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. 2002 Feb 19;99(4):2281-6.
doi: 10.1073/pnas.042355399.

Murine retroviruses activate B cells via interaction with toll-like receptor 4

John C Rassa  1 , Jennifer L Meyers, Yuanming Zhang, Rama Kudaravalli, Susan R Ross
Affiliations

Murine retroviruses activate B cells via interaction with toll-like receptor 4

John C Rassa et al. Proc Natl Acad Sci U S A. .

Abstract

Although most retroviruses require activated cells as their targets for infection, it is not known how this is achieved in vivo. A candidate protein for the activation of B cells by either mouse mammary tumor virus (MMTV) or murine leukemia virus is the toll-like receptor 4 (TLR4), a component of the innate immune system. MMTV caused B cell activation in C3H/HeN mice but not in C3H/HeJ or BALB/c (C.C3H Tlr4(lps-d)) congenic mice, both of which have a mutant TLR4 gene. This activation was independent of viral gene expression, because it occurred after treatment of MMTV with ultraviolet light or 2,2'-dithiodipyridine and in azidothymidine-treated mice. Nuclear extracts prepared from the lymphocytes of MMTV-injected C3H/HeN but not C3H/HeJ mice showed increased nuclear factor kappaB activity. Additionally, the MMTV- and Moloney murine leukemia virus envelope proteins coimmunoprecipitated with TLR4 when expressed in 293T cells. The MMTV receptor failed to coimmunoprecipitate with TLR4, suggesting that MMTV/TLR4 interaction is independent of virus attachment and fusion. These results identify retroviral proteins that interact with a mammalian toll receptor and show that direct activation by such viruses may initiate in vivo infection pathways.

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Figures

Figure 1
Figure 1
MMTV-induced activation of B cells in draining lymph nodes of C3H/HeN and C3H/HeJ mice. (A) Percentage of CD69/B220+ lymphocytes isolated from the draining popliteal lymph nodes of C3H/HeN (HeN) or C3H/HeJ (HeJ) 18–20 h after footpad injection of MMTV (FM) (average of 5–15 mice). Virus was injected with or without anti-MMTV antiserum. In some cases, virus was preincubated with normal goat serum, which had no effect on the ability of virus to stimulate B cells. LPS (10 μg) was injected into a separate set of mice. (B) Percentage of CD69/B220+ lymphocytes isolated from the draining popliteal lymph nodes of BALB/cJ (BALB) or congenic C.C3H-Tlr4lps−d (BALBlps−d) 18–20 h after footpad injection of MMTV (LA). D, draining lymph node; ND, nondraining lymph node.
Figure 2
Figure 2
MMTV-induced B cell activation is independent of viral gene expression. (A) MMTV (FM) was treated with 5 mM AT-2, DMSO vehicle, or untreated and injected into C3H/HeN mice (n = 3). Draining and nondraining lymph nodes were isolated at 18 h and 4 days postinjection, stained for CD69 and B220 expression, and analyzed by FACS. (B) Percentage of CD69/B220+ lymphocytes in AZT-treated mice injected with MMTV (LA). D, draining lymph node; ND, nondraining lymph node.
Figure 3
Figure 3
MMTV-induces NF-κB in the draining lymph nodes of C3H/HeN but not C3H/HeJ mice. C3H/HeN and C3H/HeJ mice received footpad injections of MMTV (FM), and an aliquot of cells from the draining (D) and nondraining (ND) contralateral footpads was taken for FACS analysis at 20 h postinjection. The remaining cells were used to prepare nuclear extracts. The C3H/HeN B cells showed activation (CD69+/B220+ cells, 45.9% draining, 15.0% control), whereas the C3H/HeJ B cells did not (17.9 vs. 16.4% draining vs. control). Electromobility shift analyses were carried out with an NF-κB probe in the absence or presence of anti-p50 or anti-p65 antibody. The arrow indicates the supershifted complex. (Lower) A 1,000-fold excess of cold NF-κB oligonucleotide was added to the C3H/HeN B cell extracts.
Figure 4
Figure 4
The MMTV Env interacts with TLR4. (A) 293T cells were transfected with expression vectors encoding the MMTV Env SU/transmembrane polyprotein (lanes 1–3), FLAG-tagged C3H/HeJ TLR4 (TLR4J, lanes 2 and 4), or C3H/HeN TLR4 (TLR4N, lanes 3 and 5) proteins. Extracts were made from the transfected cells, immunoprecipitated, and subjected to Western blot analysis with either goat anti-MMTV or mouse anti-FLAG antisera as indicated. (B) The MMTV SU or Env plasmids were cotransfected with TLR4N, and coimmunoprecipitation with an anti-FLAG antibody followed by Western blot analysis with goat anti-MMTV antisera was performed. (C) After cotransfection of the MMTV Env and C3H/HeN and TLR4J plasmids, cells were treated with tunicamycin or extracts were prepared and subjected to endoglycosidase H (Endo H) treatment. After immunoprecipitation with anti-FLAG antibody, the blots were probed with anti-MMTV antisera.
Figure 5
Figure 5
The Mo-MuLV Env interacts with TLR4. (A) Plasmid pHIT123 encoding the Mo-MuLV Env protein was cotransfected with TLR4N and TLR4J, and extracts were prepared and immunoprecipitated with anti-FLAG antibody. Western blots were performed with goat anti-MuLV antisera. (B) One plate of 293T cells was cotransfected with pHIT 123 and the TLR4N expression plasmid; another plate was transfected with the MMTV Env expression plasmid. Extracts were prepared from both transfections, and increasing amounts of the MMTV Env-containing extract was added to the MuLV/TLR4N extract as indicated in rows 1 and 2. As controls, extract from untransfected 293T cells was added in the same increasing increments to the MuLV/TLR4N extract (row 3). The extracts were precipitated with mouse anti-FLAG antiserum, and then Western blots were performed with anti-MuLV antisera (rows 1 and 3). Blot A was stripped and reprobed with anti-MMTV antisera (row 2).
Figure 6
Figure 6
The MTVR does not interact with TLR4. 293T cells were transfected with expression plasmids MMTV Env, TLR4N, and an MTVR/Ig fusion construct. The MTVR/Ig protein binds directly to protein A. Cell lysates were immunoprecipitated (IP) with or without anti-MMTV in the presence of protein A. Immunoprecipitations were analyzed by Western blot analysis with anti-FLAG, anti-MMTV, or anti-rabbit Ig.
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