Disruption of the murine gammaherpesvirus 68 M1 open reading frame leads to enhanced reactivation from latency

Murine gammaherpesvirus 68 (gamma HV68, or MHV-68) is a! genetically tracta ble, small animal model for the analysis of gammaherpesvirus pathogenesis. The gamma HV68 genome is colinear with the genomes of other sequence gammah erpesviruses, containing large blocks of conserved genes interspersed by a number of putative genes without clear homologs in the other gammaherpesvir uses. One of these putative unique genes, the M1 open reading frame (ORF), exhibits sequence homology to a poxvirus serine protease inhibitor, SPI-1, as well as to another gamma HV68 gene, M3, which we have recently shown enc odes an abundantly secreted chemokine binding protein. To assess the contri bution of the M1 ORF to gamma HV68 pathogenesis, we have generated a recomb inant gamma HV68 in which the M1 ORF has been disrupted through targeted in sertion of a lacZ expression cassette (M1.LacZ). Although M1.LacZ replicate d normally in tissue culture, it exhibited decreased splenic titers at days 4 and 9 postinfection in both immunocompetent and immunodeficient mice. De spite decreased levels of acute virus replication, M1.LacZ established a la tent infection comparable to wild-type (wt) gamma HV68, but exhibited an ap proximately fivefold increase in efficiency of reactivation from latency. M 1.LacZ also caused severe vasculitis of the great elastic arteries in gamma interferon receptor (IFN-gamma R)-deficient mice with a frequency comparab le to wt gamma HV68, but did not cause the mortality or splenic pathology o bserved with wt gamma HV68 infection of IFN-gamma R-deficient mice. Restora tion of M1 ORF sequences into M1.LacZ (M1 marker rescue, or M1.MR) demonstr ated that M1.LacZ phenotypic alterations in growth in vivo and latency were not due to the presence of additional mutations located elsewhere in the M 1.LacZ genome. Generation of a second M1 mutant virus containing a deletion at the 5' end of the M1 ORF (M1 Delta 511), but lacking the LacZ expressio n cassette, revealed the same latency phenotype observed with the M1.LacZ m utant. However, M1 Delta 511 was not attenuated for acute virus replication in the spleen. We conclude that (i) the induction of arteritis in gamma HV 68-infected IFN-gamma R-deficient mice cam occur in the absence of splenic pathology and mortality, (ii) replication during acute infection is not the primary determinant for the establishment of latent infection, and (iii) t he M1 ORF, or a closely linked gene, encodes a gene product that functions to suppress virus reactivation.