Cell, Vol. 19, 871-880, April 1980, Copyright 0 1980 by MIT Virus Protein Changes and RNA Termini Alterations Evolving during Persistent Infection David Rowlands,* Elizabeth Grabau, Katherine Spindler, Charlotte Jones,? Bert Semler$ and John Holland Department of Biology University of California, San Diego La Jolla, California 92093 Summary Cloned infectious vesicular stomatitis virus isolated following 5 years of persistent infection of BHK21 cells in vitro exhibits a number of peptide map changes in the G protein (spike glycoprotein), the M protein (membrane matrix protein) and the N protein (nucleocapsid structural protein). Only slight alterations have occurred in the peptide maps of the two VSV polymerase-associated proteins L and NS. Dideoxy sequencing of the 3’ ends of the cloned virus originally used to establish the persist- ent infection, and of the cloned virus recovered following 5 years of persistence, shows one base substitution in the three base junction between the 3’ leader sequence and the N protein-coding region. Repeated lytic passages of virus recovered from persistent infection led to no oligonucleotide map changes after 30 passages, but two map changes were present after 102 and remained after 133 lytic passages in BHK21 cells in vitro. Only one of these represented reversion to the original map position, and this “mutant” virus still exhibited a tempera- ture-sensitive small plaque phenotype. Finally, the mutated virus recovered after more than 5% years of persistent infection is now so slow-growing that it can establish persistent infection of BHKPl cells in the absence of DI particles (although DI particles are present constantly once the cells recover from the initial cytopathology). Introduction We recently presented oligonucleotide mapping evi- dence (Holland et al., 1979) that vesicular stomatitis virus (VSV) genome RNA undergoes extensive and continuous mutation during long-term persistence in BHK21 cells in culture. In the present report we ex- tend these findings to demonstrate that these oligo- nucleotide map changes are reflected by peptide map alterations in all five viral proteins and by several base * Present address: Animal Virus Research Institute, Pirbright, Wok- ing. Surrey, England. t Present address: Department of Cellular and Viral Molecular Biol- ogy, School of Medicine, University of Utah, Salt Lake City, Utah 84132. $ Present address: Department of Microbiology, School of Basic Health Sciences, State University at Stony Brook, Stony Brook, New York 11794. substitutions at the 3’ end of viral genome RNA, which is the initiation site for both viral transcription and replication. Persistent infection by DNA and RNA viruses of humans and animals is of considerable importance because of its known involvement in chronic or recur- rent diseases and because of its suspected involve- ment in many others (for a recent in-depth review see Stevens, Todaro and Fox, 1978). Although the molec- ular, cellular and immunological basis for long-term viral persistence has not been clearly defined for any system, three major mechanisms have been estab- lished thus far: integration of DNA viral genomes or of DNA copies of retroviral RNA genomes; the generation of temperature-sensitive mutants by RNA viruses; and the generation of defective interfering particles (DI particles) by RNA viruses. Huang and Baltimore (1970) first suggested that DI particles, because of their widespread occurrence among animal viruses, might be involved in recovery from acute infections and in facilitating persistent in- fections. Holland and Villarreal (1974) showed that DI particles are required for the establishment and main- tenance of persistent infection of BHK21 ceils by VSV. Other persistent RNA virus infections in which DI particles have been implicated include those of reo- virus (Ahmed and Graham, 1977) rabies virus (Kawai, Matsumoto and Tanabe, 1975) measles virus (Rima, Davidson and Martin, 1977), LCM virus (Welsh et al., 1975; Popescu and Lehmann-Grube, 1977) Japa- nese encephalitis virus (Schmaljohn and Blair, 1977) and others. Somewhat surprisingly (in view of the option to persist by integration), recent work has begun to link DI particles to DNA virus persistence in certain cell types. These viruses include SV40 (Nor- kin, 1979) human cytomegalovirus (Stinski, Mocarski and Thomsen, 1979) equine herpesvirus type 1 (Henry, Newcomb and O’Callaghan, 1979) and oth- ers. Perhaps this link arises from the fact that a plasmid state better facilitates continuous expression of mature virus in many cell-virus systems, and the emergence of DI particles may be necessary to atten- uate cytopathology. Fields and Raine (1974) Preble and Youngner (1973) and Youngner et al. (1976) have described persistent infections of mouse cells in culture by reo- virus, Newcastle disease virus and VSV in which tem- perature-sensitive mutants of infectious virus, rather than DI particles, are the major determinants of per- sistence. Wagner et al. (1963) have demonstrated that small plaque mutants of VSV establish persistent infections more readily than do standard large plaque virus. Ramseur and Friedman (1977) showed that interferon (as well as ts mutants) is involved in per- sistence of VSV in L cells. Nishiyama (1977) and Nishiyama, Ito and Shimokata (1978) have implicated all three of the above (DI particles, interferon and ts