*3.5. The PEI-Linear Blocks the Attachment of PRRSV Virions but Not Internalization in MARC-145 Cells*

It has been reported that PEI mediated the blockage of the attachment of HPV and HCMV to surface of susceptible cells and this probably occurs through competitively binding the same attachment sites on cells between PEI and virions [26]. Thus, we next determined whether PEI prevents the attachment of PRRSV virions to MARC-145 cells. We first tested whether the inhibition of PRRSV infection by PEI was due to the reduced virion attachment. After the inoculation of MARC-145 cells with the virus and PEI mixture, the cells were maintained at 4 ◦C without triggering endocytosis but allowed virion attachment. Then, the attached virions were quantified by RT-qPCR. The results showed that PEI reduced the PRRSV virion attachment to MARC-145 cells at both 5 and 10 MOI when PEI and the virus were added at the same time (Figure 5A). To further confirm this result, we pre-incubated cells with PEI-linear first, followed by inoculating the cells with PRRSV (5 or 10 MOI) at 4 ◦C. The RT-qPCR analysis suggests that there was reduced binding of viral particles to MARC-145 cells as well (Figure 5B).

**Figure 5.** PEI inhibits PRRSV virion attachment to MARC-145 cells but not virion internalization. (**A**). PEI inhibits PRRSV virions attachment to cells when co-incubated with PRRSV to MARC-145 cells. PRRSV RNA levels in MARC-145 cells that were incubated with a pre-chilling virus and PEI mixture containing an MOI of 5 or 10 PRRSV-JXA1 strain mixed with 6 μg/mLPEI-linear for 2 h at 4 ◦C. Then, the cells were harvested for RT-qPCR analysis. MARC-145 cells inoculated with the virus only without PEI were included as a control. (**B**). Pre-incubation of MARC-145 cells with PEI inhibits virion attachment. PRRSV RNA levels in MARC-145 cells that were pre-incubated with 6 μg PEI for 1 h, followed by chilling at 4 ◦C for 30 min and inoculation with an MOI of 5 or 10 PRRSV-JXA1 strain at 4 ◦C for 1 h. (**C**). PEI treatment of MARC-145 cells does not affect virion internalization. MARC-145 cells were inoculated with an MOI of 5 or 10 PRRSV-JXA1 for 1 h at 4 ◦C, followed by washing with cold PBS, treatment with PEI, temperature shift to 37 ◦C to trigger virion internalization via endocytosis, and removing non-internalized virions with protease K treatment. Then the cells were harvested to evaluate the PRRSV RNA level to determine internalized virions. All experiments were repeated at least three times, and the data were presented as the mean ± SD, which is further subjected to Student's *t*-test. Significant differences between indicated groups were marked by "\*" (*P* < 0.05) and "\*\*" (*P* < 0.01).

To determine whether the PEI affects PRRSV internalization, we inoculated MARC-145 cells with the virus first for attachment at 4 ◦C, followed by PEI treatment and temperature shift to 37 ◦C to trigger endocytosis-mediated internalization of the virions. The viral particles remaining on cell surface were removed by treatment with protease K. So only internalized virions were harvested for qPCR. The data suggest that PEI could not block the internalization of attached virions as shown by similar viral RNA levels in both PEI-treated and mock-treated cells (Figure 5C). Taken together, these data suggest that the 40 kDa linear PEI inhibits PRRSV via blocking virion attachment to MARC-145 cells.
