ABSTRACT
BACKGROUND: 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. 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. METHODS: Here 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. RESULTS: Analysis 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. CONCLUSION: In 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.
Subject(s)
Betaretrovirus/growth & development , Betaretrovirus/genetics , Cloning, Molecular , Oncogenic Viruses/growth & development , Oncogenic Viruses/genetics , Animals , Betaretrovirus/isolation & purification , Betaretrovirus/ultrastructure , Cell Line , DNA Mutational Analysis , Epithelial Cells/virology , Humans , Microscopy, Electron, Transmission , Oncogenic Viruses/isolation & purification , Oncogenic Viruses/ultrastructure , Polyproteins/genetics , Polyproteins/metabolism , Protein Processing, Post-Translational , Reverse Genetics , Sheep , Transfection , Viral Proteins/genetics , Viral Proteins/metabolism , Virion/ultrastructure , Virus ReplicationABSTRACT
To our knowledge, five cases of Castleman's disease involving only the central nervous system have been reported previously. We report a sixth case, which occurred in a 47-year-old woman with a 3-month history of headaches and a large superior frontal lobe mass on neuroimaging. Excisional biopsy revealed confluent lymphoid nodular areas with multiple well-developed germinal centers surrounded by concentrically layered proliferations of small B lymphocytes typical of Castleman's disease. Ultrastructural study found 100-nm virallike particles within follicular dendritic cells as well as intercellular spaces. These particles were suggestive of a D-type retrovirus. The patient underwent postoperative radiotherapy and was neurologically normal 3 months after surgery.
Subject(s)
Betaretrovirus/ultrastructure , Brain Diseases/virology , Castleman Disease/virology , Dendritic Cells/virology , Meninges/virology , Retroviridae Infections/pathology , Tumor Virus Infections/pathology , Brain Diseases/pathology , Castleman Disease/pathology , Dendritic Cells/ultrastructure , Female , Humans , Magnetic Resonance Imaging , Meninges/ultrastructure , Middle Aged , Retroviridae Infections/virology , Tumor Virus Infections/virologyABSTRACT
Epstein-Barr virus (EBV) released from the B95-8 marmoset cell line has served as a prototype for biologic and biochemical studies of EBV. Here we identify and characterize a retrovirus carried by many cultures of B95-8 cells. The experiments were stimulated by the isolation of a cDNA clone from B95-8 cells in which sequences from the EBV large internal repeat were linked to gag sequences similar to those of squirrel monkey retrovirus, human isolate, SMRV-H. However, among 413 amino acids predicted from the nucleotide sequence of the gag region of the B95-8 SMRV isolate there were 48 amino acid changes that distinguished this virus from SMRV-H originally isolated from a human lymphoid cell line by Oda et al. (1988, Virology 167, 468-476). Nucleic acid and antibody probes were developed for the B95-8 isolate of SMRV. Using such probes, we found that SMRV-B95-8 was readily transmissible, independent of EBV, as an infectious virus to human B and T cell lines. SMRV-B95-8 was highly fusogenic in the presence or absence of EBV. The ultrastructural appearance of the B95-8 retrovirus was characteristic of a type D retrovirus. Cells dually infected with EBV and SMRV-B95-8 did not demonstrate increased levels of lytic EB viral replication. SMRV-B95-8 did not by itself cause lymphocyte immortalization or enhance immortalization by EBV. Thus SMRV-B95-8 does not contribute to the major biologic properties of the B95-8 strain of EBV.
