Directed Nucleosome Sliding in SV40 Minichromosomes During the Formation of the Virus Particle Exposes DNA Sequences Required for Early Transcription

Abstract

40Simian Virus 40 (SV40) exists as chromatin throughout its life cycle, and 41 undergoes typical epigenetic regulation mediated by changes in nucleosome 42 location and associated histone modifications. In order to investigate the role of 43 epigenetic regulation during the encapsidation of late stage minichromosomes 44 into virions, we have mapped the location of nucleosomes containing acetylated 45 or methylated lysines in the histone tails of H3 and H4 present in the chromatin 46 from 48-hour post-infection minichromosomes and disrupted virions. In 47 minichromosomes obtained late in infection, nucleosomes were found carrying 48 various histone modifications primarily in the regulatory region with a major 49 nucleosome located within the enhancer and other nucleosomes at the early and 50 late transcriptional start sites. The nucleosome found in the enhancer would be 51 expected to repress early transcription by blocking access to part of the SP1 52 binding sites and the left side of the enhancer in late stage minichromosomes 53 while also allowing late transcription. In chromatin from virions, the principal 54 nucleosome located in the enhancer was shifted ~ 70 bases in the late direction 55 from what was found in minichromosomes and the level of modified histones was 56 increased throughout the genome. The shifting of the enhancer-associated 57 nucleosome to the late side would effectively serve as a switch to relieve the 58 repression of early transcription found in late minichromosomes while likely also 59 repressing late transcription by blocking access to necessary regulatory 60 3 sequences. This epigenetic switch appeared to occur during the final stage of 61 virion formation. 62 63 Introduction 64 Simian Virus 40 (SV40), a member of the Polyomavirus family of DNA 65 viruses, consists of a 5243 base-pair (bp) double-strand closed-circular genome 66(1). Like many double-strand DNA viruses, SV40 exists as chromatin with the 67 usual assortment of histones and most of the epigenetic marks typical of 68 eukaryotic chromatin when found within an infected cell (2, 3). However, unlike 69 most DNA viruses, SV40 also exists with a typical chromatin structure when 70 found within the virion (3). The organization of SV40 DNA into chromatin 71 throughout its life cycle raises the interesting possibility that epigenetics plays a 72 critical role in the regulation of the various stages of the SV40 life cycle. In 73 support of this hypothesis, we have previously shown that cellular treatments that 74 affect the level of certain histone modifications in the SV40 virus produced during 75 the course of an infection can result in changes in the infectivity of the virus (4). 76Moreover, we have also shown that the organizations of nucleosomes in SV40 77 minichromosomes at 30 minutes and 48 hours post-infection differs significantly 78 from the organization of nucleosomes in the chromatin from SV40 virions (5). 79Since SV40 virion chromatin differs in organization from late stage 80 minichromosomes and also carries informati...

Differential SP1 Interactions in SV40 Chromatin from Virions and Minichromosomes

“…Second, the location of nucleosomes within the regulatory region in chromatin from virions, 30 minute minichromosomes, and 48 hour minichromosomes also vary significantly with the virions and 30 minute minichromosomes more (open) than the 48 hour minichromosomes (Kumar et al, 2017;Kumar et al, 2018).…”

“…The chromatin found in disrupted SV40 virions has been shown to be relatively heterogeneous with respect to histone modifications (Milavetz et al, 2012) and nucleosome organization (Kumar et al, 2017;Kumar et al, 2018).…”

“…For SV40 there is evidence that this is true. SV40 is regulated by many of the same transcription factors binding to their cognate DNA that function in eukaryotic cells (Tooze, 1981;Wasylyk et al, 1983;Wildeman, 1988) and SV40 chromatin contains the same chromatin structural elements found in cellular chromatin including a nucleosome-free regulatory region, specifically positioned nucleosomes, and histone modifications (Balakrishnan and Milavetz, 2017a, b;Milavetz, 1989, 1996;Kumar et al, 2017;Kumar et al, 2018;Scott and Wigmore, 1978;Varshavsky et al, 1979).…”

“…Because the SV40 genome is organized as chromatin in virions, the chromatin present in the virions serves as a potential link between the chromatin structure present late in infection that serves as the substrate for encapsidation and the chromatin structure that is required to initiate a subsequent infection when the virion infects a cell. In order to test whether the formation of virions was associated with changes in chromatin structure, we compared the chromatin structure of SV40 minichromosomes late in infection (the putative encapsidation substrates) to the structure of the chromatin found in infectious virions (Kumar et al, 2018).…”

Differential SP1 interactions in SV40 chromatin from virions and minichromosomes

“…For SV40 there is evidence that this is true. SV40 is regulated by many of the same transcription factors binding to their cognate DNA that function in eukaryotic cells (Tooze, 1981;Wasylyk et al, 1983;Wildeman, 1988) and SV40 chromatin contains the same chromatin structural elements found in cellular chromatin including a nucleosome-free regulatory region, specifically positioned nucleosomes, and histone modifications (Balakrishnan and Milavetz, 2017a, b;Kube andMilavetz, 1989, 1996;Kumar et al, 2017;Kumar et al, 2018;Scott and Wigmore, 1978;Varshavsky et al, 1979).…”