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. 1999 Jul;73(7):5970-80.
doi: 10.1128/JVI.73.7.5970-5980.1999.

Replication of murine cytomegalovirus in differentiated macrophages as a determinant of viral pathogenesis

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Replication of murine cytomegalovirus in differentiated macrophages as a determinant of viral pathogenesis

L K Hanson et al. J Virol. 1999 Jul.

Abstract

Blood monocytes or tissue macrophages play a pivotal role in the pathogenesis of murine cytomegalovirus (MCMV) infection, providing functions beneficial to both the virus and the host. In vitro and in vivo studies have indicated that differentiated macrophages support MCMV replication, are target cells for MCMV infection within tissues, and harbor latent MCMV DNA. However, this cell type presumably initiates early, antiviral immune responses as well. In addressing this paradoxical role of macrophages, we provide evidence that the proficiency of MCMV replication in macrophages positively correlates with virulence in vivo. An MCMV mutant from which the open reading frames M139, M140, and M141 had been deleted (RV10) was defective in its ability to replicate in macrophages in vitro and was highly attenuated for growth in vivo. However, depletion of splenic macrophages significantly enhanced, rather than deterred, replication of both wild-type (WT) virus and RV10 in the spleen. The ability of RV10 to replicate in intact or macrophage-depleted spleens was independent of cytokine production, as this mutant virus was a poor inducer of cytokines compared to WT virus in both intact organs and macrophage-depleted organs. Macrophages were, however, a major contributor to the production of tumor necrosis factor alpha and gamma interferon in response to WT virus infection. Thus, the data indicate that tissue macrophages serve a net protective role and may function as "filters" in protecting other highly permissive cell types from MCMV infection. The magnitude of virus replication in tissue macrophages may dictate the amount of virus accessible to the other cells. Concomitantly, infection of this cell type initiates the production of antiviral immune responses to guarantee efficient clearance of acute MCMV infection.

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Figures

FIG. 1
FIG. 1
Construction of the MCMV mutants RV10 and RV10Rev. The open boxes indicate wild-type MCMV sequences within the HindIII J and I fragments. Solid lines denote deleted sequences. Shaded boxes indicate the locations of the e1–β-glucuronidase cassette. Plasmid pJIβglu-10 contains MCMV and e1–β-glucuronidase sequences in the vector pGem4Z; pDJI contains the denoted MCMV sequences in the vector pcDNA3. The numbers refer to sizes (in kilobases) of the indicated fragments.
FIG. 2
FIG. 2
Growth of RV10 and WT MCMV in fibroblasts and macrophages in vitro. Monolayers of NIH 3T3 cells (A) or IC-21 macrophages (B) were infected at a multiplicity of 0.1 PFU/cell. Cell-free and cell-associated viruses were collectively titerate at the indicated times postinfection by plaque assay on NIH 3T3 cells. Data shown are representative of three individual experiments.
FIG. 3
FIG. 3
Expression of RV10 and RV10Rev immediate-early genes in fibroblasts and macrophages. NIH 3T3 fibroblasts or IC-21 macrophages were mock infected (M) or were infected with RV10Rev (Rev) or RV10 at a multiplicity of 1 PFU/cell. The virus inocula were purified virions banding at the 40 to 50% sorbitol interface of a 20 to 70% sorbitol gradient. Five micrograms of total RNA harvested at 4 h after infection was run on a formaldehyde gel for Northern blot analyses. The loading of equal amounts of RNA was confirmed by ethidium bromide staining of the 18S and 28S rRNA. (A) Analysis of the major immediate-early gene region. The Northern blot was hybridized to a probe corresponding to the HindIII L region of MCMV in order to detect RNA from ie1 (2.7 kb), ie2 (1.7 kb), or ie3 (2.7 kb). (B) Analysis of the m142 and m143 immediate-early gene region. The Northern blot shown in panel A was stripped and hybridized with a probe corresponding to m142 and m143 (bases 198832 to 201744 of the MCMV genome) within HindIII-I. The faint band visible between the m142 and m143 transcripts represents incompletely stripped ie1/ie3 signal.
FIG. 4
FIG. 4
Wild-type MCMV and RV10Rev attachment to or penetration into NIH 3T3 fibroblasts and IC-21 macrophages. (A) Penetration assay. NIH 3T3 cells were infected with WT MCMV at room temperature for 1 h, were shifted to 37°C, and at the indicated times after the temperature shift were washed with acid-glycine buffer. Monolayers were then overlaid with semisolid complete medium and incubated for 5 days to quantitate the number of plaques which formed in each treatment group. The percentage of bound virus which penetrated was calculated as follows: (the number of plaques formed with acid-glycine washing)/(the number of plaques formed without acid-glycine washing) × 100. (B and C) Viral DNA stably attached to or penetrated into fibroblasts and macrophages. NIH 3T3 fibroblasts (B) or IC-21 macrophages (C) were infected as described above at room temperature or at 4°C with RV10Rev (4 PFU/cell). At the indicated times after the shift to 37°C, monolayers were washed with acid–glycine buffer and returned for a total of 4 h of incubation in complete medium. Cells were then harvested for extraction of viral DNA and Southern blotting. The DNA was hybridized with a probe corresponding to HindIII-L. The C denotes control cells that were not washed with acid-glycine but were incubated for a total of 4 h after the shift to 37°C.
FIG. 5
FIG. 5
RV10 attachment to or penetration into NIH 3T3 fibroblasts and IC-21 macrophages. Experiments were performed essentially as described in the legend for Fig. 4B. In this experiment, monolayers of NIH 3T3 fibroblasts or IC-21 macrophages were infected with RV10 or RV10Rev (Rev), were shifted to 37°C, and at 120 min after the shift were washed with acid-glycine buffer. Infected cells were incubated for a total of 4 h in complete medium. Digestion of RV10 DNA with HindIII and BamHI yields bands of 2.8, 2.6, and 1.1 kb which hybridize to the HindIII-I probe. RV10Rev DNA digested with the same enzymes yields bands of 4.1, 2.8, and 2.6 kb which hybridize to this probe.
FIG. 6
FIG. 6
Lethality of RV10 and WT MCMV for SCID mice. (A and B) Mice (four or five per group) were infected i.p. with 104 PFU of the indicated virus and observed daily for mortality. The experiments were performed twice.
FIG. 7
FIG. 7
Growth of RV10 and WT MCMV in spleen and liver tissues. BALB/c mice were inoculated i.v. with 3 × 105 PFU of the indicated virus. Spleen (A) and liver (B) tissues were harvested from three individual mice at the indicated times postinfection. Virus titers in 20% (wt/vol) tissue homogenates were determined by plaque assay on NIH 3T3 cells. Data points are the average values for three individual mice, and error bars represent standard deviations.
FIG. 8
FIG. 8
Depletion of splenic macrophages by L-Cl2MBP treatment. Frozen tissue sections of spleens from mice injected i.v. with L-Cl2MBP (A) or PBS (B) and infected with 3 × 105 PFU of WT MCMV were stained for the macrophage-specific marker acid phosphatase. Mice received virus 48 h after L-Cl2MBP or PBS treatment, and spleens were harvested at 3 days postinfection. Acid phosphatase-positive cells in the red pulp, marginal zone, and white pulp stain bright red.
FIG. 9
FIG. 9
Growth of RV10 and WT MCMV in macrophage-depleted and intact tissues. Mice were injected i.v. with either L-Cl2MBP or PBS and 48 h later were infected with 3 × 105 PFU of the indicated virus. Spleens (A) and livers (B) were harvested from four or five individual mice at the indicated times postinfection. Virus titers in 20% (wt/vol) homogenates were determined by plaque assay on NIH 3T3 cells. Data points are the average values for individual mice, and error bars represent standard deviations. Rev designates infection with RV10Rev, the suffix -P designates treatment with PBS, and the suffix -L designates treatment with L-Cl2MBP.
FIG. 10
FIG. 10
Cytokine levels produced by splenocytes in response to RV10 or WT MCMV infection of normal or macrophage-depleted mice. Mice were administered L-Cl2MBP (L) or PBS (P) i.v., and 48 h later, they were inoculated i.v. with mock virus preparation or with RV10Rev or RV10 (3 × 105 PFU). Thirty-six hours later, spleens were harvested from three individual mice, and suspensions containing 5 × 106 splenocytes/ml were incubated overnight. After 24 h, supernatants were collected and cytokines were quantitated by sandwich ELISA assays. IL-12 data are for IL-12 p40. Values represent the mean titers and error bars denote standard deviations for three mice. A single asterisk denotes a significant difference (P ≤ 0.05) between L-Cl2MBP- and PBS-treated mice infected with RV10Rev. A double asterisk denotes a significant difference (P ≤ 0.05) between RV10- and RV10Rev-infected mice treated with PBS. The data are representative of two separate experiments.

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