A retrograde trafficking inhibitor of ricin and Shiga-like toxins inhibits infection of cells by human and monkey polyomaviruses

Abstract

Polyomaviruses are ubiquitous pathogens that cause severe disease in immunocompromised individuals. JC polyomavirus (JCPyV) is the causative agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML), whereas BK polyomavirus (BKPyV) causes polyomavirus-induced nephropathy and hemorrhagic cystitis. Vaccines or antiviral therapies targeting these viruses do not exist, and treatments focus on reducing the underlying causes of immunosuppression. We demonstrate that retro-2(cycl), an inhibitor of ricin and Shiga-like toxins (SLTs), inhibits infection by JCPyV, BKPyV, and simian virus 40. Retro-2(cycl) inhibits retrograde transport of polyomaviruses to the endoplasmic reticulum, a step necessary for productive infection. Retro-2(cycl) likely inhibits polyomaviruses in a way similar to its ricin and SLT inhibition, suggesting an overlap in the cellular host factors used by bacterial toxins and polyomaviruses. This work establishes retro-2(cycl) as a potential antiviral therapy that broadly inhibits polyomaviruses and possibly other pathogens that use retrograde trafficking.

Importance: The human polyomaviruses JC polyomavirus (JCPyV) and BK polyomavirus (BKPyV) cause rare but severe diseases in individuals with reduced immune function. During immunosuppression, JCPyV disseminates from the kidney to the central nervous system and destroys oligodendrocytes, resulting in the fatal disease progressive multifocal leukoencephalopathy. Kidney transplant recipients are at increased risk of BKPyV-induced nephropathy, which results in kidney necrosis and loss of the transplanted organ. There are currently no effective therapies for JCPyV and BKPyV. We show that a small molecule named retro-2(cycl) protects cells from infection with JCPyV and BKPyV by inhibiting intracellular viral transport. Retro-2(cycl) treatment reduces viral spreading in already established infections and may therefore be able to control infection in affected patients. Further optimization of retro-2(cycl) may result in the development of an effective antiviral therapy directed toward pathogens that use retrograde trafficking to infect their hosts.

FIG 1

Retro-2^cycl prevents infection with three polyomaviruses. (A) Dose-dependent effect of retro-2^cycl treatment on infection. Cells were preincubated with the indicated concentrations of retro-2^cycl prior to inoculation with virus. Infections were scored and normalized to a DMSO-treated sample. (B) Retro-2^cycl does not block infection with adenovirus pseudovirus. Vero cells were pretreated with equivalent concentrations of retro-2^cycl prior to infection with an Ad5-GFP pseudovirus. Cells were scored and normalized to a DMSO-treated sample. (C) Retro-2^cycl prevents virus spreading in a multicycle growth assay. Cells were infected with virus for 72 h. Cells were then maintained in 0.1 mM retro-2^cycl. Cells were scored for infection every 3 days. (D) Retro-2^cycl-treated cultures release less infectious virus into culture medium. Tissue culture medium was harvested every 3 days and used to infect naive cells not treated with retro-2^cycl. The data represent the mean of three replicates, and error bars indicate the standard deviation.

FIG 2

Structure of retro-2^cycl and inhibitory activities of retro-2 analogs. (A) The structure of retro-2^cycl was solved by X-ray diffraction, and it was verified to be a DHQ. (B) Retro-2^cycl protects cells from polyomavirus infection. Cells were pretreated with retro-2, the indicated analog, the vehicle control, or BFA (71 nM, 20 ng/ml) and infected with JCPyV. Infected cells were scored and normalized to the vehicle-treated control. The data represent the average of triplicate samples. Error bars indicate the standard deviation.

FIG 3

Retro-2^cycl inhibits polyomavirus infectivity at early time points during infection. (A) Cells were chilled prior to incubation with JCPyV, BKPyV, or SV40 to synchronize infections, and retro-2^cycl (0.1 mM) was added at the indicated time points. After 72 h, infected cells were scored and normalized to the vehicle control. The data represent the mean of three replicates, and error bars indicate the standard deviation. (B) Effect of retro-2^cycl on virus binding to cells. Cells were detached and pretreated with the vehicle control or retro-2^cycl for 30 min at 37°C. Cells were then inoculated with Alexa Fluor 633-labeled JCPyV, BKPyV, SV40, or CTxB for 1 h on ice. Samples were then washed and read by flow cytometry. CTB, CTxB.

FIG 4

Retro-2^cycl inhibits polyomavirus ER trafficking. (A) Colocalization was assessed with a PLA. Error bars denote the standard deviation. (B) Cells were pretreated with the indicated drug (500 ng/ml BFA or 0.1 mM retro-2^cycl) for 0.5 h prior to inoculation with JCPyV, BKPyV, or SV40 at an MOI of 100. Cells were incubated for 8 h with the indicated drugs, fixed, and permeabilized. Cells were then stained with a mouse monoclonal antibody to PDI and a rabbit polyclonal antibody to VP1 prior to detection by PLA. Fluorescent foci indicate areas of colocalization. BKV, BKPyV.

FIG 5

Retro-2^cycl inhibits VP2 exposure of polyomaviruses. (A) VP2 is exposed at late time points during infection. Cells were pretreated with the indicated drugs and then inoculated with JCPyV, BKPyV, or SV40 at an MOI of 10 for 10 h before fixation and staining for VP2. VP2 puncta are green, and nuclei are blue. Scale bars, 10 µm. (B) VP2 is exposed in the ER. Cells were incubated with SV40 for 10 h, fixed, and then stained for VP1 (green), VP2 (red), and PDI (purple), and the nuclei were stained with BOBO-3 (blue). On the right, enlargements of the boxed area of the fluorescence micrograph show individual antibody staining. (C) Quantitation of panel A. Cells from triplicate samples were scored for the presence of VP2. Error bars show the standard deviations.