The Hemagglutinin Protein of Highly Pathogenic H5N1 Influenza Viruses Overcomes an Early Block in the Replication Cycle To Promote Productive Replication in Macrophages

Abstract

Macrophages are known to be one of the first lines of defense against influenza virus infection. However, they may also contribute to severe disease caused by the highly pathogenic avian (HPAI) H5N1 influenza viruses. One reason for this may be the ability of certain influenza virus strains to productively replicate in macrophages. However, studies investigating the productive replication of influenza viruses in macrophages have been contradictory, and the results may depend on both the type of macrophages used and the specific viral strain. In this work, we investigated the ability of H1 to H16 viruses to productively replicate in primary murine alveolar macrophages and RAW264.7 macrophages. We show that only a subset of HPAI H5N1 viruses, those that cause high morbidity and mortality in mammals, can productively replicate in macrophages, as measured by the release of newly synthesized virus particles into the cell supernatant. Mechanistically, we found that these H5 strains can overcome a block early in the viral life cycle leading to efficient nuclear entry, viral transcription, translation, and ultimately replication. Studies with reassortant viruses demonstrated that expression of the hemagglutinin gene from an H5N1 virus rescued replication of H1N1 influenza virus in macrophages. This study is the first to characterize H5N1 influenza viruses as the only subtype of influenza virus capable of productive replication in macrophages and establishes the viral gene that is required for this characteristic. The ability to productively replicate in macrophages is unique to H5N1 influenza viruses and may contribute to their increased pathogenesis.

Influenza virus replication in macrophages: balancing protection and pathogenesis

“…The results demonstrated that HPAI H5N1 IAV is unique in its ability to replicate productively in macrophages. In contrast to what has been shown for human AMs, primary mouse AMs were also shown to support productive replication of HPAI H5N1, but not an H1N1 virus [39]. A comparison of the replication of 23 different IAV strains of different HA subtypes in RAW264.7 cells also showed that HPAI H5N1 viruses and the H1N1 WSN strain were the only viruses that could productively replicate.…”

“…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.…”

“…When the replicative capacity of a panel of eight H5N1 viruses was tested in RAW264.7 cells, only those associated with high virulence in mammals were capable of productive replication, demonstrating a correlation between replication in macrophages and disease severity [39]. Further, a reassortant CA/09 virus expressing the HA protein from a HPAI H5 virus was capable of productive replication in macrophages and was associated with higher morbidity and mortality in mice relative to the WT CA/09 virus, which does not replicate in macrophages [51].…”

“…While IAV-infected M1 macrophages expressed the highest levels of pro-inflammatory cytokines such as TNF-a, IAV infection of M2 macrophages was able to override the anti-inflammatory cytokine profile of these cells and cause them to secret TNF-a and other M1-like cytokines. Importantly, this programming override to a more pro-inflammatory phenotype was only seen during the infection of macrophages with WSN and HPAI H5N1, strains that have been shown to replicate productively in macrophages [32,39]. However, a requirement for productive replication for the M2 to M1 phenotype switch has not been investigated directly.…”

Early host response and immune signaling to 2009 pandemic influenza A (H1N1) viruses in primary cell culture models.

“…Previous studies looking at IAV-MDM interactions used the 1ug/ml TPCK trypsin concentration [222,353,391] and since we did not see a difference between 0.5 and 1ug/ml we decided to move forward using 1ug/ml so we could best compare our results to others. Here we compared influenza isolates derived in eggs to those derived in cell cultures to determine if there was a difference in activation and their ability to infect MDM.…”