Persistent equine arteritis virus infection in HeLa cells.
The horse-adapted virulent Bucyrus (VB) strain of equine arteritis virus (EAV) established persistent infection in high-passage-number human cervix cells (HeLa-H cells; passages 170 to 221) but not in low-passage-number human cervix cells (HeLa-L cells; passages 95 to 115) or in several other cell lines that were evaluated. However, virus recovered from the 80th passage of the persistently infected HeLa-H cells (HeLa-H-EAVP80) readily established persistent infection in HeLa-L cells. Comparative sequence analysis of the entire genomes of the VB and HeLa-H-EAVP80 viruses identified 16 amino acid substitutions, including 4 in the replicase (nsp1, nsp2, nsp7, and nsp9) and 12 in the structural proteins (E, GP2, GP3, GP4, and GP5). Reverse genetic studies clearly showed that substitutions in the structural proteins but not the replicase were responsible for the establishment of persistent infection in HeLa-L cells by the HeLa-H-EAVP80 virus. It was further demonstrated that recombinant viruses with substitutions in the minor structural proteins E and GP2 or GP3 and GP4 were unable to establish persistent infection in HeLa-L cells but that recombinant viruses with combined substitutions in the E (Ser53-->Cys and Val55-->Ala), GP2 (Leu15-->Ser, Trp31-->Arg, Val87-->Leu, and Ala112-->Thr), GP3 (Ser115-->Gly and Leu135-->Pro), and GP4 (Tyr4-->His and Ile109-->Phe) proteins or with a single point mutation in the GP5 protein (Pro98-->Leu) were able to establish persistent infection in HeLa-L cells. In summary, an in vitro model of EAV persistence in cell culture was established for the first time. This system can provide a valuable model for studying virus-host cell interactions, especially virus-receptor interactions.
Zhang
Jianqiang
J
Department of Veterinary Science, Maxwell H Gluck Equine Research Center, University of Kentucky, Lexington, Kentucky 40546-0099, USA.
Timoney
Peter J
PJ
MacLachlan
N James
NJ
McCollum
William H
WH
Balasuriya
Udeni B R
UB
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D018174:Arterivirus Infections / Q000662:veterinary* / Q000821:virology; D001483:Base Sequence; D002353:Carrier State / Q000662:veterinary* / Q000821:virology; D020732:Cytogenetic Analysis / Q000662:veterinary; D018274:Electroporation / Q000662:veterinary; D007240:Equartevirus / Q000145:classification / Q000235:genetics* / Q000254:growth & development / Q000276:immunology; D019084:Fluorescent Antibody Technique, Indirect / Q000662:veterinary; D006367:HeLa Cells; D006734:Horse Diseases / Q000821:virology*; D006736:Horses; D006801:Humans; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000662:veterinary; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D017423:Sequence Analysis, RNA / Q000662:veterinary; D017361:Viral Nonstructural Proteins / Q000378:metabolism
D000911:Antibodies, Monoclonal; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins