Inducible nitric oxide synthase expression inhibition by adenovirus E1A

Abstract

Nitric oxide (NO) is an antiviral effector of the innate immune system. Viruses that can interfere with NO synthesis may be able to replicate more rapidly than viruses that cannot limit NO synthesis. We show that the adenovirus E1A protein inhibits NO production by decreasing expression of the inducible NO synthase (NOS2). The amino-terminal portion of E1A decreases transactivation of the NOS2 5'-flanking region, limiting the DNA binding activity of NF-kappaB and inhibiting NOS2 expression. E1A is thus able to deactivate a critical component of the host defense against viral infection. Viral inhibition of NO production is a mechanism that may enable certain viruses to evade the host innate immune system.

Fig. 1.

Viral E1A decreases NOS2 expression. (A) NO inhibits adenovirus replication. HeLa cells were infected with a WT adenovirus at a moi of 10, and treated with DETA-NONOate (diethylenetriamine NONOate) at 0, 24, and 48 h, and the amount of virus at 60 h was measured by staining cells with antibody to hexon and counting stained cells per high-power field (hpf) (results are presented as mean ± SD; n = 3). (B) Viral E1A decreases NOS2 steady-state protein levels in infected cells. RAW cells were infected with a WT adenovirus or mutant adenovirus lacking E1A (ΔE1A) at a moi of 20 or 200 for 40 h. Cell lysates were immunoblotted for NOS2 (Upper) and E1A (Lower). (This experiment was repeated four times with similar results.) (C) Viral E1A decreases NOS2 steady-state protein levels in infected cells. RAW cells were infected with adenovirus at a moi of 200, and then treated 4 h later with LPS and N^ω-nitro-l-arginine methyl ester (NAME). Cell lysates were immunoblotted for NOS2. (D) Viral E1A inhibits NO production from infected cells. RAW cells were infected with adenovirus for 24 h, and then treated with LPS for 16 h. Some cells received the NOS inhibitor NAME. The [] in the supernatant was measured by using the Griess assay (mean ± SD; n = 3).

Fig. 2.

Ectopic E1A decreases NOS2 expression. (A) 4-HT induces expression of an E1A fusion polypeptide in the nucleus. (Left and Center) RAW cells were stably transfected with a plasmid construct expressing a fusion polypeptide consisting of E1A and an estrogen receptor fragment (ER). Cells were treated with control or 4-HT for 16 h, and imaged by fluorescence microscopy for E1A or DNA (DNA stained with Hoechst 33342; Lower). The 4-HT treatment causes expression of E1A-fusion polypeptide in the nucleus (Center). This experiment was repeated in three different stably transfected cell clones with similar results. (Right) RAW cells were infected with WT adenovirus type 5 and imaged by fluorescence microscopy for E1A and DNA. (B) Ectopic E1A decreases NOS2 steady-state protein levels. RAW cells were stably transfected with a plasmid expressing ER alone or E1A-ER, treated for 6 h with 4-HT to induce ER or E1A-ER expression in the nucleus, and then treated for 16 h with LPS to induce NOS2 expression. Cell lysates were immunoblotted for NOS2 and nuclear extracts were immunoblotted for E1A. Total protein was stained with Coomassie. (This experiment was repeated three times with similar results.) (C) Ectopic E1A expression decreases steady-state NOS2 mRNA levels. RAW cells were stably transfected as above, and treated as above with medium, 4-HT, LPS, or 4-HT and LPS. Total RNA was analyzed by Northern blotting with a probe for NOS2 mRNA or for ribosomal phosphoprotein 36B4 mRNA as a control. (This experiment was repeated three times with similar results.) (D) Ectopic E1A expression decreases NOS2 transcription. RAW cells were stably transfected with a vector expressing ER (Upper) or E1A-ER (Lower), treated with 4-HT for 16 h, and then treated with LPS for 4 h. Transcription of NOS2 was analyzed by nuclear run-on assays (center). As controls, transcription of aldolase (left) and GAPDH (right) was also measured.

Fig. 3.

E1A inhibits NOS2 promoter transactivation. (A) E1A inhibits transactivation of the entire NOS2 promoter. RAW cells were transiently cotransfected with a vector expressing E1A and with a NOS2 reporter construct consisting of the NOS2 5′-flanking region driving expression of luciferase. Cells were treated with LPS for 16 h, and the amount of luciferase was measured in a luminometer (Upper; mean ± SD; n = 3). (Lower) E1A immunoblot and total protein stain of the cell lysates. (B) E1A inhibits transactivation of a minimal NOS2 promoter. RAW cells were transiently cotransfected with the full-length E1A expression vector and various deletion mutants of the NOS2 5′-flanking region reporter construct diagrammed Left. Cotransfected cells were then treated with medium alone or with LPS, and luciferase activity was measured (Right). LPS increases NOS2 promoter transactivation in RAW cells in the absence of E1A. However, LPS fails to increase NOS2 promoter activity in RAW cells also expressing E1A. E1A inhibits LPS activation of the NOS2 promoter, even if only 262 bp of the NOS2 promoter region remain (mean ± SD; n = 3).

Fig. 4.

E1A inhibits NF-κB binding activity. RAW cells were stably transfected with constructs expressing the estrogen receptor alone (ER), or expressing the E1A-ER fusion polypeptide. Transfected cells were then treated with medium, LPS, 4-HT, or both. Nuclear extracts were analyzed for binding activity to a radiolabeled oligonucleotide derived from the NOS2 5′-flanking region containing a κB element. (Left) 4-HT induction of E1A inhibits κB binding activity. (Right) Antibody to p50 or p65 decreases the intensity of the κB DNA–protein complex, and antibody to p50 supershifts the complex, suggesting that E1A regulates NF-κB binding to the κB element. (These experiments were repeated three times with similar results.)

Fig. 5.

Amino-terminal domains of E1A inhibit NOS2 promoter transactivation. RAW cells were cotransfected with the full-length NOS2 promoter-reporter construct and with vectors expressing deletion mutants of E1A. Transfected cells were stimulated with medium alone or with LPS, and the amount of luciferase in cell lysates was measured. WT E1A blocks transactivation of the NOS2 promoter. However, E1A lacking the amino-terminal domains (N, CR1, and CR2) fails to block transactivation of the NOS2 promoter.