Characterization of human herpesvirus 6A/B U94 as ATPase, helicase, exonuclease and DNA-binding proteins.

Frédéric Trempe,Guillaume Morissette,Shella Gilbert-Girard,Nina Wallaschek,Vanessa Collin,Annie Gravel,Benedikt B Kaufer,Louis Flamand,Isabelle Dubuc

doi:10.1093/nar/gkv503

Frédéric Trempe, Guillaume Morissette + Show 7 more

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https://doi.org/10.1093/nar/gkv503

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Abstract

Human herpesvirus-6A (HHV-6A) and HHV-6B integrate their genomes into the telomeres of human chromosomes, however, the mechanisms leading to integration remain unknown. HHV-6A/B encode a protein that has been proposed to be involved in integration termed U94, an ortholog of adeno-associated virus type 2 (AAV-2) Rep68 integrase. In this report, we addressed whether purified recombinant maltose-binding protein (MBP)-U94 fusion proteins of HHV-6A/B possess biological functions compatible with viral integration. We could demonstrate that MBP-U94 efficiently binds both dsDNA and ssDNA containing telomeric repeats using gel shift assay and surface plasmon resonance. MBP-U94 is also able to hydrolyze adenosine triphosphate (ATP) to ADP, providing the energy for further catalytic activities. In addition, U94 displays a 3′ to 5′ exonuclease activity on dsDNA with a preference for 3′-recessed ends. Once the DNA strand reaches 8–10 nt in length, the enzyme dissociates it from the complementary strand. Lastly, MBP-U94 compromises the integrity of a synthetic telomeric D-loop through exonuclease attack at the 3′ end of the invading strand. The preferential DNA binding of MBP-U94 to telomeric sequences, its ability to hydrolyze ATP and its exonuclease/helicase activities suggest that U94 possesses all functions required for HHV-6A/B chromosomal integration.

Highlights

The human beta-herpesvirus subfamily is composed of four viruses: human cytomegalovirus (CMV), human herpesvirus (HHV)-6A (HHV-6A), HHV-6B and HHV-7.Based on epidemiological, biological and molecular characteristics, Human herpesvirus-6A (HHV-6A) and HHV-6B were recently recognized as distinct viruses rather than viral variants [1,2]
adenosine triphosphate (ATP) hydrolysis by maltose-binding protein (MBP)-Rep68, MBP-U94 proteins of HHV-6A (U94A) and MBPU94B occurred as efficiently in the absence of DNA. These results indicate that the MBP-U94 proteins possess ATPase activity measured by the hydrolyzing ATP into ADP, which is independent of the presence of a DNA substrate
Based on estimates from several independent studies, ∼0.5–1% of world population harbor HHV6 in every single cell of their body (reviewed in [8,9,10,23]). This suggests that HHV-6 was able to infect a sperm cell or an ovum and integrate its genome into gametes at some point in time

Summary

Introduction

The human beta-herpesvirus subfamily is composed of four viruses: human cytomegalovirus (CMV), human herpesvirus (HHV)-6A (HHV-6A), HHV-6B and HHV-7.Based on epidemiological, biological and molecular characteristics, HHV-6A and HHV-6B were recently recognized as distinct viruses rather than viral variants [1,2]. Several independent investigators have confirmed these findings (reviewed in [8,9]) This integration can occur in both somatic and germ cells. Chromosomal integration into germ cells can result in individuals that harbor HHV-6A/B in every single cell of their body. Fifty percent of their descendants will inherit this condition, which we referred to as inherited chromosomally-integrated HHV-6 (iciHHV-6). It is estimated that between 0.5–1% of the world population are iciHHV-6+ and carry an integrated copy of the HHV-6A/B genome in every cell of their body [8,9,10]. The underlying mechanisms of HHV-6A/B integration remain completely unknown

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