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We report the development of recombinant New World (Junín; JUNV) and Old World (lymphocytic choriomeningitis virus; LCMV) mammarenaviruses that encode an HA-tagged matrix protein (Z). These viruses permit the robust affinity purification of Z from infected cells or virions, as well as the detection of Z by immunofluorescent microscopy. Importantly, the HA-tagged viruses grow with wild-type kinetics in a multi-cycle growth assay. Using these viruses, we report a novel description of JUNV Z localization in infected cells, as well as the first description of colocalization between LCMV Z and the GTPase Rab5c. This latter result, when combined with our previous findings that LCMV genome and glycoprotein also colocalize with Rab5c, suggest that LCMV may target Rab5c-positive membranes for preassembly of virus particles prior to budding. The recombinant viruses reported here will provide the field with new tools to better study Z protein functionality and identify key Z protein interactions with host machinery.

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.