Abstract
Merkel cell polyomavirus (MCV) causes one of the most aggressive human skin cancers, but laboratory studies on MCV replication have proven technically difficult. We report the first recombinase-mediated MCV minicircle (MCVmc) system that generates high levels of circularized virus, allowing facile MCV genetic manipulation and characterization of viral gene expression kinetics during replication. Mutations to Fbw7, Skp2, β-TrCP and hVam6p interaction sites, or to the stem loop sequence for the MCV-encoded miRNA precursor, markedly increase viral replication, whereas point mutation to an origin-binding site eliminates active virus replication. To further increase the utility of this system, an mScarlet fusion protein was inserted into the VP1 c-terminus to generate a non-infectious reporter virus for studies on virus kinetics. When this reporter virus genome is heterologously expressed together with MCV VP1 and VP2, virus-like particles are generated. The reporter virus genome is encapsidated and can be used at lower biosafety levels for one-round infection studies. Our findings reveal that MCV has multiple, self-encoded viral restriction mechanisms to promote viral latency over lytic replication, and these mechanisms are now amenable to examination using a recombinase technology.
Highlights
Received: 7 February 2022Merkel cell polyomavirus (MCV) causes most cases of Merkel cell carcinoma (MCC), an uncommon human skin cancer associated with immunosuppression [1] and aging [2].MCC is highly aggressive with a 54% 5-year survival rate [3]
MCV is a near-ubiquitous component of viral skin flora; it causes MCC if the viral genome becomes integrated into the host genome and acquires mutations ablating replication functions [4,5]
To gain a better understanding of MCV biology, we generated an MCV minicircle (MCVmc) that enaTo gain a better understanding of MCV biology, we generated an MCVmc that enabled bled the production of a covalently closed circular genome free of bacterial sequences and the production of amanipulation covalently (Figure closed 1)
Summary
Received: 7 February 2022Merkel cell polyomavirus (MCV) causes most cases of Merkel cell carcinoma (MCC), an uncommon human skin cancer associated with immunosuppression [1] and aging [2].MCC is highly aggressive with a 54% 5-year survival rate [3]. Merkel cell polyomavirus (MCV) causes most cases of Merkel cell carcinoma (MCC), an uncommon human skin cancer associated with immunosuppression [1] and aging [2]. MCV is a near-ubiquitous component of viral skin flora; it causes MCC if the viral genome becomes integrated into the host genome and acquires mutations ablating replication functions [4,5]. MCV is a nonenveloped, double-stranded DNA virus with a 5.4 kb genome partitioned into early (ER) and late regions (LR) by a non-coding control region (NCCR) [4,11]. In addition to the replication origin, the NCCR contains promoter elements regulating early and late gene expression [12]. The ER encodes several proteins designated tumor antigens (T-Ags), such as large T-Ag (LT), small T-Ag (sT), 57kT [5] and alternative LT open reading frame (ALTO) [13]. Despite the simplicity of its two-gene genome (ER and LR), actual MCV gene expression and replication is Accepted: 20 February 2022
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