Co-infection with the friend retrovirus and mouse scrapie does not alter prion disease pathogenesis in susceptible mice

Abstract

Prion diseases are fatal, transmissible neurodegenerative diseases of the central nervous system. An abnormally protease-resistant and insoluble form (PrP(Sc)) of the normally soluble protease-sensitive host prion protein (PrP(C)) is the major component of the infectious prion. During the course of prion disease, PrP(Sc) accumulates primarily in the lymphoreticular and central nervous systems. Recent studies have shown that co-infection of prion-infected fibroblast cells with the Moloney murine leukemia virus (Mo-MuLV) strongly enhanced the release and spread of scrapie infectivity in cell culture, suggesting that retroviral coinfection might significantly influence prion spread and disease incubation times in vivo. We now show that another retrovirus, the murine leukemia virus Friend (F-MuLV), also enhanced the release and spread of scrapie infectivity in cell culture. However, peripheral co-infection of mice with both Friend virus and the mouse scrapie strain 22L did not alter scrapie disease incubation times, the levels of PrP(Sc) in the brain or spleen, or the distribution of pathological lesions in the brain. Thus, retroviral co-infection does not necessarily alter prion disease pathogenesis in vivo, most likely because of different cell-specific sites of replication for scrapie and F-MuLV.

Figure 1. The Friend Murine Leukemia Virus (F-MuLV) strongly enhances PrP levels and prion infectivity released into the cell culture supernatant.

(A) Reverse transcriptase (RT) levels in NIH3T3-22L cells alone (22L) and NIH3T3 cells coinfected with the F-MuLV strain FB29 (22L+F-MuLV). (B) NIH3T3-22L (lane 1) and NIH3T3-22L/F-MuLV cells (lane 2) were assayed for F-MuLV expression using the DJ-39462 antibody for the viral envelope (Envgp70, Panel 1) and the DJ-39461 antibody for the viral capsid (CAp30, panel 2). PrP was detected using SAF-32 (Total PrP, panel 3) and exosomes using anti-EF1α (panel 4). The proteins recognized are indicated on the right side of the figure. Molecular mass markers are shown on the left side of the panel and lane numbers are indicated on the bottom of the figure. (C) NIH3T3-22L cells (mock) or NIH3T3-22L cells co-infected with the Friend viruses Fr57 or FB29 were analyzed by western blot for F-MuLV expression as well as PrP and exosome release as in panel B. Note that the F-MuLV Fr57 strain, like FB29, strongly enhances PrP and exosome release. Molecular mass markers are shown on the left side of the panel and lane numbers are indicated on the bottom of the figure. The proteins recognized are indicated on the right side of the figure. (D) Coculture assay. Uninfected target N2a#58 neuroblastoma cells grown on the bottom surface of a six well plate and a 0.4 mm pore size insert containing the 100 K pellet from the supernatants of NIH3T3-22L, NIH3T3-22L/MoMuLV or NIH3T3-22L/F-MuLV were cocultured for 4 days. The N2a#58 target cells were then passaged 5 times over 2 weeks and assayed for PrP^Sc. Ct = control. (E) Total PrP (-PK) and PrP^Sc (+PK) levels in N2a58 cells co-cultured with NIH3T3-22L/MoMuLV or NIH3T3-22L/F-MuLV supernatants were analyzed by dot blot. Ct cells = 22L infected and uninfected N2a#58 cells not exposed to supernatant. Lane numbers are shown at the bottom of the panel.

Figure 2. Co-infection of mice with 22L scrapie and F-MuLV does not affect scrapie disease incubation time.

Survival curve of (B10 X A.BY)F1 mice inoculated intraperitoneally (i.p.) with 22L mouse scrapie alone (closed circles) or co-infected with 22L scrapie i.p. and FV intravenously (i.v., open circles). dpi = days post-infection.

Figure 3. PrP^Sc accumulates to equivalent levels in the brain and spleen of mice infected with 22L or 22L plus F-MuLV.

(A) Western blot of PK-treated spleen homogenates from (B10 X A.BY)F1 mice either inoculated i.v. with FV alone (F-MuLV), i.p. with mouse 22L mouse scrapie alone (22L), or co-infected with 22L mouse scrapie i.p. and FV i.v. (22L+F-MuLV). Samples from 4 individual mice are shown for both 22L and 22L+FV and a single sample for FV infected mice. Lanes 2–5 show dilutions of a standard comprised of equal amounts of spleen homogenate from individual mice inoculated with 22L+FV (Pooled Sample) used to calculate the relative amount of PrP^Sc in the spleen of infected mice. (B) The relative amount of PrP^Sc in each lane in (A) was determined by quantitative comparison to a standard curve derived from equal amounts of spleen homogenate (as shown in panel A) or brain homogenate (data not shown) derived from individual mice inoculated with 22L+FV.

Figure 4. PrP^Sc localization in the spleen is the same in mice infected with 22L or 22L plus FV.

PrP^Sc in spleens from (B10 X A.BY)F1 mice inoculated i.v. with FV alone (A), i.p. with mouse 22L mouse scrapie alone (B), or co-infected with 22L mouse scrapie i.p. and FV i.v. (C). Sections were stained using the anti-PrP mouse monoclonal antibody D13. Magnification = 200×.

Figure 5. Vacuolation and PrPS^c deposition in the brain is the same in mice infected with 22L or 22L plus FV.

(A–C) Hemotoxylin and eosin staining of the cerebellum from (B10 X A.BY)F1 mice inoculated (A) i.v. with FV alone, (B) i.p. with mouse 22L mouse scrapie alone, or (C) co-infected with 22L mouse scrapie i.p. and FV i.v.. Magnification = 200×. (D–F) PrP^Sc deposition in the cerebellum of (B10 X A.BY)F1 mice inoculated (D) i.v. with FV alone, (E) i.p. with mouse 22L mouse scrapie alone, or (F) co-infected with 22L mouse scrapie i.p. and FV i.v.. Sections were stained using the anti-PrP mouse monoclonal antibody D13 following hydrated autoclaving at high pressure and temperature. Red stain indicates the presence of PrP^Sc. Magnification = 200×.

Figure 6. Analysis of follicular dendritic cells (FDCs) for Friend virus infection.

Flow cytometry was used to detect cell surface FV glycosylated gag using the monoclonal antibody mAb34 (Viral antigen). FDCs are B220⁺ cells gated for lack of expression of CD11c, CD8, CD19, CD4, CD3, CD11b and Gr-1, but staining positive for MHCII. The representative plot of FDCs from an acutely infected mouse shows no viral antigen staining above background (Naïve) levels.