Murine Cytomegalovirus with a Transposon Insertional Mutation at Open Reading Frame m155 Is Deficient in Growth and Virulence in Mice

Abstract

A pool of murine cytomegalovirus (MCMV) mutants was previously generated by using a Tn3-based transposon mutagenesis approach (X. Zhan, M. Lee, J. Xiao, and F. Liu, J. Virol. 74:7411-7421, 2000). In this study, one of the MCMV mutants, Rvm155, which contained the transposon insertion in open reading frame m155, was characterized in vitro for its replication in tissue culture and in vivo for its growth and virulence in immunodeficient SCID mice. Compared to the wild-type strain and a rescued virus that restored the m155 region, the mutant is significantly deficient in growth in many organs of the infected animals. At 21 days postinfection the titers of Rvm155 in the salivary glands, lungs, spleens, livers, and kidneys of the intraperitoneally infected SCID mice were lower than the titers of the wild-type virus and the rescued virus by 50-, 1,000-, 500-, 100-, and 500-fold, respectively. Moreover, the viral mutant was attenuated in killing the SCID mice, as none of the SCID mice that were intraperitoneally infected with Rvm155 died until 38 days postinfection while all the animals infected with the wild-type and rescued viruses died at 27 days postinfection. Our results provide the first direct evidence that a disruption of m155 expression leads to attenuation of viral virulence and growth in animals. Moreover, these results suggest that m155 is a viral determinant for optimal MCMV growth and virulence in vivo.Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that causes mild or subclinical diseases in immunocompetent adults but may lead to severe complications in neonates and immunocompromised individuals (18, 23). Disseminated HCMV infection, common in AIDS patients, is usually associated with gastroenteritis, pneumonia, and retinitis (8,22). HCMV infection continues to be a major cause of morbidity and mortality in bone marrow and solid-organ transplant recipients (18, 23). Studies on the functions of viral genes in HCMV replication in vivo are essential for understanding viral pathogenesis and developing new strategies to combat the viral infection. However, there are presently no suitable animal models for HCMV infection. HCMV only propagates in human cells and grows slowly due to a long lytic replication cycle (18,23). These properties of HCMV have hampered the studies of HCMV pathogenesis and gene function.Infection of the mouse with murine cytomegalovirus (MCMV) provides a valuable in vivo model for studying the biology of CMV infection. This is because infection of mice by MCMV resembles in many ways its human counterpart with respect to pathogenesis (11,12,18,23). For example, tropism for the spleen and liver is believed to be important in the infection of both HCMV and MCMV, as the spleen and liver are the sites for primary acute infections as well as persistent and latent infections (18,23). The MCMV genome is 230 kb long and is predicted to encode more than 170 open reading frames, 78 of which have extensive homology to those of HCMV (4,25). A clear understanding of the biology of MCMV and the ...

Effective inhibition in animals of viral pathogenesis by a ribozyme derived from RNase P catalytic RNA

“…The improved survival of animals receiving M1-1 is probably because of the lower viral load found in the livers of these animals. This notion is consistent with the observation that a high level of viral lytic replication and production usually leads to severe damage of hepatic tissues and liver failure and contributes significantly to MCMV virulence and killing of SCID mice (1,2,5). Thus, our results suggest that inhibiting viral infection in the spleen and liver may significantly block viral systemic infection in other organs and increase host survival.…”

“…infection, animals were killed and the spleens, livers, lungs, and salivary glands were harvested (5). The viral titers in each of the organs from animals receiving either M1-2 or M1-TK were similar to those in animals that received only PBS (Fig.…”

“…Immunodeficient animals, such as SCID mice, have been shown to be extremely susceptible to MCMV infection (1,5,6). To determine the effect of M1GS ribozymes on the replication and infection of MCMV in vivo, we applied hydrodynamic transfection (24-26) of plasmid LXSN-M1GS DNA to SCID mice that had been i.p.…”

“…NIH 3T3 cells expressing different ribozymes were constructed, and the expression of these ribozymes was assayed using Northern analyses, following procedures described previously (see SI Materials and Methods) (5,20).…”

“…Murine cytomegalovirus (MCMV) infection of mice resembles its human counterpart with respect to pathogenesis, thus providing an animal model for studying CMV infection in vivo and for screening novel agents and developing new antiviral approaches (1)(2)(3)(4). For example, the CB17 SCID mice, which lack functional T and B lymphocytes, are highly susceptible to MCMV infection (5,6). Analysis of viral replication in these mice can be used for studying whether new therapeutic approaches block CMV opportunistic infection and prevent viral-associated diseases in immunocompromised hosts.…”