Involvement of Microtubular Network and Its Motors in Productive Endocytic Trafficking of Mouse Polyomavirus

Abstract

Infection of non-enveloped polyomaviruses depends on an intact microtubular network. Here we focus on mouse polyomavirus (MPyV). We show that the dynamics of MPyV cytoplasmic transport reflects the characteristics of microtubular motor-driven transport with bi-directional saltatory movements. In cells treated with microtubule-disrupting agents, localization of MPyV was significantly perturbed, the virus was retained at the cell periphery, mostly within membrane structures resembling multicaveolar complexes, and at later times post-infection, only a fraction of the virus was found in Rab7-positive endosomes and multivesicular bodies. Inhibition of cytoplasmic dynein-based motility by overexpression of dynamitin affected perinuclear translocation of the virus, delivery of virions to the ER and substantially reduced the numbers of infected cells, while overexpression of dominant-negative form of kinesin-1 or kinesin-2 had no significant impact on virus localization and infectivity. We also found that transport along microtubules was important for MPyV-containing endosome sequential acquisition of Rab5, Rab7 and Rab11 GTPases. However, in contrast to dominant-negative mutant of Rab7 (T22N), overexpression of dominant-negative mutant Rab11 (S25N) did not affect the virus infectivity. Altogether, our study revealed that MPyV cytoplasmic trafficking leading to productive infection bypasses recycling endosomes, does not require the function of kinesin-1 and kinesin-2, but depends on functional dynein-mediated transport along microtubules for translocation of the virions from peripheral, often caveolin-positive compartments to late endosomes and ER – a prerequisite for efficient delivery of the viral genome to the nucleus.

Hydrophobic domains of mouse polyomavirus minor capsid proteins promote membrane association and virus exit from the ER

“…In this assay, the appearance of fluorescent spots indicates that the proximity of target proteins is < 40 nm. We selected the time 5 hpi, since, at this time, a subpopulation of the WT virus has previously been detected in ER . The PLA spots per cell were quantified in three independent experiments in 40 cells per experiment for each virus variant.…”

Viral escape from endosomes and host detection at a glance

“…Endocytosis is highly complex and dynamic, and involves recycling, trafficking, maturation and fusion of endocytic vesicles (Grant and Donaldson, 2009). Viruses that are running the endocytic gauntlet need to make sure they escape the endosomal compartment before being recycled back into the extracellular space (Zila et al, 2014;Mainou and Dermody, 2012;Grove and Marsh, 2011) or subjected to degradation in the harsh environment of the lysosome (see poster). To this end, enveloped viruses (e.g.…”

ICAM-1 Binding Rhinoviruses A89 and B14 Uncoat in Different Endosomal Compartments

“…Although a number of viruses traffic through the ERC during entry (55)(56)(57), their infection was not inhibited by Rab11-S25N, which is indicative of the Twenty-four hours later, the respective virus (at 100 TCID 50 /cell) was allowed to internalize for 60 min; unbound virus was washed away with cold PBSϩ containing 25 mM NH 4 Cl for 15 min, and replication was allowed to proceed for 6.5 h at 37°C in infection medium containing NH 4 Cl. Cells were fixed, quenched, permeabilized, and blocked, and de novo-synthesized viral protein was detected with the respective antibodies, followed by suitable fluorescent secondary antibodies.…”