Visualization of the lymphocytic choriomeningitis mammarenavirus (LCMV) genome reveals the early endosome as a possible site for genome replication and viral particle  pre-assembly

Abstract

We report a fluorescence in situ hybridization (FISH) assay that allows the visualization of lymphocytic choriomeningitis mammarenavirus (LCMV) genomic RNAs in individual cells. We show that viral S segment genomic and antigenomic RNA, along with viral nucleoprotein, colocalize in subcellular structures we presume to be viral replication factories. These viral RNA structures are highly dynamic during acute infection, with the many small foci seen early coalescing into larger perinuclear foci later in infection. These late-forming perinuclear viral RNA aggregates are located near the cellular microtubule organizing centre and colocalize with the early endosomal marker Rab5c and the viral glycoprotein in a proportion of infected cells. We propose that the virus is using the surface of a cellular membrane-bound organelle as a site for the pre-assembly of viral components, including genomic RNA and viral glycoprotein, prior to their transport to the plasma membrane, where new particles will bud.

Keywords: LCMV; RNA FISH; Rab5c; arenavirus; viral RNA genome; viral replication complex.

Fig. 1.

Visualization of S genome and antigenomic RNAs by multiple, singly-labelled FISH probes. (a) Diagram showing the transcription and replication scheme of the LCMV S genomic RNA. Briefly, the S genome serves as the template for the viral polymerase to generate full-length, antigenome replicative intermediates. The S genome and S antigenome serve as templates for the transcription of the NP and GPC mRNAs, respectively. FISH probe sets (each containing 48 individual 20mer probes bearing a single fluorophore at their 3′ terminus) were used to specifically visualize either the S genomic or S antigenomic RNA. (b) Maximum intensity projection of either mock- or LCMV-infected cells (48 h p.i.) stained with S genome FISH probes labelled with Cy3. (c) Maximum intensity projection of either mock- or LCMV-infected cells (48 h p.i.) stained with S antigenome FISH probes labelled with Cy3. (d) Single Z stack of either mock- or LCMV-infected cells (48 h p.i.) stained for S genome (Cy5) and LCMV nucleoprotein [1–1.3 (from M. Buchmeier, University of California Irvine) (primary antibody) as previously described [26]; goat, anti-mouse AlexaFluor 488 (secondary antibody)]. (e) Fluorescence line scan of S genome and NP signals along the line indicated in the inset of the merged image in (d). (f) Single Z stack of either mock- or LCMV-infected cells (48 h p.i.) stained with S genome (Cy5) and S antigenome (Cy3) FISH probes. (g) Fluorescence line scan of S genome and S antigenome signals along the line indicated in the inset of the merged image in (f). Scale bar=10 µm.

Fig. 2.

Dynamics of S genome and S antigenome during acute LCMV infection. Cells were infected with LCMV at an m.o.i. of 0.01 or not (mock) and fixed at multiple time points following infection. Maximum intensity projections of cells stained with S genome (Cy5) and S antigenome (Cy3) FISH probes are presented. Scale bar=10 µm.

Fig. 3.

LCMV S segment genome selectively colocalizes with Rab5c and viral glycoprotein later during acute infection. (a) At 48 h p.i., perinuclear S genome aggregates localize near the microtubule organizing centre (MTOC), as visualized by gamma-tubulin [GTU-88, Sigma-Aldrich (primary); goat anti-mouse AlexaFluor 488 (secondary)] and S genome (Cy5-labelled FISH probes). A maximum intensity projection of a representative cell is shown. (b) Single Z stack of either mock- or LCMV-infected cells at the indicated time points after infection were stained for S genome (Cy5-labelled FISH probes) and Rab5c [sc-365667, Santa Cruz Biotechnology (primary); goat anti-rabbit AlexaFluor 488 (secondary)]. (c) Fluorescence line scan of S genome and Rab5c along the line indicated in the inset of the merged image at 48 h p.i. (b) is shown. (d) The Pearson’s correlation coefficient between the S genome and Rab5c fluorescence signals in individual infected cells at either 24 or 48 h p.i. was calculated in softWoRx software and the scores of individual cells were graphed. (e) Single Z stacks of either mock- or LCMV-infected cells that were stained for S genome (Cy5-labelled FISH probes) and viral glyocoprotein (GPC) [mouse anti-GPC, 33.6 (from M. Buchmeier, University of California Irvine) (primary) at 1 : 500; goat anti-mouse AlexaFluor 488 (secondary)] are shown. (f) A fluorescence line scan of S genome and GPC along the line indicated in the inset of the merged image at 48 h p.i. (e) is shown. (g) The Pearson’s correlation coefficient between the S genome and GPC fluorescence signals in individual infected cells at either 12, 24, or 48 h p.i. was calculated in softWoRx software and the scores of individual cells were graphed.