*3.6. The 40 kDa Linear PEI Blocks the Attachment of PRRSV Virions to PAMs via Acting on the Virus but Not Cells*

Since PAMs are the major target of PRRSV in vivo, we investigated whether PEI has a similar inhibition mechanism to PRRSV in PAMs as that of MARC-145 cells. Similar to MARC-145 cells, thw co-administration of PRRSV and PEI to PAMs reduced the binding of PRRSV virions to PAMs (Figure 6A). Moreover, it is notable that the susceptibility of PAMs to PRRSV is much higher than MARC-145 cells, since the inoculation of PRRSV at 0.01 MOI to PAMs was sufficient for RT-qPCR analysis of attached virions but it required at least an MOI of 5 of the virus to inoculate the MARC-145 cells for RT-qPCR analysis of attached virions (below the detection limit in MARC-145 cells if below 1 MOI). Meanwhile, if PAMs were pre-incubated with PEI-linear, followed by inoculation with PRRSV-JXA1 at 4 ◦C, there was a significant increase in virion binding compared with PAMs without

PEI pretreatment (Figure 6B), which is different from that of MARC-145 cells. Moreover, if PAMs were inoculated with PRRSV before PEI treatment, followed by a temperature shift to trigger endocytosis, the analysis of the internalized PRRSV virions showed that PEI also promoted the internalization of attached virions as well (Figure 6C). Therefore, the data suggest that PEI-linear may act on the virus rather than cells to prevent the binding of the virus to PAMs. Meanwhile, the pre-treatment of PAMs with PEI even promotes PRRSV attachment and internalization to PAMs. Taken together, these data suggest that the 40 kDa PEI inhibits PRRSV attachment and is capable of conferring the broad inhibition of heterogeneous *PRRSV-2* isolates.

**Figure 6.** PEI inhibits the attachment of PRRSV virion to PAMs but promotes PRRSV internalization into PAMs. (**A**). PEI prevents virion attachment to cells when co-incubated with PAMs. PRRSV RNA levels in PAMs that were incubated with a pre-chilling virus and PEI mixture containing an MOI of 0.1 or 0.5 PRRSV-JXA1 virus and 6 μg/mL PEI-linear for 2 h at 4 ◦C to allow attachment. Cells inoculated with the virus only were included as a control. (**B**). Pre-incubation of PAMs with PEI enhances virion attachment to PAMs. PRRSV RNA levels in PAMs that were pre-incubated with 6 μg PEI-linear for 1 h, followed by chilling at 4 ◦C for 30 mins and inoculation with an MOI of 0.1 or 0.5 of PRRSV-JXA1 virus at 4 ◦C for 1 h. (**C**). PEI treatment of PAMs enhances virion internalization into PAMs. PRRSV RNA levels in PAMs that were inoculated with an MOI of 0.1 or 0.5 PRRSV-JXA1 for 1 h at 4 ◦C, followed by washing with cold PBS, treatment with 6 μg PEI and temperature shift to 37 ◦C to trigger virion internalization via endocytosis. 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).

#### **4. Discussion**

PEI, also known as polyaziridine, is a polymer with a repeating structure that is composed of the amine group along with a two-carbon aliphatic CH2CH2 spacer. It was reported that PEI could be in linear, branched, and dendrimeric forms [34], while only linear and branched molecules of PEI are available from commercial suppliers. PEI has been used for many applications, usually due to its polycationic character. For cell biology research, PEI has been used as a transfection reagent [35]. Also, PEI is explored for its antiviral activity. For HPV, the 25 kDa linear PEI blocks the primary attachment of HPV16 and HCMV to susceptible cells [26]. It appears that PEI preincubation with cells blocks HPV and HCMV binding to their primary receptor HSPG [26]. Moreover, the antiviral activity of PEI has been investigated in vivo as well. Intranasal administration of the 25 kDa linear PEI suppresses influenza virus infection in mice [36]. The 3610 Da branched PEI combined with liposomes was shown to strongly enhance antiviral efficiency against herpes simplex virus type 2 in a mouse model [37].

Currently, the inhibition mechanism of PEIs against viral infections is still not fully understood and appears to depend on blocking virion attachment to susceptible cells. As a result, PEI potentially interferes with electrostatic interactions between viral surface proteins and host receptors [26]. However, it was reported that branched PEI with a molecular mass of 70 kDa accelerated HIV-1 infection despite its inhibition of HIV-1 attachment to cells [27], which may be partially due to the promotion of the exposure of co-receptor and/or viral entry into cells via the influence of cell membrane fluidity [27].

In our study, the 40 kDa linear PEI was shown to be effective against PRRSV infection through the inhibition of PRRSV virion attachment to MARC-145 cells, the most frequently used cell line for PRRSV study in vitro. Meanwhile, PEI treatment of MARC-145 cells does not affect the internalization of attached PRRSV virions on the cell surface. This inhibition mechanism appears to be consistent with a previous observation for HPV and HCMV [26]. Conversely, the inhibition of PRRSV in PAMs by the PEI was weaker than that in MARC-145 cells when the same dose of PEI was used. Notably, based on our data, although PEI prevented the attachment of PRRSV virions to PAMs when the PEI and virus were co-administrated simultaneously, further analysis suggests that the pre-treatment of PAMs with PEI enhanced the attachment of PRRSV virions. Moreover, PEI enhanced the internalization of PRRSV virions, which appears to be similar to the scenario in HIV but different from PRRSV in MARC-145 cells. Therefore, these data suggest that the exact procedures of the attachment and internalization of PRRSV virions in MARC-145 cells and PAMs may be different, which could also be supported by the different susceptibility of both cells to PRRSV as well. Moreover, how such a difference affects PRRSV pathogenesis or antibody-mediated viral neutralization (mainly via blocking virion attachment to susceptible cells) remains unclear. Meanwhile, when acting as a transfection agent, PEI condenses DNA into positively charged particles, which bind to anionic cell surface residues to deliver the PEI/DNA complex into the cells via endocytosis [38,39]. Once inside the endosome, amines of PEI result in an influx of counter-ions and a lowering of the osmotic potential. Therefore, osmotic swelling disrupts the vesicle to release the PEI/DNA complex into the cytoplasm [38,39]. It has been known that PRRSV virions enter PAMs via clathrin-mediated endocytosis [32]. It is possible that PEI promotes endocytosis after PRRSV virion attachment or disrupts the endosome vesicle to release PRRSV RNA into the cytoplasm in PAMs to enhance the virion internalization. However, why such a scenario in PAMs is different from MARC-145 cells remains unclear.

On the one hand, PK-15 cells (immortalized swine kidney cell line is not susceptible for PRRSV) stably expressing porcine CD163 (the essential receptor for PRRSV infection) support the complete PRRSV replication cycle [40]. On the other hand, PK-15 cells stably expressing CD169 (a putative receptor involved in PRRSV internalization) support the internalization of PRRSV virions but not the uncoating of nucleocapsid and fusion with the endocytic vesicle membrane [41]. These data imply that the endocytosis-mediated internalization and uncoating procedure of PRRSV particles in PAMs and immortalized cell lines such as PK-15 and MARC-145 is different. Such a difference may contribute to the different inhibitory effect of the 40 kDa linear PEI on PRRSV virions.

The cytotoxicity of different PEI forms was evaluated in this study as well. A previous report suggested that branched (25 kDa) and linear (750 kDa) PEI can both induce membrane damage and initiate apoptosis in human cell lines [42]. Based on our observation, the cytotoxicity of PEI appears to be cell type and structure dependent. Both linear PEIs demonstrated better tolerance in MARC-145 cells than primary PAMs, while branched PEI had a much better tolerance than linear PEI in PAMs. However, the reason for the differences remains elusive. This implies that a certain modification or optimization of PEI molecular structure may be explored to reduce cytotoxicity while maintaining comparable antiviral activity.

Mechanically, PEI blocks the binding of virus to its receptors to prevent the attachment of virions to susceptible cells as demonstrated for HPV16 and HCMV [26]. This is also consistent with our observation that PEI prevents PRRSV attachment to MARC-145 cells. The difference in PEI inhibition of PRRSV in MARC-145 and PAM cells suggests that the interaction between PRRSV virions and receptors in these two types of cells might be different. Several membrane proteins have been identified as potential receptors for PRRSV infection in permissive cells, such as heparin sulfate (HS) [9], vimentin [10], CD151 [11], porcine CD163 [12], sialoadhesin (CD169) [13], DC-SIGN (CD209) [14] and MYH9 [15]. However, how these receptors cooperate to mediate the PRRSV infection remains elusive.

For HIV, branched PEI enhanced HIV-1 infection partially due to its promotion of the exposure of co-receptor and/or viral entry into cells via the influence of cell membrane fluidity [27]. Since PRRSV might use several receptors in PAMs in vivo, it is possible that PEI may promote the exposure of co-receptor or receptors and influence the cell membrane fluidity of PAMs.

Moreover, it is also notable that the inhibitory effect of the 40 kDa linear PEI on different *PRRSV-2* isolates is variable, especially in MARC-145 cells. This is somehow unusual as the PEI inhibition of PRRSV should be non-specific and such an inhibition on different PRRSV isolates should be similar. It is unclear whether PEI blocks the interaction of PRRSV virions with the receptors mentioned above indiscriminately or whether PEI preferably blocks the interaction of PRRSV virions with certain receptors from the list above. It is possible that there is a different receptor preference among various PRRSV isolates, as evidenced by the observation that the replacement of the fifth scavenger receptor cysteine-rich domain (SRCR5) of porcine CD163 with the SRCR5 domain of the human CD163-like homolog (CD163Li) only confers resistance to PRRSV-1 but not to PRRSV-2 [43]. Therefore, it is possible that, due to an uneven PEI blockage to the interaction of PRRSV virions to different receptors along with a strain-specific receptor preference of various PRRSV isolates, the PEI cannot confer a consistent inhibition among different PRRSV isolates. Moreover, it appears that different steps and variable outcomes of PEI mediated inhibition between MARC-145 cells and PAMs suggest that there is a difference in the attachment and internalization of PRRSV virions on these two cells. The impact of such a difference on our understanding of PRRSV virion attachment and internalization as well as virus neutralization and pathogenesis in vivo require further investigation.

In conclusion, the 40 kDa linear PEI demonstrates an inhibitory effect against different PRRSV-2 isolates both in MARC-145 cells and PAMs with tolerable cytotoxicity. Further study is needed to investigate whether PEI can confer the inhibition of PRRSV infection in swine. Meanwhile, the potential different internalization procedure of PRRSV in PAMs and MARC-145 cells warrants further investigation.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1999-4915/11/9/876/s1, Figure S1: Calculation of median cytotoxic concentration (CC50) for two linear polyethylenimines (PEIs) in PAMs, Figure S2: Evaluation of cytotoxicity of branched PEI in PAMs and calculation of CC50, Figure S3: Calculation of median effective concentration (EC50) of PEI in MARC-145 cells.

**Author Contributions:** Data curation, J.W., J.L., N.W., Q.J., M.L. and C.W.; Funding acquisition, Y.N. and C.W.; Methodology, Y.N.; Project administration, C.W.; Resources, E.-M.Z.; Supervision, E.-M.Z. and C.W.; Writing—original draft, J.W., J.L. and Y.N.; Writing—review and editing, Y.N., Y.Z. and C.W.

**Funding:** This work was supported by a grant from the National Key Research and Development Program of China awarded to Y.N. and C.W. (Grant No. 2017YFD0501004), a grant from the Key Research and Development Program of Shaanxi Province (Grant No. 2017NY-156) and the Fundamental Research Funds for the Central Universities awarded to C.W.

**Acknowledgments:** We thank Gaiping Zhang of Henan Agriculture University for providing the NADC30-like isolate.

**Conflicts of Interest:** The authors declare no conflict of interest.
