Dissemination of lymphocytic choriomeningitis virus from the gastric mucosa requires G protein-coupled signaling

Abstract

The gastric mucosa is an important portal of entry for lymphocytic choriomeningitis virus (LCMV) infections. Within hours after intragastric (i.g.) inoculation, virus appears in the gastric epithelia, then in the mesenteric lymph nodes and spleen, and then in the liver and brain. By 72 h i.g.-inoculated virus is widely disseminated and equivalent to intravenous (i.v.) infection (S. K. Rai, B. K. Micales, M. S. Wu, D. S. Cheung, T. D. Pugh, G. E. Lyons, and M. S. Salvato. Am. J. Pathol. 151:633-639, 1997). Pretreatment of mice with a G protein inhibitor, pertussis toxin (PTx), delays LCMV dissemination after i.g., but not after i.v., inoculation. Delayed infection was confirmed by plaque assays, by reverse transcription-PCR, and by in situ hybridization. The differential PTx effect on i.v. and i.g. infections indicates that dissemination from the gastric mucosa requires signals transduced through heterotrimeric G protein complexes. PTx has no direct effect on LCMV replication, but it modulates integrin expression in part by blocking chemokine signals. LCMV infection of macrophages up-regulates CD11a, and PTx treatment counteracts this. PTx may prevent early LCMV dissemination by inhibiting the G protein-coupled chemotactic response of macrophages infected during the initial exposure, thus blocking systemic virus spread.

FIG. 1

PTx does not affect LCMV mRNA production in splenocyte cell culture. LCMV Gp mRNA was detected by RT-PCR, starting with total RNA from splenocyte cultures. Agarose gel electrophoresis indicates that the expected 1-kb LCMV Gp product is not detected in uninfected splenocytes (lane 1) but is detected in samples from in vitro LCMV-infected splenocytes (lane 2), despite pretreatment (lane 3) or posttreatment (lane 4) with PTx (0.1 μg/ml). Lane N is a negative control for PCR. The 447-bp DHFR gene product was used as an internal control. Lane M is a 1-kb DNA ladder supplied by Gibco BRL (the upper gel depicts only the 1-kb marker fragment, whereas the lower gel depicts 0.5-kb and smaller marker fragments).

FIG. 2

In vivo effects of PTx on virus. LCMV Gp mRNA was detected by RT-PCR of total RNA isolated from kidneys and livers of i.g.- or i.v.-infected mice. Gel electrophoresis of the RT-PCR product of total RNA isolated from the kidneys of i.g.- and i.v.-infected mice 3 days after infection is shown. The expected 1-kb LCMV Gp product is detected in samples from an i.g.-infected mouse (lane 1), an i.v.-infected mouse (lane 2), and a PTx-pretreated, i.v.-infected mouse (lane 4) but not in that from a PTx-pretreated, i.g.-infected mouse (lane 3). Lane 5 is from the kidney of an uninfected mouse. Lane P is a 1-kb LCMV Gp fragment. Lane N is a negative control for PCR. The 447-bp DHFR gene product was used as an internal control. Lane M is a 1-kb DNA ladder supplied by Gibco BRL (the upper gel depicts only the 1-kb marker fragment, whereas the lower gel depicts 0.5-kb and smaller marker fragments).

FIG. 3

In situ hybridization of various mouse tissue sections with an LCMV Gp probe. (a) Stomach sections at 24 h showing infection of the gastric epithelium in the body of the stomach. Magnification, ×100. (b and c) Spleen and liver sections, respectively, at 48 h after infection. Magnification, ×100. IG, i.g. inoculation; IV, i.v. inoculation.

FIG. 4

Absolute leukocyte counts in peripheral blood from LCMV-infected mice with or without PTx pretreatment. Leukocytes were counted under a 20×-objective light microscope in the presence of trypan blue to eliminate dead cell counts. Data are represented as leukocytes/milliliter of whole blood at days 7 and 10 after toxin administration. IV, i.v. inoculation; IG, i.g. inoculation.

FIG. 5

Phenotypic analysis of mouse PBMC at different time points (days 0, 3, 7, and 10) after PTx treatment in vivo. All five of the adhesion molecules monitored show decreased expression on PBMC 3 to 7 days after PTx treatment and return to near normal by 10 days.