Microtubule-dependent transport of arenavirus matrix protein demonstrated using live-cell imaging microscopy

Abstract

Lassa virus (LASV), belonging to the family Arenaviridae, causes severe haemorrhagic manifestations and is associated with a high mortality rate in humans. Thus, it is classified as a biosafety level (BSL)-4 agent. Since countermeasures for LASV diseases are yet to be developed, it is important to elucidate the molecular mechanisms underlying the life cycle of the virus, including its viral and host cellular protein interactions. These underlying molecular mechanisms may serve as the key for developing novel therapeutic options. Lymphocytic choriomeningitis virus (LCMV), a close relative of LASV, is usually asymptomatic and is categorized as a BSL-2 agent. In the present study, we visualized the transport of viral matrix Z protein in LCMV-infected cells using live-cell imaging microscopy. We demonstrated that the transport of Z protein is mediated by polymerized microtubules. Interestingly, the transport of LASV Z protein showed characteristics similar to those of Z protein in LCMV-infected cells. The live-cell imaging system using LCMV provides an attractive surrogate measure for studying arenavirus matrix protein transport in BSL-2 laboratories. In addition, it could be also utilized to analyze the interactions between viral matrix proteins and the cellular cytoskeleton, as well as to evaluate the antiviral compounds that target the transport of viral matrix proteins.

Generation of Reporter-Expressing New World Arenaviruses: A Systematic Comparison

“…Nonetheless, despite not being viable in a virus context, these directly C-terminally reporter fused NPs are well-expressed and easily detectable, and thus, still have significant potential value, for instance in imaging studies examining inclusion body dynamics and/or protein trafficking. In these cases, the fluorescent versions of NP could be transfected either alone, together with other components of the ribonucleoprotein complex, or even together with a TCRV-wt infection, such as has been previously done for studies of LCMV Z trafficking [ 75 ].…”

“…Interestingly, these differences in viral growth between the directly fused constructs and the corresponding constructs containing a T2A site do not seem to be explained by differences in budding efficiency or the interactions of Z with either NP or L, as seen from the VLP and minigenome data. While plasmid-based expression of LCMV Z directly fused to RFP at its C-terminus has been previously used in microscopy studies exploring the trafficking of this protein [ 75 ], this is the first indication that such fusions are also viable in a viral context. It is, however, worth noting that budding of TCRV Z is somewhat unusual among arenaviruses, in that it occurs without canonical proline-rich late domains that normally play a major role in the recruitment of ESCRT proteins [ 80 ].…”

The Integrity of the YxxL Motif of Ebola Virus VP24 Is Important for the Transport of Nucleocapsid-Like Structures and for the Regulation of Viral RNA Synthesis

“…For live-cell imaging, Huh-7 cells were seeded onto 4-well (2 ϫ 10 4 cells)/8well (1 ϫ 10 4 cells) -Slide (ibidi) and cultivated in DMEM-PS-Q with 10% FBS. The transfection was performed in 50 l Opti-MEM without phenol red (Life Technologies) (75,76). The inoculum was removed at 1 h p.t., and a volume of 250 l of CO 2 -independent Leibovitz's medium was added (Life Technologies).…”

Recruitment of dynein and kinesin to viral particles

“…The NW JUNV and MACV arenaviruses group also interact with KIF13A. Thus, the MT‐dependent motor protein regulates the trafficking of LASV Z proteins, and KIF13A promotes viral infection through a conserved mechanism of anterograde transport within the Arenaviridae family 53,107 …”