Construction of a molecular clone of ovine enzootic nasal tumor virus.

Scott R Walsh,Sarah K Wootton,María Carla Rosales Gerpe

doi:10.1186/s12985-016-0660-x

Scott R Walsh, Sarah K Wootton + Show 1 more

Open Access

https://doi.org/10.1186/s12985-016-0660-x

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Abstract

BackgroundEnzootic nasal tumor virus (ENTV-1) is an ovine betaretrovirus that has been linked to enzootic nasal adenocarcinoma (ENA), a contagious tumor of the ethmoid turbinates of sheep. Transmission experiments performed using virus isolated from cell free nasal tumor homogenates suggest that ENTV-1 is the causative agent of ENA; however, this etiological relationship has not been conclusively proven due to the fact that the virus cannot be propagated in vitro nor is there an infectious molecular clone of the virus.MethodsHere we report construction of a molecular clone of ENTV-1 and demonstrate that transfection of this molecular clone into HEK 293T cells produces mature virus particles.ResultsAnalysis of recombinant virus particles derived from the initial molecular clone revealed a defect in the proteolytic processing of Gag; however, this defect could be corrected by co-expression of the Gag-Pro-Pol polyprotein from the highly related Jaagsiekte sheep retrovirus (JSRV) suggesting that the polyprotein cleavage sites in the ENTV-1 molecular clone were functional. Mutagenesis of the molecular clone to correct amino acid variants identified within the pro gene did not restore proteolytic processing; whereas deletion of one proline residue from a polyproline tract located in variable region 1 (VR1) of the matrix resulted in production of CA protein of the mature (cleaved) size strongly suggesting that normal virion morphogenesis and polyprotein cleavage took place. Finally, electron microscopy revealed the presence of spherical virus particles with an eccentric capsid and an average diameter of about 100 nm.ConclusionIn summary, we have constructed the first molecular clone of ENTV-1 from which mature virus particles can be produced. Future experiments using virus produced from this molecular clone can now be conducted to fulfill Koch’s postulates and demonstrate that ENTV-1 is necessary and sufficient to induce ENA in sheep.

Highlights

Enzootic nasal tumor virus (ENTV-1) is an ovine betaretrovirus that has been linked to enzootic nasal adenocarcinoma (ENA), a contagious tumor of the ethmoid turbinates of sheep
A protein of 26 kDa was detected in the lane containing Jaagsiekte sheep retrovirus (JSRV) lysate (Fig. 1c, lane 2) as well as in the lane containing bacterially expressed ENTV-1 capsid (ECa) protein (Fig. 1c, lane 4). This 26 kDa protein was absent in the ENTV-1 molecular clone (Fig. 1c, lane 3) and instead, a protein of approximately 78 kDa, which is the expected size of the unprocessed Gag polyprotein, was detected. These results suggest that the ENTV-1 molecular clone is able to direct production of virus particles but that there is a defect in proteolytic processing
The ENTV-1 molecular clone produces unprocessed immature virions ENTV-1 cannot be propagated in conventional cell culture systems, it was necessary to generate a molecular clone in order to produce virus for experimental purposes and to show that ENTV-1 alone is sufficient to induce nasal tumors in sheep

Summary

Introduction

Enzootic nasal tumor virus (ENTV-1) is an ovine betaretrovirus that has been linked to enzootic nasal adenocarcinoma (ENA), a contagious tumor of the ethmoid turbinates of sheep. Protease-mediated processing of the Gag-Pro-Pol polyprotein is an essential step in the replication of retroviruses [1]. Strict regulation of the protease is required to prevent premature activation, which would inhibit virus assembly, budding and infectivity [3,4,5,6]. This is especially important for betaretroviruses as the core. Inactive protease is detrimental to retroviral replication because in the absence of protease processing the virion will not convert to the mature metastable conformation required for the virus to become infectious. There is a class of antiretroviral drugs designed to inhibit the protease and inhibit virus replication [12, 13]

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