Expression and replication of virus-like circular DNA in human cells

Sebastian Eilebrecht,Charlotte Sauerland,Victor Sarachaga,Claudia Tessmer,Ekaterina Nikitina,Karin Gunst,Agnes Hotz-Wagenblatt,Timo Bund,Deblina Chakraborty,Amelie Burk,Imke Grewe,Konstantina Falida,Corinna Whitley

doi:10.1038/s41598-018-21317-w

Abstract

The consumption of bovine milk and meat is considered a risk factor for colon- and breast cancer formation, and milk consumption has also been implicated in an increased risk for developing Multiple Sclerosis (MS). A number of highly related virus-like DNAs have been recently isolated from bovine milk and sera and from a brain sample of a MS patient. As a genetic activity of these Acinetobacter-related bovine milk and meat factors (BMMFs) is unknown in eukaryotes, we analyzed their expression and replication potential in human HEK293TT cells. While all analyzed BMMFs show transcriptional activity, the MS brain isolate MSBI1.176, sharing homology with a transmissible spongiform encephalopathy-associated DNA molecule, is transcribed at highest levels. We show expression of a replication-associated protein (Rep), which is highly conserved among all BMMFs, and serological tests indicate a human anti-Rep immune response. While the cow milk isolate CMI1.252 is replication-competent in HEK293TT cells, replication of MSBI1.176 is complemented by CMI1.252, pointing at an interplay during the establishment of persistence in human cells. Transcriptome profiling upon BMMF expression identified host cellular gene expression changes related to cell cycle progression and cell viability control, indicating potential pathways for a pathogenic involvement of BMMFs.

Highlights

The consumption of red meat has been consistently reported to be a risk factor for developing colorectal cancer[1,2,3,4]
We focused on CMI3.168 and CMI1.252, a small and a large cow milk isolate, the latter one containing two large ORFs - replicationassociated protein (Rep) and the uncharacterized ORF-2 - as well as on the Multiple Sclerosis (MS) brain isolates MSBI1.176 and MSBI2.176
The prediction of the secondary structure of each of these antisense transcripts resulted in a defined hairpin-structure, which is similar in CMI1.252, CMI3.168 and MSBI1.176, but not in MSBI2.176 (Fig. 1b)

Summary

Introduction

The consumption of red meat has been consistently reported to be a risk factor for developing colorectal cancer[1,2,3,4]. Recent studies linked an increased breast cancer risk to the long-term consumption of red meat and cow milk[5,6,7,8]. In the case of red meat, carcinogenic aromatic carbohydrates and nitrosamine derivatives arising during the process of broiling or frying have been suggested to be responsible for the increased cancer risk[18,19,20]. This suggests such carcinogens at least not being solely responsible for an increased risk for developing cancer These observations together with the above mentioned epidemiological data have led to a proposed model that species-specific infectious agents of bovine origin - bovine milk and meat factors (BMMFs) – significantly contribute to the etiology of these common human diseases[10,11,23,24]. We used transcriptome profiling in order to investigate BMMF-host cell interactions at the gene expression level, which may give indications for a mode of action during a potential involvement of BMMFs in pathogenesis

Methods

Results

Conclusion