The vesicular stomatitis virus matrix protein inhibits NF-κB activation in mouse L929 cells

Abstract

A previous study found that NF-κB activation is delayed in L929 cells infected with wild-type (wt) strains of VSV, while activation occurred earlier in cells infected with mutant strain T1026R1 (R1) that encodes a mutation in the cytotoxic matrix (M) protein. The integrity of the other R1 proteins is unknown; therefore our goal was to identify the viral component responsible for preventing NF-κB activation in L929 cells. We found that the M protein inhibits viral-mediated activation of NF-κB in the context of viral infection and when expressed alone via transfection, and that the M51R mutation in M abrogates this function. Addition of an IκB kinase (IKK) inhibitor blocked NF-κB activation and interferon-β mRNA expression in cells infected with viruses encoding the M51R mutation in M. These results indicate that the VSV M protein inhibits activation of NF-κB by targeting an event upstream of IKK in the canonical pathway.

Keywords: Canonical pathway; IKK inhibitor; Interferon; L929 cells; M51 mutation; Matrix protein; NF-kappa B (NF-κB); VSV; Vesicular stomatitis virus.

Fig. 1

Nuclear Localization and DNA-binding activity of NF-κB occurs rapidly in cells infected with viruses containing the M51R mutation in M. (A) Cells were TNF-α treated, mock infected, or infected at an MOI of 10 for the indicated time. The p65 subunit of NF-κB was then visualized by confocal microscopy. The experiment was repeated three times; representative images are shown (B) The percent of total cells containing nuclear NF-κB was determined. At least 200 cells per sample were counted and these data represent the mean of two independent experiments. (C) Cells were infected at an MOI of 5, nuclear extracts prepared, and the DNA-binding activity of NF-κB determined using the TransAM NF-κB p65 Chemi Kit (Active Motif). Binding activity was expressed as fold mock and was calculated by dividing measured DNA binding activity of NF-κB in an infected sample by the activity found in mock-infected cells. Data represent the mean values from at least three independent experiments, performed in duplicate.

Fig. 2

Viruses containing a wt M protein block virus-induced activation of NF-κB. (A) Nuclear extracts were isolated from cells infected with a single viral strain, or coinfected with R1 (MOI of 5 for each virus, 5 hpi). The NF-κB DNA binding activity was measured using the TransAM NF-κB p65 Kit (colorimetric, Active Motif). Each sample was standardized to mock infected samples. Data represent the mean of five independent experiments, performed in duplicate. (B) Luciferase transcriptional reporter activity in stably transfected cells following viral infection (MOI of 25 for each virus). A representative experiment of three is shown.

Fig. 3

The VSV M protein blocks virus-mediated activation of NF-κB. An expression vector encoding the indicated VSV gene was Nucleofected into cells. After approximately 48 hours post-nucleofection cells were mock or R1-infected (MOI of 5 for 5 hours), and the DNA-binding activity in nuclear extracts was determined using the TransAM Kit (colorimetric). The data was calculated by dividing DNA binding activity of NF-κB in a sample by the activity found in cells nucleofected with the empty vector pJC119 and mock infected. Three independent experiments were performed. The mean of a representative experiment done in quadruplicate is shown.

Fig. 4

An inhibitor of IKK reduces NF-κB activation and IFN-β mRNA expression in cells infected with viruses containing mutant M proteins. L929 cells were untreated or treated with Bay during adsorption and infection with the indicated virus at an MOI of 5. (A) Nuclear lysates were prepared at 5 hpi and NF-κB activation was measured using the TransAM Kit (colorimetric). Each sample was standardized to mock infected samples. The data represent the mean of two independent experiments, performed in duplicate. (B) Total RNA was collected from infected cells at 5 hpi and reverse transcribed into cDNA and submitted to Real-Time PCR analysis of mouse IFN-β mRNA production. Samples were done in triplicate and were normalized to HPRT gene expression in respective samples. Data is represented as fold change relative to mock-infected cells. The mean of one representative experiment of three is shown. (C) Whole cell protein lysates were collected after 8 hours of infection and analyzed by western blot analysis. A representative experiment of three is shown. Relative intensities of the VSV G band are shown below the panel.