The Antiviral Compound PSP Inhibits HIV-1 Entry via PKR-Dependent Activation in Monocytic Cells

Abstract

Actin depolymerization factor (ADF) cofilin-1 is a key cytoskeleton component that serves to lessen cortical actin. HIV-1 manipulates cofilin-1 regulation as a pre- and post-entry requisite. Disruption of ADF signaling is associated with denial of entry. The unfolded protein response (UPR) marker Inositol-Requiring Enzyme-1α (IRE1α) and interferon-induced protein (IFN-IP) double-stranded RNA- activated protein kinase (PKR) are reported to overlap with actin components. In our published findings, Coriolus versicolor bioactive extract polysaccharide peptide (PSP) has demonstrated anti-HIV replicative properties in THP1 monocytic cells. However, its involvement towards viral infectivity has not been elucidated before. In the present study, we examined the roles of PKR and IRE1α in cofilin-1 phosphorylation and its HIV-1 restrictive roles in THP1. HIV-1 p24 antigen was measured through infected supernatant to determine PSP's restrictive potential. Quantitative proteomics was performed to analyze cytoskeletal and UPR regulators. PKR, IRE1α, and cofilin-1 biomarkers were measured through immunoblots. Validation of key proteome markers was done through RT-qPCR. PKR/IRE1α inhibitors were used to validate viral entry and cofilin-1 phosphorylation through Western blots. Our findings show that PSP treatment before infection leads to an overall lower infectivity. Additionally, PKR and IRE1α show to be key regulators in cofilin-1 phosphorylation and viral restriction.

Keywords: HIV; IRE1α; PKR; PSP; UPR; cofilin-1; proteomics.

Figure 1

Diagram depicting HIV-1 entry during infection in CD4+ cells. (A) Early interaction between HIV-1 and CD4 receptor initiates activation of guanine nucleotide exchange factors (GEFs) and subsequently downstream signaling through RhoA/ROCK/LIMK-1. This results in phosphorylation and inactivation of cofilin-1. Actin dynamics will shift towards the polymerization state leading to CXCR4/CCR5 co-receptor clustering as the first requirement for viral entry. Filamin A serves as a linkage factor between CD4 and co-receptors. (B.1) HIV-1 associates with CXCR4/CCR5 in proximity and triggers downstream dephosphorylation and activation of cofilin-1. This process is carried out by G-protein coupled receptor signaling, specifically through Gα subunit-mediated phosphatases such as SSH3. (B.2) Cofilin-1 will begin the breakdown of actin filaments and subsequently lead to viral fusion as the final entry requirement.

Figure 2

PSP lowers HIV entry in THP1 cells. Percentages of HIV entry in PSP-treated cells analyzed through viral load of HIV p24 antigen and compared with unpaired t test. Data are represented as mean ± SEM. Statistically significant difference (**), p < 0.01 is shown, n = 3.

Figure 3

Inhibition of PKR and IRE1α activity facilitates HIV-1 entry. Percentages of HIV entry in PSP-treated cells supplemented with either 56.09 nM of C16-PKR or 221.8 nM of 4µ8C-IRE1α pharmaceutical blockers. Experiments were analyzed through viral loads of the HIV p24 antigen and compared with one-way ANOVA with Tukey multiple comparisons tests. Data are represented as mean ± SEM. Statistically significant difference (***), p < 0.001 and (****), p < 0.0001 are shown, n = 3.

Figure 4

Western blot results related to UPR, IFN, and cytoskeletal biomarkers in PSP-treated THP1 monocytic cells. (A) Representative Western blot data for the protein expression levels of UPR: (B) GRP78; (C) IRE1α; IFN: (D) PKR; (E) p-PKR; ADF: (F) Cofilin1; (G) p-Cofilin1; and ABP: (H) Gelsolin signaling. β-Actin was used as a loading control and for normalization of data. Images were quantified using the ImageJ software (NIH, version 1.52a) by comparing the integrated density value with the control group. Mean ± SEM and significant difference (*), p ≤ 0.05, (**), p ≤ 0.01, (***), p ≤ 0.001, (****), p ≤ 0.0001 are shown and were determined using one-way ANOVA with Tukey multiple comparisons test, n = 3. All Western blot images can be found in Figure S2.

Figure 5

PKR inhibition decreases cofilin-1 phosphorylation and the expression levels relating to gelsolin. The activation of PKR was suppressed after 56.09 nM of C16 inhibitor in THP1 monocytic cells with or without PSP treatment. (A) Representative Western blot data for the protein expression levels of cytoskeletal: (B) pCofilin1; (C) Cofilin1; (D) Gelsolin; and IFN-IP: (E) pPKR; (F) PKR. β-Actin was used as a loading control and for normalization of data. Images were quantified using the ImageJ software (NIH, version 1.52a) by comparing the integrated density value with the control group. Mean ± SEM and significant difference (*), p ≤ 0.05, (**), p ≤ 0.01, (***), p ≤ 0.001, (****), p ≤ 0.0001 are shown and were determined using one-way ANOVA with Tukey multiple comparisons test, n = 3. All Western blot images can be found in Figure S2.

Figure 6

IRE1α modulates downstream phosphorylation activity of PKR and cofilin-1. Endoribonuclease activity of IRE1α was suppressed after 221.8 nM of 4µ8C inhibitor in THP1 monocytic cells with or without PSP treatment. (A) Representative Western blot data for the protein expression levels of cytoskeletal: (B) pCofilin1; (C) Cofilin1; (D) Gelsolin; IFN-IP: (E) p-PKR; (F) PKR and UPR: (G) XBP1s. β-Actin was used as a loading control and for normalization of data. Images were quantified using the ImageJ software (NIH, version 1.52a) by comparing the integrated density value with the control group. Mean ± SEM and significant difference (*), p ≤ 0.05, (**), p ≤ 0.01, (***), p ≤ 0.001, (****), p ≤ 0.0001 are shown and were determined using one-way ANOVA with Tukey multiple comparisons test, n = 3. All Western blot images can be found in Figure S2.

Figure 7

Validation of western blot results using RT-qPCR analysis for cytoskeletal and IFN-IP markers. Relative gene expression analyzed through RT-qPCR approach. The mRNA expression levels for the ADF: (A) Cofilin-1; ABP: (B) Gelsolin; and IFN-IP: (C) PKR are shown. All data were normalized using 18S as a housekeeping gene in response to PSP treatment. Mean ± SEM and significant difference (*), p ≤ 0.05, (**), p ≤ 0.01, (***), p ≤ 0.001 are shown and were determined using one-way ANOVA, with Tukey multiple comparisons test, n = 3.

Figure 8

Model depicting PSP signaling through a UPR/IFN-induced pathway. HIV-1 infection results in a chronic ER stress response, while simultaneously dephosphorylating cofilin-1 through SSH3 phosphatase. This results in actin depolymerization for viral entry. Prior to infection, PSP treatment induces actin polymerization via an acute UPR. PKR mediates downstream phosphorylation of IRE1α signals and reverses HIV-induced actin remodeling while infection persists. Legend colors: Green—PSP-mediated signaling and events. Cream- HIV-downstream pathways.