Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Sun, 16 Dec 2018 07:19:03 Short Communication Emergence of infectious simian virus 40 whose AT tract in the replication origin/early promoter region is substituted by cellular or viral DNAs Simon Keiser, 1 Katharina Schmidt, 1 Tobias Bethge, 1,2 Julia Steiger, 1 Hans H. Hirsch, 2 Walter Schaffner 1 and Oleg Georgiev 1 Correspondence Oleg Georgiev oleg.georgiev@imls.uzh.ch Received 20 August 2014 Accepted 2 November 2014 1 Institute of Molecular Life Sciences, University of Zu ¨ rich, Winterthurer Str. 190, CH-8057 Zu ¨ rich, Switzerland 2 Transplantation & Clinical Virology, Dept. of Biomedicine, University of Basel, CH-4003 Basel, Switzerland In simian virus 40 (SV40) and several other polyomaviruses, the TATA box of the early promoter is embedded in an AT tract that is also an essential part of the replication origin. We generated an ‘AT trap’, an SV40 genome lacking the AT tract and unable to grow in CV-1 monkey cells. Co- transfection of the AT trap with oligonucleotides containing AT tracts of human polyomaviruses, a poly(A : T) tract or variants of the SV40 WT sequence all restored infectious virus. In a transfection of the AT trap without a suitable oligonucleotide, an AT-rich segment was incorporated, stemming either from bovine (calf serum) or monkey (host cell) DNA. Similarly, when cells were grown with human serum, a human DNA segment was captured by SV40 to substitute for the missing AT stretch. We conclude that the virus is quite opportunistic in accepting heterologous substitutes, and that even low-abundance DNA from serum can be incorporated into the viral genome. Mouse polyomavirus and simian virus 40 (SV40) are the founding members of the large polyomavirus family (DeCaprio & Garcea, 2013; White et al., 2013; Ehlers & Wieland, 2013). In humans, 12 members are now known, including the first-discovered BK polyomavirus (BKPyV; Gardner et al., 1971) and JC polyomavirus (JCPyV; Padgett et al., 1971), both of which are closely related to SV40 (Broekema & Imperiale, 2012) and can cause severe diseases in immunocompromised individuals. In the course of disease progression, variant viruses with increased patho- genicity often emerge. Such viruses display alterations in their non-coding control region (NCCR), typically duplica- tions and/or deletions that enhance early gene expression and thus replication in the affected tissue (Moens & Van Ghelue, 2005; Hirsch et al., 2013). The genome of polyomaviruses is organized as a circular dsDNA of approx. 5 kb, which is divergently transcribed into early and late RNAs. The NCCR includes the origin of DNA replication (ori), early and late promoters, and a strong transcription enhancer (Fig. 1a). In SV40, which is particularly well characterized, the minimal replication origin has been narrowed to a segment of 64 bp (nt 5211– 0031), which overlaps the early promoter (Deb et al., 1986). The origin also harbours GAGGC sequence motifs, the binding sites for the large T antigen (LTag), an early protein that drives viral DNA replication (An et al., 2012). One conspicuous feature of the ori/early promoter region is a stretch of 17 As and Ts (Fig. 1a, red box). This AT tract is essential for viral replication (Fromm & Berg, 1982; Bergsma et al., 1982; Stillman et al., 1985; Gerard & Gluzman, 1986) and gene expression: the sequence TATTTAT at the right- hand end serves as a TATA box for early transcription (Benoist & Chambon, 1980; Wasylyk et al., 1983) (Fig. 1a, b). This sequence motif of consensus TATAWAWR, typ- ically located about 25 bp upstream of the transcription initiation site(s), is the binding site for the general transcription factor TBP. The TATA box is usually flanked upstream and/or downstream by more GC-rich core promoter elements termed BREu and BREd, which bind the general transcription factor TFIIB (Kadonaga, 2012). The AT tract region of SV40 deviates both in sequence and spacing from an optimal promoter arrangement, probably due to constraints imposed by the dual role in replication and transcription (Fig. 1b). However, any deficits seem to be largely compensated by the strong enhancer-promoter region further upstream. In the SV40 laboratory strains, commonly referred to as ‘wild type’, this harbours, among other elements, two 72 bp enhancer repeats (Banerji et al., 1981; Moreau et al., 1981) and three 21 bp repeats, the latter being essential promoter components for early (and late) gene transcription (Barrera-Saldana et al., 1985; Gidoni et al., 1985)(Fig. 1a). The AT tract in its entire length could be viewed as a TATA box with an AT-rich preface, or as multiple TATA boxes to account for some of the minor early Journal of General Virology (2015), 96, 601–606 DOI 10.1099/vir.0.071274-0 071274 G 2015 The Authors Printed in Great Britain 601