Pseudovirus mimics cell entry and trafficking of the human polyomavirus JCPyV

Abstract

The normally asymptomatic human polyomavirus, JCPyV, is the causative agent of a rare but fatal demyelinating disease known as progressive multifocal leukoencephalopathy (PML). Individuals at risk for developing PML include those with AIDS, with other underlying immunosuppressive diseases, and in patients treated with immunomodulatory regimens. Drugs to prevent viral reactivation in the setting of immunosuppression or immunomodulation could be used to sustain lives. Development of such drugs has been impeded by the difficulty of growing and studying the virus. We sought to develop a more efficient method for screening drugs that inhibit viral infection. Pseudovirus models have been developed which may be of use in pharmaceutical research. The use of pseudoviruses as models for viral infection is dependent on them using similar pathways for infection as virus. We screened known inhibitors of viral entry for their ability to block pseudovirus infection. Here we show that the pseudovirus based on the human polyomavirus JCPyV recapitulates virus binding, entry and trafficking. This system can be used for high-throughput screening of antiviral drugs.

Keywords: JC polyomavirus; Natalizumab; Progressive multifocal leukoencephalopathy; Pseudovirus; Virus entry.

Figure 1. JCPsV maintains the tropism of authentic virus

Cell lines from different species were infected with JCPsV. At 72 hours post infection luciferase expression was measured to determine whether JCPsV maintained the tropism of authentic virus. In cell lines which were negative, we verified that the reporter plasmid could be express using transfection and concluded that PsV was unable to deliver the reporter plasmid to the nucleus (Data not shown). (Ha = Hamster)

Figure 2. Entry of JCPsV is specifically blocked by neutralizing antibody against JCPyV

JCPsV was pretreated with rabbit preimmune serum, anti-BK or anti-JC for 1 hour and then used to infect SVG cells. Infection was measured 72 hours post infection (RLU). Anti-JC blocked JCPsV infection of SVGA cells (p = 0.04). The experiment was performed in triplicate and bars represent the standard error.

Figure 3. Removal of sialic acid inhibits JCPsV entry

3A: Cells were pretreated for 45 minutes at 37°C with PBS (pH5 with Mg²⁺ and Ca²⁺) or .8U/100uL Type II neuraminadase (NA) in PBS (pH5 + Mg₂ and Cl₂). After infections JCPsV was removed and replaced with complete media and antibody to neutralize unbound JCPsV. Secreted luciferase was measured 72 hours post infection. Neuraminadase blocked JCPsV infection of SVG cells (p = 0.006). Error bars represent the standard error in three experiments. 3B: JCsV behaves as JCPyV and hemagglutinates type O red blood cells. The ablility of two dilutions of JCPsV genome equivalents (GE) was compared to purified wild type JCPyV and controls (33% Iodixanol in PBS, PBS, purified JCPyV or mock pseudovirus). 10⁸ JCPsV GE hemagglutinated as well as 50 uL of purified virus.

Figure 4. Disruption of clathrin-dependent endocytocis significantly reduces JCPsV infection

4A: SVGA cells were untreated or pretreated with 5 μM chlorpromazine for 2 hours prior to infection. After infection, JCPsV was removed and replaced with drug in complete media and neutralizing antibody. Secreted luciferase was measured 72 hours post infection in three separate experiments and bars represent the standard error. Chlorpromazine treatment significantly reduced JCPsV infection by approximately half (p = 0.001). 4B: To confirm this effect, we counted the number of GFP+ cells and confirmed that chlorpromazine treatment and observed a significant reduction in the number of infected cells. (p = 4×10⁻⁸) The experiment was performed in triplicate and bars represent the standard error. 4C: To control for toxicity and cell viability, an MTS assay performed in the same well as the luciferase reading. The absorbance at 490 nm was averaged for the three experiments and bars represent the standard error.

Figure 5. JCPsV requires endosomal acidification for infection

5A: SVGA cells were pretreated with 20 mM NH₄Cl for (4 hours), 10 nM monesin, 10 nM bafilomycinA1 (BafA1) or DMSO control for 1 hr. After infection, PsV was removed and replaced with drug in complete media and neutralizing antibody. Secreted luciferase was measured 72 hours post infection. Blocking endosomal acidification significantly reduced JCPsV infection using NH₄Cl. NH₄Cl p value: 0.002; Monesin p value: 0.004; BafA1 p value: 0.01. The experiment was performed in triplicate and bars represent the standard error. 5B: To control for toxicity and cell viability an MTS assay performed in the same well as the luciferase reading. The absorbance at 490 nm was averaged for the three experiments and bars represent the standard error.

Figure 6. Chemical inhibitors of ER function prevent JCPsV Infection

6A: SVGA cells were pretreated with DMSO, 25 nM Thapsigargin or 2.5 mM DTT for 1hr. After infection, PsV was removed and replaced with drug in complete media and neutralizing JC antibody. Secreted luciferase was measured 72 hours post infection (RLU). 6B: To control for toxicity and cell viability, an MTS assay performed in the same well as the luciferase reading. The absorbance at 490 nm was averaged for the three experiments and bars represent the standard error. Because DTT treatment interferes with the MTS assay, RLU was used to measure infectivity and cell viability was visually confirmed. Both inhibitors of ER function inhibited JCPsV infection. Thaps p value: 0.001; 5 mM DTT p value: 0.0002