Avian reovirus major mu-class outer capsid protein influences efficiency of productive macrophage infection in a virus strain-specific manner

Abstract

We determined that the highly pathogenic avian reovirus strain 176 (ARV-176) possesses an enhanced ability to establish productive infections in HD-11 avian macrophages compared to avian fibroblasts. Conversely, the weakly pathogenic strain ARV-138 shows no such macrophagotropic tendency. The macrophage infection capability of the two viruses did not reflect differences in the ability to either induce or inhibit nitric oxide production. Moderate increases in the ARV-138 multiplicity of infection resulted in a concomitant increase in macrophage infection, and under such conditions the kinetics and extent of the ARV-138 replication cycle were equivalent to those of the highly infectious ARV-176 strain. These results indicated that both viruses are apparently equally capable of replicating in an infected macrophage, but they differ in the ability to establish productive infections in these cells. Using a genetic reassortant approach, we determined that the macrophagotropic property of ARV-176 reflects a post-receptor-binding step in the virus replication cycle and that the ARV-176 M2 genome segment is required for efficient infection of HD-11 cells. The M2 genome segment encodes the major mu-class outer capsid protein (muB) of the virus, which is involved in virus entry and transcriptase activation, suggesting that a host-specific influence on ARV entry and/or uncoating may affect the likelihood of the virus establishing a productive infection in a macrophage cell.

FIG. 1

ARV-176 shows a predilection for macrophages. Diluted stocks of ARV-138 and ARV-176 were used to infect QM5 fibroblasts (a and b, respectively). Infected cell monolayers were incubated at 37°C and fixed at 16 hpi before being immunostained using a reovirus-specific rabbit polyclonal antiserum and goat anti-rabbit immunoglobulin G conjugated with alkaline phosphatase to detect viral foci of infection. Virus dilutions that gave equivalent numbers of foci in QM5 fibroblasts were then used to infect HD-11 macrophages (c and d) that were similarly fixed and immunostained at 9 hpi.

FIG. 2

Relative infectivities of ARV-176 and ARV-138 in macrophages versus fibroblasts. Both viruses were serially diluted and used to infect monolayers of QM5 fibroblasts (gray bars). Virus dilutions that gave equivalent numbers of foci of infection in QM5 cells were similarly used to infect HD-11 macrophages (black bars). Infected monolayers were immunostained to detect viral foci of infection as described for Fig. 1. The average number of foci of infection per field at a magnification of ×100 was determined after correcting for the relative virus dilution, as described in Materials and Methods. Results are presented as means and standard errors from a representative experiment.

FIG. 3

Neither ARV-176 nor ARV-138 differentially affects NO production by infected macrophages. HD-11 cell monolayers were incubated for 6 h in the presence (black bars) or absence (gray bars) of LPS at 500 ng/ml followed by infection with equivalent concentrations of QM5 focus-forming units of ARV-176 or ARV-138 per milliliter. At 12 hpi the supernatants were harvested and assayed for the presence of nitrite using standard protocols. Nitrite concentrations were determined relative to a standard sodium nitrite curve. Uninf., uninfected cells.

FIG. 4

ARV-138 is capable of complete infection of HD-11 macrophage monolayers. HD-11 macrophages were left uninfected (b) or were infected with increasing concentrations of standardized QM5 focus-forming units of ARV-176 (a) or ARV-138 (c) per milliliter. Monolayers were fixed and immunostained as described for Fig. 1 to reveal viral foci of infection. The ARV-138 inoculum in panel c corresponds to a 32-fold increase in the QM5 focus-forming units compared to the ARV-176 inoculum in panel a.

FIG. 5

ARV-176 and ARV-138 protein synthesis and replication are similar in fibroblasts and macrophages. (A) Cell monolayers of quail QM5 fibroblasts or HD-11 macrophages were left uninfected (U) or infected with ARV-176 (176) or ARV-138 (138) using the minimum concentrations of virus inocula required for complete infection of the monolayer (see Fig. 4). Cultures were pulse-labeled for 1 h with [³⁵S]methionine at 12 and 16 hpi, and the radiolabeled cell lysates were fractioned by SDS-PAGE and detected by autoradiography. The locations of the major λ, μ, and ς classes of viral proteins are indicated on the right. (B) Monolayers of quail QM5 fibroblasts or HD-11 macrophage cells were infected with ARV-176 (gray bars) or ARV-138 (black bars) as described for panel A. Infected cultures were harvested at 48 hpi, and the yield of infectious progeny virions was determined by a plaque assay on QM5 cell monolayers. Results are presented as means and standard errors from a representative experiment.

FIG. 6

Genome segment profiles of parental and reassortant ARV virions. Viral double-stranded RNA was isolated from concentrated virus stocks of the parental and reassortant viruses, and individual genome segments were resolved on SDS–12.5% PAGE gels. Gels were stained with ethidium bromide and visualized under UV illumination. Images were captured and enhanced with Adobe Photoshop, and negative images were printed. The parental ARV-176 and ARV-138 genome segment profiles are shown by themselves in the left-hand panel and included as markers on the gels that contain the reassortant genomes. Only a selected number of reassortants used in this study are shown.

FIG. 7

Genome segment assignments and relative infectivities of ARV reassortants. Parental (black bars) and reassortant (gray bars) clones are identified at the extreme left. For each reassortant, the parental identity of each genome segment, as determined by its relative mobility by SDS-PAGE, is indicated (3 for ARV-138 and 7 for ARV-176). Reassortant virus stocks were standardized to give equal and countable numbers of focus-forming units in quail QM5 fibroblasts. The standardized inocula were then used to infect QM5 or HD-11 cell monolayers. The infected QM5 and HD-11 cell monolayers were fixed and immunostained, and the average number of foci per field was determined as described for Fig. 2. The relative infectivity was calculated as the ratio of HD-11/QM5 foci, and values were normalized to the relative infectivity of ARV-138 arbitrarily set to a value of 1. Results are presented as means and standard errors from three or more separate experiments.

FIG. 8

The ARV-176 M2 genome segment is required for enhanced macrophage infection. Virus stocks of ARV-176 (a and b), ARV-138 (c and d), and the ARV-138 M2 monoreassortant R402 (e and f) were standardized to give equivalent concentrations of focus-forming units in quail QM5 fibroblasts (a, c, and e). The same virus dilutions were used to infect HD-11 macrophage monolayers (b, d, and f). Cells were immunostained as described for Fig. 1 to reveal viral foci of infection.