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Rabies virus infection induces type I interferon production in an IPS-1 dependent manner while dendritic cell activation relies on IFNAR signaling.

Faul EJ, Wanjalla CN, Suthar MS, Gale M, Wirblich C, Schnell MJ - PLoS Pathog. (2010)

Bottom Line: However, IPS-1 is essential for both BMDC activation and IFN production.In the absence of either receptor, there is a significant decrease in BMDC activation at 12h post infection.However, only RIG-I-/- cells exhibit a delay in type I IFN production.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
As with many viruses, rabies virus (RABV) infection induces type I interferon (IFN) production within the infected host cells. However, RABV has evolved mechanisms by which to inhibit IFN production in order to sustain infection. Here we show that RABV infection of dendritic cells (DC) induces potent type I IFN production and DC activation. Although DCs are infected by RABV, the viral replication is highly suppressed in DCs, rendering the infection non-productive. We exploited this finding in bone marrow derived DCs (BMDC) in order to differentiate which pattern recognition receptor(s) (PRR) is responsible for inducing type I IFN following infection with RABV. Our results indicate that BMDC activation and type I IFN production following a RABV infection is independent of TLR signaling. However, IPS-1 is essential for both BMDC activation and IFN production. Interestingly, we see that the BMDC activation is primarily due to signaling through the IFNAR and only marginally induced by the initial infection. To further identify the receptor recognizing RABV infection, we next analyzed BMDC from Mda-5-/- and RIG-I-/- mice. In the absence of either receptor, there is a significant decrease in BMDC activation at 12h post infection. However, only RIG-I-/- cells exhibit a delay in type I IFN production. In order to determine the role that IPS-1 plays in vivo, we infected mice with pathogenic RABV. We see that IPS-1-/- mice are more susceptible to infection than IPS-1+/+ mice and have a significantly increased incident of limb paralysis.

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Infection of APCs is non-productive.(A) NA, BSR, Raw264.7, and JAWSII cells were infected with RABV at an MOI = 10. A one-step growth curve was generated by titering infectious RABV in the supernatant of the infected cells at various hours post infection as indicated. (B) Western blot analysis. BSR or JAWSII cells were infected with RABV (MOI of 10) and lysed 12, 24 or 48 h later. Proteins were separated by SDS-PAGE and subjected to Western blotting with mouse polyclonal antibodies specific for RABV (strain N2c), or a mouse monoclonal against actin (Actin). The RABV proteins N, P, and M are indicated.
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ppat-1001016-g002: Infection of APCs is non-productive.(A) NA, BSR, Raw264.7, and JAWSII cells were infected with RABV at an MOI = 10. A one-step growth curve was generated by titering infectious RABV in the supernatant of the infected cells at various hours post infection as indicated. (B) Western blot analysis. BSR or JAWSII cells were infected with RABV (MOI of 10) and lysed 12, 24 or 48 h later. Proteins were separated by SDS-PAGE and subjected to Western blotting with mouse polyclonal antibodies specific for RABV (strain N2c), or a mouse monoclonal against actin (Actin). The RABV proteins N, P, and M are indicated.

Mentions: In order to account for the increased amounts of type I IFN produced following RABV infection of macrophages and DCs when compared to the amount produced by fibroblast and neuronal cells, we did a one-step growth curve following infection of the various cell types. Supernatants from infected cells was titered on BSR cells, which are insensitive to type I IFN [4]. We detected that, although all four cell types were infected, only BSR and NA cells produce infectious virus (Figure 2A). There are two possible explanations for the defect in viral production observed here: either a block in viral replication or a defect in viral assembly. In order to compare viral transcription and replication in fibroblast and dendritic cell lines we used quantitative PCR. In fibroblast cells, we saw that the amount of RABV-N messenger RNA (mRNA) transcripts increased an average of 1.95 logs from 8 hours post infection (hpi) to 48 hpi. Similarly, the quantity of RABV-N genomic RNA transcripts (gRNA) increased an average of 1.2 logs from 8 hpi to 48 hpi. This data indicates that following infection of fibroblast cells both viral transcription (mRNA) and replication (gRNA) occurs. On the other hand, when looking at the quantity of RABV-N found in dendritic cells following infection we see that there was no increase in the number of mRNA or gRNA viral transcripts when comparing 8hpi to 48 hpi. Thus, it appears that RABV is able to enter APCs, but only limited viral transcription occurs following entry.


Rabies virus infection induces type I interferon production in an IPS-1 dependent manner while dendritic cell activation relies on IFNAR signaling.

Faul EJ, Wanjalla CN, Suthar MS, Gale M, Wirblich C, Schnell MJ - PLoS Pathog. (2010)

Infection of APCs is non-productive.(A) NA, BSR, Raw264.7, and JAWSII cells were infected with RABV at an MOI = 10. A one-step growth curve was generated by titering infectious RABV in the supernatant of the infected cells at various hours post infection as indicated. (B) Western blot analysis. BSR or JAWSII cells were infected with RABV (MOI of 10) and lysed 12, 24 or 48 h later. Proteins were separated by SDS-PAGE and subjected to Western blotting with mouse polyclonal antibodies specific for RABV (strain N2c), or a mouse monoclonal against actin (Actin). The RABV proteins N, P, and M are indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2908622&req=5

ppat-1001016-g002: Infection of APCs is non-productive.(A) NA, BSR, Raw264.7, and JAWSII cells were infected with RABV at an MOI = 10. A one-step growth curve was generated by titering infectious RABV in the supernatant of the infected cells at various hours post infection as indicated. (B) Western blot analysis. BSR or JAWSII cells were infected with RABV (MOI of 10) and lysed 12, 24 or 48 h later. Proteins were separated by SDS-PAGE and subjected to Western blotting with mouse polyclonal antibodies specific for RABV (strain N2c), or a mouse monoclonal against actin (Actin). The RABV proteins N, P, and M are indicated.
Mentions: In order to account for the increased amounts of type I IFN produced following RABV infection of macrophages and DCs when compared to the amount produced by fibroblast and neuronal cells, we did a one-step growth curve following infection of the various cell types. Supernatants from infected cells was titered on BSR cells, which are insensitive to type I IFN [4]. We detected that, although all four cell types were infected, only BSR and NA cells produce infectious virus (Figure 2A). There are two possible explanations for the defect in viral production observed here: either a block in viral replication or a defect in viral assembly. In order to compare viral transcription and replication in fibroblast and dendritic cell lines we used quantitative PCR. In fibroblast cells, we saw that the amount of RABV-N messenger RNA (mRNA) transcripts increased an average of 1.95 logs from 8 hours post infection (hpi) to 48 hpi. Similarly, the quantity of RABV-N genomic RNA transcripts (gRNA) increased an average of 1.2 logs from 8 hpi to 48 hpi. This data indicates that following infection of fibroblast cells both viral transcription (mRNA) and replication (gRNA) occurs. On the other hand, when looking at the quantity of RABV-N found in dendritic cells following infection we see that there was no increase in the number of mRNA or gRNA viral transcripts when comparing 8hpi to 48 hpi. Thus, it appears that RABV is able to enter APCs, but only limited viral transcription occurs following entry.

Bottom Line: However, IPS-1 is essential for both BMDC activation and IFN production.In the absence of either receptor, there is a significant decrease in BMDC activation at 12h post infection.However, only RIG-I-/- cells exhibit a delay in type I IFN production.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
As with many viruses, rabies virus (RABV) infection induces type I interferon (IFN) production within the infected host cells. However, RABV has evolved mechanisms by which to inhibit IFN production in order to sustain infection. Here we show that RABV infection of dendritic cells (DC) induces potent type I IFN production and DC activation. Although DCs are infected by RABV, the viral replication is highly suppressed in DCs, rendering the infection non-productive. We exploited this finding in bone marrow derived DCs (BMDC) in order to differentiate which pattern recognition receptor(s) (PRR) is responsible for inducing type I IFN following infection with RABV. Our results indicate that BMDC activation and type I IFN production following a RABV infection is independent of TLR signaling. However, IPS-1 is essential for both BMDC activation and IFN production. Interestingly, we see that the BMDC activation is primarily due to signaling through the IFNAR and only marginally induced by the initial infection. To further identify the receptor recognizing RABV infection, we next analyzed BMDC from Mda-5-/- and RIG-I-/- mice. In the absence of either receptor, there is a significant decrease in BMDC activation at 12h post infection. However, only RIG-I-/- cells exhibit a delay in type I IFN production. In order to determine the role that IPS-1 plays in vivo, we infected mice with pathogenic RABV. We see that IPS-1-/- mice are more susceptible to infection than IPS-1+/+ mice and have a significantly increased incident of limb paralysis.

Show MeSH
Related in: MedlinePlus