were in the G 1 phase). The level of expression of the middle T and small T proteins was lower than that of large T at all times, due, at least in part, to a splicing preference for the large-T 5 splice site at nucleotide 411. A large increase in the level of both early and late transcripts coincided closely with the detection in mid-S phase of viral genome amplification. Thereafter, both classes of transcripts continued to further accumulate up to the end of the experiments (48 hpi). In addition, during the late phase, "giant" multigenomic transcripts were synthesized from the early as well as the late promoter. Thus, a major type of transcriptional control appears to be applied similarly to the transcription of both early and late genes. This view differs from that in the literature, which highlights the enhancement of late transcription and the repression of early transcription. However, despite this parallel transcriptional control, additional regulations are applied which result in higher levels of late compared to early transcripts, as previously described. In the accompanying article, a key role for middle T and/or small T in this late-phase enhancement of early and late transcription is demonstrated (16). Other novel findings, e.g., the synthesis of a very abundant short early promoter proximal RNA, are also described.Polyomaviruses have served as important model systems for the understanding of various aspects of cellular organization and function, such as chromatin structure, the role of enhancers in gene expression, splicing mechanism, and the composition of the DNA replication machinery, to name a few. This is in part a consequence of the DNA nature and small size of the genomes of this virus family, which render viral replication highly dependent on, and hence compatible with, cellular processes. As a result, and with the added interest in their capacity to induce neoplastic transformation, these viruses have been the subjects of extensive studies.Surprisingly at this late stage of analysis, there has been no comprehensive study of the temporal relationships between all the various steps of the lytic cycle. Current references to the timing of these steps invariably quote the 1981 review by Acheson (2), which offers a thorough integration of the studies available at the time. However, these studies were done at different times, in differ...

Role of Middle T-Small T in the Lytic Cycle of Polyomavirus: Control of the Early-to-Late Transcriptional Switch and Viral DNA Replication

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Induction of the Human Papillomavirus Type 31 Late Promoter Requires Differentiation but Not DNA Amplification

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