Human Metapneumovirus SH and G Glycoproteins Inhibit Macropinocytosis-Mediated Entry into Human Dendritic Cells and Reduce CD4 ⁺ T Cell Activation

Abstract

Human metapneumovirus (HMPV) is a major etiologic agent of respiratory disease worldwide. HMPV reinfections are common in healthy adults and children, suggesting that the protective immune response to HMPV is incomplete and short-lived. We used gene-deletion viruses to evaluate the role of the attachment G and small hydrophobic SH glycoproteins on virus uptake by primary human monocyte-derived dendritic cells (MDDC) in vitro and on subsequent MDDC maturation and activation of autologous T cells. HMPV with deletion of G and SH (⌬SHG) exhibited increased infectivity but had little effect on MDDC maturation. However, MDDC stimulated with ⌬SHG induced increased proliferation of autologous Th1-polarized CD4 ؉ T cells. This effect was independent of virus replication. Increased T cell proliferation was strictly dependent on contact between virus-stimulated MDDC and CD4 ؉ T cells. Confocal microscopy revealed that deletion of SH and G was associated with an increased number of immunological synapses between memory CD4؉ T cells and virus-stimulated MDDC. Uptake of HMPV by MDDC was found to be primarily by macropinocytosis. Uptake of wild-type (WT) virus was reduced compared to that of ⌬SHG, indicative of inhibition by the SH and G glycoproteins. In addition, DC-SIGN-mediated endocytosis provided a minor alternative pathway that depended on SH and/or G and thus operated only for WT. Altogether, our results show that SH and G glycoproteins reduce the ability of HMPV to be internalized by MDDC, resulting in a reduced ability of the HMPV-stimulated MDDC to activate CD4 ؉ T cells. This study describes a previously unknown mechanism of virus immune evasion. IMPORTANCEHuman metapneumovirus (HMPV) is a major etiologic agent of respiratory disease worldwide. HMPV reinfections are common in healthy adults and children, suggesting that the protective immune response to HMPV is incomplete and short-lived. We found that HMPV attachment G and small hydrophobic SH glycoproteins reduce the ability of HMPV to be internalized by macropinocytosis into human dendritic cells (DC). This results in a reduced ability of the HMPV-stimulated DC to activate Th1-polarized CD4 ؉ T cells. These results contribute to a better understanding of the nature of incomplete protection against this important human respiratory virus, provide new information on the entry of HMPV into human cells, and describe a new mechanism of virus immune evasion.

Structural Insights into the Human Metapneumovirus Glycoprotein Ectodomain

“…In addition, because of the nonprotective and cross-protective nature of antibodies directed against G and F, respectively (10–14, 48), transient immunity leading to reinfection could be explained if G can reduce the immunological footprint associated with F. This is consistent with the increased CD4 + T cell activation observed with HMPV lacking the G and SH proteins (22). Interestingly, an avian metapneumovirus isolate bearing a long (585-residue) G protein was found to replicate efficiently without signs of disease in domestic turkeys, suggesting decreased activation of the innate immune response (49–51).…”

Aberrant T cell immunity triggered by human Respiratory Syncytial Virus and human Metapneumovirus infection

“…Consistent with the latter, the SH and/or G proteins reduced CD4 C T cell proliferation in a co-culture assay of hMPV-infected MDDC with CD4 C T cells when compare with T cells co-cultured with DSH/G hMPV-infected DCs. 146 Likewise, in vitro studies show that human and mouse hMPV-infected DCs lose the capacity to activate and expand na€ ıve T cells, although to a lesser degree than hRSV-infected DCs. 26,[146][147][148] Furthermore, the neutralizing antibodies detected in mice after hMPV infection, appear to be dependent on CD4 C T cells, because no detectable neutralizing antibodies were observed in hMPV-infected mice depleted of CD4 C T cells.…”

“…23,25 However, reports performed in humans and mouse dendritic cells (DCs) have described that both viruses infect these cells affecting DCs capacity of promote an adequate immunological memory due to their interference with na€ ıve T cell priming. [26][27][28][29] The ineffectiveness of the natural infection to induce long-term immunity has hampered vaccine generation and currently there is no licensed vaccine available to prevent the bronchiolitis and pneumonia caused either by hRSV or hMPV. [30][31][32][33][34] However, several candidate vaccines are in different stages of development for preventing the diseases caused by these viral agents.…”

DC-SIGN and L-SIGN Are Attachment Factors That Promote Infection of Target Cells by Human Metapneumovirus in the Presence or Absence of Cellular Glycosaminoglycans

“…A previous study implicated DC-SIGN-mediated recognition of HMPV in entry into DCs (26), although direct binding interactions were not investigated. We first examined the ability of recombinant human DC-SIGN or L-SIGN to bind to HMPV by an ELISA.…”

“…In many studies, recognition by cell surface CLR has been associated with enhanced susceptibility to viral infection (reviewed in reference 25). Recently, Le Nouen et al reported that macropinocytosis represented the predominant route for infectious entry of HMPV into monocyte-derived dendritic cells (MDDCs) and presented data implicating DC-SIGN-mediated endocytosis as an alternative pathway for HMPV entry and infection (26).…”