Mammarenaviral Infection Is Dependent on Directional Exposure to and Release from Polarized Intestinal Epithelia

Warner, Nikole L.; Jokinen, Jenny D.; Beier, Juliane I.; Sokoloski, Kevin J.; Lukashevich, Igor S.

Parallel analysis of MOPV infection reveals a pattern of viral release similar to that observed for LCMV. As shown in (Figure 5A) , the apical infection of polarized Caco-2 cells primarily resulted in the release of infectious viral particles from the apical surface; however, basolateral release was observed. Similar to LCMV, and different from ML-29, basolateral infection of polarized Caco-2 cells resulted in the more-or-less equivalent release of viral progeny apically and basolaterally. Supernatants were collected from both the insert, and well of the Transwells, to determine viral release from the apical or basolateral surfaces independent from one another. Viral titer was measured using standard plaque assay. Release from the Apical surface (black) and the Basolateral surface (red) is plotted with respect to time, with initial viral load subtracted. Values shown are the means of 3 biological replicates with the error bar representing the standard deviation of the mean. If viral PFUs were not observed, data received a place-holder value to signify samples were tested, but no data (ND) was collected. # indicates that one or more biological replicates was below limit of detection Analysis of LCMV attachment indicated that for both LCMV-ARM and LCMV-WE~5% of the input virus adsorbed to the polarized Caco-2 cells (Figure 6A,B) . Comparisons of apical-and basolateral-bound viral levels indicated that LCMV-ARM exhibited preferential binding to the basolateral surface of the polarized Caco-2 cells ( Figure 6A ). LCMV-WE, in contrast, did not exhibit preferential binding to either surface ( Figure 6B ). Collectively, these data indicate a potential difference between the two LCMV strains in regards to cell attachment. Assessment of ML-29 binding indicated a strong attachment preference to the apical surface of the cells. As shown in (Figure 6C ), approximately 4-fold more virus attached to the apical surface relative to the basolateral surface of polarized Caco-2 cells. These data are in apparent congruence with the observations reported in Figure 4 , where basolateral infection were less efficient as compared to parallel apical infections. Quantitative analysis of MOPV attachment reveals similar observations to LCMV-WE. Specifically, as shown in ( Figure 6D ) the attachment of MOPV virus was equivalent amongst the apical and basolateral surfaces.