Infection of Mouse Macrophages by Seasonal Influenza Viruses Can Be Restricted at the Level of Virus Entry and at a Late Stage in the Virus Life Cycle

Abstract

Airway epithelial cells are susceptible to infection with seasonal influenza A viruses (IAV), resulting in productive virus replication and release. Macrophages (M⌽) are also permissive to IAV infection; however, virus replication is abortive. Currently, it is unclear how productive infection of M⌽ is impaired or the extent to which seasonal IAV replicate in M⌽. Herein, we compared mouse M⌽ and epithelial cells for their ability to support genomic replication and transcription, synthesis of viral proteins, assembly of virions, and release of infectious progeny following exposure to genetically defined IAV. We confirm that seasonal IAV differ in their ability to utilize cell surface receptors for infectious entry and that this represents one level of virus restriction. Following virus entry, we demonstrate synthesis of all eight segments of genomic viral RNA (vRNA) and mRNA, as well as seven distinct IAV proteins, in IAV-infected mouse M⌽. Although newly synthesized hemagglutinin (HA) and neuraminidase (NA) glycoproteins are incorporated into the plasma membrane and expressed at the cell surface, electron microscopy confirmed that virus assembly was defective in IAV-infected M⌽, defining a second level of restriction late in the virus life cycle. IMPORTANCE Seasonal influenza A viruses (IAV) and highly pathogenic avian influenza viruses (HPAI) infect macrophages, but only HPAIreplicate productively in these cells. Herein, we demonstrate that impaired virus uptake into macrophages represents one level of restriction limiting infection by seasonal IAV. Following uptake, seasonal IAV do not complete productive replication in macrophages, representing a second level of restriction. Using murine macrophages, we demonstrate that productive infection is blocked late in the virus life cycle, such that virus assembly is defective and newly synthesized virions are not released. These studies represent an important step toward identifying host-encoded factors that block replication of seasonal IAV, but not HPAI, in macrophages. In humans, infection with seasonal influenza A viruses (IAV) is generally restricted to the respiratory tract. IAV infection of airway epithelial cells (AEC) is initiated following recognition of cell surface sialic acid (SIA) by the viral hemagglutinin (HA) glycoprotein (reviewed in reference 1). In addition to HA-SIA binding, it is likely that particular membrane-associated glycoproteins and/or glycolipids facilitate virus entry into AEC; however, the identity of such an entry receptor(s) is currently unknown. IAV infection of AEC results in productive virus replication, characterized by synthesis of viral RNA (vRNA) and mRNA, production of viral proteins, virion assembly, and budding of viral progeny from the surfaces of infected cells. Productive infection of AEC results in amplification of IAV in the airways, promoting virus dissemination and disease.In addition to AEC, macrophages (M⌽) are one of the first cells in the respiratory tract to detect and respond to IAV. As for AEC, binding ...

Influenza virus replication in macrophages: balancing protection and pathogenesis

“…While RAW264.7 cells support a modest amount of productive replication of RG-Braz-HA over the first 24 h post-infection, the PR8 virus, despite enhanced entry efficiency in the presence of zanamivir, does not productively replicate, confirming the presence of a replication block downstream of nuclear entry. In contrast to RAW264.7 cells, mouse PEC macrophages did not support productive replication of either virus strain, with the block being downstream of RNA and protein synthesis and upstream of virus budding [27]. This result appears to be consistent with that of Marvin et al and suggests that mouse PEC macrophages may also serve as a good model for the restriction of IAV replication in human macrophages.…”

“…In this case, virus nucleoprotein (NP) accumulated in 20 % of infected cells, but virus release was undetected [26]. Other studies support the findings of early experiments [11,[27][28][29]. However, certain seasonal H1N1 and H3N2 strains are capable of limited productive replication in mouse bone marrow-derived macrophages (BMDMs) or human MDMs, respectively [30][31][32][33].…”

“…The differences in infection efficiency were unrelated to sialic acid binding, as both viruses were found to bind equally well to cell surface receptors. The infectivity of PR8 was enhanced in the presence of the neuraminidase inhibitor zanamivir [27]. This suggests a role for the viral NA protein in entry into macrophages.…”

“…The infectivity of reverse genetics-derived PR8 was poor in RAW264.7 cells at 2 h post-infection, while a reassortant PR8 virus expressing the HA gene from influenza A/Brazil/11/78 H1N1 (RGBraz-HA) was able to efficiently infect RAW264.7 cells [27]. The differences in infection efficiency were unrelated to sialic acid binding, as both viruses were found to bind equally well to cell surface receptors.…”

“…PR8 also failed to productively replicate in RAW264.7 cells. The restriction of PR8 in RAW264.7 cells has been linked to inefficient attachment and entry [27] and to a block downstream of virus internalization but upstream of nuclear entry [32,39]. The differences in the results presented by these investigators may be attributed to differences in the preparation or source of the exact PR8 virus used in the studies.…”