Occludin Regulates HIV-1 Infection by Modulation of the Interferon Stimulated OAS Gene Family

Abstract

HIV-1-associated blood brain barrier (BBB) alterations and neurocognitive disorders are frequent clinical manifestations in HIV-1 infected patients. The BBB is formed by cells of the neurovascular unit (NVU) and sealed together by tight junction proteins, such as occludin (ocln). Pericytes are a key cell type of NVU that can harbor HIV-1 infection via a mechanism that is regulated, at least in part, by ocln. After viral infection, the immune system starts the production of interferons, which induce the expression of the 2'-5'-oligoadenylate synthetase (OAS) family of interferon stimulated genes and activate the endoribonuclease RNaseL that provides antiviral protection by viral RNA degradation. The current study evaluated the involvement of the OAS genes in HIV-1 infection of cells of NVU and the role of ocln in controlling OAS antiviral signaling pathway. We identified that ocln modulates the expression levels of the OAS1, OAS2, OAS3, and OASL genes and proteins and, in turn, that the members of the OAS family can influence HIV replication in human brain pericytes. Mechanistically, this effect was regulated via the STAT signaling. HIV-1 infection of pericytes significantly upregulated expression of all OAS genes at the mRNA level but selectively OAS1, OAS2, and OAS3 at the protein level. Interestingly no changes were found in RNaseL after HIV-1 infection. Overall, these results contribute to a better understanding of the molecular mechanisms implicated in the regulation of HIV-1 infection in human brain pericytes and suggest a novel role for ocln in controlling of this process.

Keywords: Blood brain barrier; HIV-1; Interferon; OAS; Occludin; Pericytes; RNaseL.

Fig. 1

Expression pattern of the OAS proteins in cells of the NVU. Expression of OAS1 (A), OAS2 (B), OAS3 (C), and OASL (D) in primary human brain pericytes, astrocytes, EC, immortalized human microglial cells, and SH-SY5Y neuroblastoma cell line as measured by immunoblotting. GAPDH was used as a loading control. Values are mean ± SEM. ****p < 0.0001, ***p = 0.0002, **p = 0.003, *p < 0.0449, n=4 independent samples per group

Fig. 2

Ocln regulates IFN genes and alters the STAT signaling pathway. Pericyte ocln levels were overexpressed by transfection with PCMV3-OCLN expression vector, followed by the assessment of the mRNA of INFα5 (A), INFα2 (B), and INFβ (C). INFγ expression was non-detectable. mRNA and protein of STAT1, STAT1 Tyr 701 phosphorylation (F), and STAT2 (G) were measured. (E) Cells were co-transfected with PCMV3-OCLN plasmid and a luciferase construct under the control of the STAT1 promoter and cell lysates were analyzed using the dual-luciferase reporter assay. In addition, IRF9 (D) were assessed by q-PCR. Graphs indicate the mean ± SEM from three independent experiments. ****p<0.0001, ***p=0.0002, **p=0.003, n= 4–9 per group

Fig. 3

Ocln upregulation enhances OAS expression in human brain pericytes. Pericytes were transfected with ocln overexpression vector PCMV3-OCLN or with PCMV3 control vector, and the expression of mRNA and protein for ocln (A), OAS1 (B), OAS2 (C), OAS3 (D), and OASL (E) was evaluated by q-PCR and immunoblotting respectively. GAPDH was used as a housekeeping gene and loading control. Values are mean ± SEM. ****p<0.0001, ***p=0.0002, **p=0.003, *p<0.05, n=4–6 independent samples per group

Fig. 4

OAS expression is specifically regulated by ocln and not altered by silencing of tight junction protein ZO-1. Pericytes were transfected with ocln-siRNA, ZO-1 siRNA, or control-siRNA, and the expression of genes encoding for ocln (A), ZO-1 (B), OAS1 (C), OAS2 (D), OAS3 (E), and OASL (F) was evaluated by q-PCR. GAPDH was used as a housekeeping gene. Values are mean ± SEM. ****p<0.0001, ***p=0.0002, **p=0.003, *p<0.05, n=4–6 independent samples per group

Fig. 5

Ocln expression is specifically regulated by OASL and not by other members of the OAS family. Pericytes were transfected with OAS1 siRNA, OAS2 siRNA, OAS3 siRNA, OASL siRNA, or control-siRNA (A), and ocln expression was evaluated by q-PCR (B) and immunoblotting (C). GAPDH was used as a housekeeping gene and loading control. Values are mean ± SEM. ****p<0.0001, ***p=0.0002, **p=0.003, *p<0.05, n=4–8 independent samples per group

Fig. 6

The OAS family members regulate each other´s expression. Pericytes were transfected as in Fig. 4, and mRNA and protein levels of OAS1 (A), OAS2 (B), OAS3 (C), and OASL (D) were evaluated by q-PCR and immunoblotting, respectively. GAPDH was used as a housekeeping gene and loading control. Values are mean ± SEM. ****p<0.0001, ***p=0.0002, **p=0.003, *p<0.05, n=4–6 independent samples per group

Fig. 7

Impact of HIV-1 infection on OAS expression. Pericytes were either mock-infected or infected with 60 ng/mL HIV-1 p24 for 24, 48, or 72 h and mRNA and protein expression of OAS1 (A), OAS2 (B), OAS3 (C), and OASL (D) was measured by q-PCR and immunoblotting, respectively. GAPDH was used as a housekeeping gene and loading control. Values are mean ± SEM. ****p<0.0001, ***p=0.0002, **p=0.003, *p<0.05, n=4–6 independent samples per group

Fig. 8

Ocln and OAS genes differentially regulate HIV-1 replication. Human brain pericytes were mock transfected or transfected with control siRNA (A) or with ocln siRNA, OAS1 siRNA, OAS2 siRNA, OAS3 siRNA or OASL siRNA (B). Next, cells were either mock-infected or infected with 60 ng/ml HIV-1 p24 for 12 h. p24 levels were analyzed 48h post infection in cell culture media by ELISA. Values are mean ± SEM. ****p<0.0001, ***p=0.0002, **p=0.003, *p<0.0449, n=4–10 per group

Fig. 9

RNaseL expression pattern in cells of NVU. Expression of RNaseL in primary human brain pericytes, astrocytes, EC, immortalized human microglial cells, and SH-SY5Y neuroblastoma cell line as measured by immunoblotting (A). Pericytes were either mock-infected or infected as in Fig. 6, and mRNA and protein expression of RNaseL was measured by qPCR and immunoblotting (B). Pericytes were transfected with ocln overexpressing vector PCMV3-OCLN or with PCMV3 control vector, and the expression of RNaseL was evaluated by qPCR and immunoblotting (C). GAPDH was used as a housekeeping gene and loading control. Values are mean ± SEM. ****p<0.0001, ***p=0.0002, **p=0.003, *p<0.05, n=4–6 independent samples per group

Fig. 10

Proposed model of ocln-mediated regulation of the OAS genes and protection against HIV-1 infection in human brain pericytes. Ocln increases OAS1, OAS2, OAS3, and OASL expression levels by regulating the STAT signaling pathway. Our data indicate that ocln enhances STAT1 expression and phosphorylation levels. STAT1 and STAT2 form a dimer that recruits IRF9, also upregulated by ocln, and the complex moves to the nucleus where they bind to specific DNA elements, and initiate transcription of interferon stimulated OAS genes which restrain HIV replication. Furthermore, OASL can alter ocln expression in positive feedback. Following HIV-1 infection, there is also an increase in the expression of mRNA OAS1, OAS2, OAS3 and OASL and protein levels of OAS1, OAS2 and OAS3. TxF; transcription factor, IRNAR1; IFN receptor subunit 1, IRNAR2; IFN receptor subunit 2