Analysis of lymphocytic choriomeningitis virus gene expression in acutely and persistently infected mice.

Abstract

Department of Immunology, Scripps Clinic and Research Foundation, La Jolla, CA 92037, USA Lymphocytic choriomeningitis virus (LCMV) is a desirable model for the study of virus persistence because (i) it establishes a natural state of persistence in wild mice and in experimentally infected laboratory animals, (ii) there is an extensive body of knowledge in genetically defined hosts concerning why the immune response to the virus does not lead to viral clearance [1, 2] and (iii) the virus is relatively non-cyto- pathic in cells but can selectively impair specialized cellular functions [1, 5]. There are two major requirements for in vivo establishment of a state of viral per- sistence: (i) failure of host defence mechanisms to eliminate the virus and (ii) a non- cytopathic cell/virus relationship. We have been studying the molecular mechanisms by which LCMV persists focusing on the in vivo interactions of the virus with host ceils. We have constructed and partially sequenced a cDNA library from LCMV (Arm- strong strain) virion RNA -- virtually all of the S and 50% of the L segments have been sequenced. We have identified potential protein coding regions within the viral genomic sequences and produced antisera to synthetic peptides from these regions to identify nucleoprotein, glycoprotein, and the L (probable polymerase) protein coding assign- ments. These clones have been used to characterize LCMV gene expression during acute and persistent infections in mice and tissue culture. Since the sequencing and protein encoding potential of the L segment is not fully known we have focused our analysis primarily on the S RNA segment. Groups of four BALB/c mice persistently infected following intracerebral inocula- tion of 30 PFU of LCMV as neonates were sacrificed at 5, 9, 13, and 21 days and 5 months of age, and organs were used for extraction of total cellular RNA. Similar pro- cedures were followed for acutely infected adult BALB/c mice with samples taken at 1, 3, and 5 days after initiating infection (these mice die 7 to 9 days post-viral inocu- lation from an acute immune-mediated inflammatory response). Frozen organs were placed in guanidinium thiocyanate, a potent protein denaturant which inactivates RNase, and homogenized with Tekmar tissumizer. RNA was purified by pelleting through a cushion of 5.7M cesium chloride followed by ethanol precipitation. Equi- valent amounts of total intracellular RNA were size-fractionated by denaturing (form- aldehyde) agarose gel electrophoresis, transferred to nitrocellulose, and hybridized with nick-translated 32p-labelled LCMV cDNA probes.