Influenza Virus Overcomes Cellular Blocks To Productively Replicate, Impacting Macrophage Function

Abstract

Whether influenza virus replication in macrophages is productive or abortive has been a topic of debate. Utilizing a panel of 28 distinct human, avian, and swine influenza viruses, we found that only a small subset can overcome cellular blocks to productively replicate in murine and primary human macrophages. Murine macrophages have two cellular blocks. The first block is during viral entry, where virions with relatively acid-stable hemagglutinin (HA) proteins are rendered incapable of pH-induced triggering for membrane fusion, resulting in lysosomal degradation. The second block is downstream of viral replication but upstream of late protein synthesis. In contrast, primary human macrophages only have one cellular block that occurs after late protein synthesis. To determine the impact of abortive replication at different stages of the viral life cycle or productive replication on macrophage function, we assessed cytotoxicity, nitric oxide or reactive oxygen species production, and phagocytosis. Intriguingly, productive viral replication decreased phagocytosis of IgG-opsonized bioparticles and Fc receptor CD16 and CD32 surface levels, a function, to our knowledge, never before reported for an RNA virus. These data suggest that replication in macrophages affects cellular function and plays an important role in pathogenesis during infection in vivo IMPORTANCE: Macrophages are a critical first line of defense against respiratory pathogens. Thus, understanding how viruses evade or exploit macrophage function will provide greater insight into viral pathogenicity and antiviral responses. We previously showed that only a subset of highly pathogenic avian (HPAI) H5N1 influenza virus strains could productively replicate in murine macrophages through a hemagglutinin (HA)-mediated mechanism. These studies expand upon this work and demonstrate that productive replication is not specific to unique HPAI H5N1 viruses; an H1N1 strain (A/WSN/33) can also replicate in macrophages. Importantly, we identify two cellular blocks limiting replication that can be overcome by an avian-like pH of activation for nuclear entry and a yet-to-be-identified mechanism(s) to overcome a postnuclear entry block. Overcoming these blocks reduces the cell's ability to phagocytose IgG-opsonized bioparticles by decreasing Fc receptor surface levels, a mechanism previously thought to occur during bacterial and DNA viral infections.

Keywords: influenza; macrophage; replication.

FIG 1

A/WSN/33 replicates in Raw264.7 macrophages and requires HA and NA. MDCK cells and Raw264.7 macrophages were infected with CA/09, WSN, or VN/1203 (A and B) or reverse genetics viruses (C and D) (MOI of 2 [A and C] or 0.01 [B and D]). Cells were fixed at 4 hpi and stained for nucleoprotein (NP; green). Nuclei were stained with DAPI (blue). (A and C) Representative images are shown. The percentage of nuclear NP-positive cells was calculated, and means ± standard deviations are shown in the bottom left corner (A), or the percentage of nuclear NP in Raw264.7 macrophages standardized to the percentage of nuclear NP in MDCK cells to account for differences in infection (percentage of MDCK cells) is shown (C). (B and D) Viral replication was measured by TCID₅₀ analysis using MDCK cells in the viral supernatant. Results are reported as the viral titer at 2 hpi (residual virus after infection) subtracted from the viral titer at 24 hpi. Data are averages from 2 to 5 independent experiments performed at least in duplicate. Error bars indicate standard deviations (SD). The bar is 50 μm.

FIG 2

(A) HA activation pH values for WSN reverse genetics viruses. Raw264.7 macrophages and MDCK cells were incubated in the presence of 10 μg/ml pHrodo green 10-kDa dextran at 37°C for 30 min. Cells were washed and fluorescence was read on a plate reader. The pH of endosome compartments was calculated compared to a standard curve generated from cells incubated in the presence of pH-adjusted buffers. (B) Influenza pH of WSN activation mutants containing CA/09 genes and parental viruses were rescued by reverse genetics. To determine the pH of activation, Vero cells were infected with the indicated viruses. After 6 hpi (VN/1203 virus) or 16 hpi (WSN and CA/09 viruses), cells were incubated in the presence of TPCK trypsin followed by incubation with pH-adjusted PBS. The pH of activation was reported as the highest pH at which fusion occurs (indicated by arrows).

FIG 3

CA/09 colocalizes with LAMP1-positive compartments in Raw264.7 macrophages. MDCK cells (A) and Raw264.7 macrophages (B) were plated on coverslips. Cells were infected the next day (MOI of 2) on ice for 1 h. Cells were washed and warm medium was added (time point 0). Cells were fixed at the indicated time points and stained for NP (green) and LAMP1 (red). Nuclei were stained with DAPI. The percentage of NP colocalized with LAMP1 was determined in MDCK cells (C) and Raw264.7 macrophages (D) by using the mean fluorescence intensity of the LAMP1 channel at NP surfaces compared to the secondary-only control using Imaris software. Data are averages from 2 independent experiments (B and C). Panels A and B are representative images. Error bars indicate SD, and an asterisk represents significance (P < 0.005) as measured by two-tailed t test. The bar is 15 μm.

FIG 4

(A) HA activation pH value for VN/1203 HA2-K58I. Influenza pH of activation mutant rgVN/1203 HA2-K58I (HA2-K58I) and parental virus was rescued by reverse genetics. To determine the pH of activation, Vero cells were infected with the indicated viruses. After 6 hpi, cells were incubated in the presence of TPCK trypsin, followed by incubation of pH-adjusted PBS. pH of activation was reported as the highest pH at which fusion occurs (indicated by arrows). (B) Raw264.7 macrophages and MDCK cells were infected with the indicated viruses and fixed and stained for NP protein 4 hpi. The percentage of nuclear NP-positive cells was calculated and standardized to the percentage of nuclear NP in MDCK cells to account for differences in infection (percentage of MDCK cells). Viral replication in Raw264.7 macrophages was measured by TCID₅₀ analysis in the viral supernatant. (C) Results are reported as the viral titer at 2 hpi (residual virus after infection) and was subtracted from the viral titer at 24 hpi. Results are averages from 2 independent experiments. *, P < 0.05.

FIG 5

Viral strains that reach the nucleus are blocked at protein translation. Raw264.7 macrophages and MDCK cells were infected with the indicated viruses (MOI of 2) and incubated for 4 h (for NP staining) or 20 h (for NS1 staining). Cells were stained for NP and NS1 (green), and the percent positive (± standard deviations) cells was calculated (bottom right corner). Nuclei were stained with DAPI (blue). The bar is 50 μm. Representative images of 3 independent experiments are shown.

FIG 6

Influenza virus replication in primary human macrophages. Primary human macrophages were generated after isolating monocytes from blood as described in Materials and Methods. (A) Cells were infected and viral replication was determined at 24, 48, and 72 hpi. (B) NP staining (green). Nuclei were visualized using DAPI (blue) at 4 hpi. Images in panel B are representative images. The percentage of nuclear NP-positive cells was calculated, and means ± standard deviations are shown in the bottom left corner. The bar is 50 μm. Data are averages from at least 3 independent experiments performed in at least duplicate from at least 2 donors. Error bars indicate SD.

FIG 7

CA/09 infection in primary human macrophages produces RNA synthesis and protein translation. A549 cells (A) and primary human macrophages (B and C) were infected with CA/09 or WSN. (A and B) Total RNA was extracted and vRNA, cRNA, and mRNA species were determine as described in Materials and Methods. Data are standardized to GAPDH levels and input levels at 30 min postinfection. WSN levels for the 12-h time point were set to 1. (C) Cells were fixed 20 h postinfection and stained for NS1 protein (green). Nuclei were stained with DAPI (blue). The percent NS1-positive cells was calculated and is indicated in the panels. The bar is 50 μm. Data are averages from 2 independent experiments from 2 different donors.

FIG 8

Influenza virus replication in macrophages affects macrophage phagocytosis. Raw264.7 macrophages and primary human macrophages were infected with the indicated viruses (MOI of 1). At 24 hpi, the amount of cytotoxicity (A), nitrite levels (B), and ROS production (C) were determined as described in Materials and Methods. T:0, time point 0; EtOH, ethanol. (D) Phagocytosis of macrophages was measured by the percentage of FITC-positive cells (indicating IgG-opsonized pHrodo-S. aureus bioparticle uptake) measured by flow cytometry. FcγRIII/FcγRII cell surface levels in Raw264.7 macrophages (E and G) and primary human macrophages (F and H) were measured by flow cytometry at 24 hpi. Std., standardized. Data in panels D, E, F, G, and H are presented as the percentage of uninfected cells, which was set at 100%. Error bars indicate SD. *, P < 0.05; **, P < 0.005.

FIG 9

Model for influenza virus replication in Raw264.7 and primary human macrophages. (1) In Raw264.7 macrophages, influenza viruses enter the cell (13) and either degrade in the lysosome or enter the nucleus. (2) A second block occurs prior to protein translation. (3) Select viruses can productively replicate. All viruses enter the nucleus in human macrophages, but most viruses encounter a block after nuclear entry despite productive translation of viral protein.