WSU Virology

we suggest that RABV hijacks a specific mechanism that enables the neuron to transport cargos over long distances.

Rabies virus (RABV) is a neurotropic virus that depends on long distance axonal transport in order to reach the central nervous system (CNS).

Measuring both the area and average intensities of the RABV particles in each group, we found that the p75NTR positive RABV particles were larger in size, (average area of 1.34±0.09 µm2 vs. 0.81±0.07 µm2, p<0.0005), and had stronger intensity of GFP signal, when normalized to the average intensity of RABV particles in each experiment (1.24±0.11 vs. 0.61±0.1, p<0.001) (Fig. 8K–L).

ence there are likely to be additional ways for RABV to merge into the p75NTR-RABV endosome

Other membrane-associated components have also been implicated in RABV binding [20]. By binding one of its receptors, RABV could enter the cell and activate downstream signaling which would allow it to hijack and manipulate axonal transport machineries. Although p75NTR is known to be involved in the retrograde transport of neurotrophic factors, little is known regarding its direct contribution to viral transport.

Possibly, RABV binding to dynein tethers projecting microtubules (MT) in the cell cortex thereby facilitating its retrograde trafficking from the cell periphery. Following this tethering, RABV particles can merge into the RABV-p75NTR endosomes and travel to the neuron cell body.

Within the VF, viral core particles transcribe and release viral mRNAs that possess a dimethylated cap 1 structure at the 5′ terminus but lack a poly(A) tail (19).

Consistent with our findings, the authors noted that the rough endoplasmic reticulum (RER) made numerous contacts with VF, which they suggested may indicate a role for RER in the transport of newly synthesized viral proteins to the VF, as is the case for rubella virus (64).

To address this, we monitored protein expression levels of eIF4E, eIF4A1, and eIF4G over the course of an infection. As others have found (39), we were unable to detect any difference in the levels of total protein in mock versus infected cells from 0 to 20 h p.i. (Fig. 3C and data not shown). Together, these data suggest that cellular translation proteins are redistributed to the VF.

Most reovirus strains form filamentous VF through an association with stabilized microtubules. However, the T3D strain used in these experiments contains a temperature-sensitive mutation in the viral protein μ2 that prevents this association, resulting in the production of globular VF at 37°C (27, 28). Therefore, to evaluate if PMY labeling occurs within filamentous viral factories, we infected cells with the serotype 1 Lang (T1L) strain. As we found for T3D-infected cells, the PMY labeling localized to T1L VF at 18 h postinfection

It was unclear if this was a result of increased expression levels or as a consequence of redistribution of the proteins to the factories. To address this, we monitored protein expression levels of eIF4E, eIF4A1, and eIF4G over the course of an infection. As others have found (39), we were unable to detect any difference in the levels of total protein in mock versus infected cells from 0 to 20 h p.i. (Fig. 3C and data not shown). Together, these data suggest that cellular translation proteins are redistributed to the VF.

Our finding that σNS interacts with eIF3A and pS6R suggests that translational machinery is recruited to the factory by viral proteins. This is consistent with the finding of others that σNS cosediments with 40S and 60S ribosomes (62) and suggests that σNS is directly involved in viral translation.

FIG 3  Cellular translation initiation factors colocalize to viral factories. (A, B) CV-1 cells were infected with T3D or T1L at an MOI of 1. At 18 h p.i., RPM-labeled cells were coimmunostained for μNS and eIF4E (A) or eIF3A (B). Scale bars, 10 µm. (C) CV-1 cells were infected with T3D, MOI of 3, for the times indicated. Protein levels were assessed by immunoblotting. M = mock.