Reduced expression of human metapneumovirus matrix protein impacts virus assembly and ribonucleoprotein localization

Human metapneumovirus (HMPV) causes severe respiratory tract infections in all cohorts, particularly in vulnerable groups such as children, older adults, and the immunocompromised. The matrix (M) protein of HMPV, like the M proteins of other members of the order Mononegavirales, is involved in virus assembly and budding. However, other functions of the HMPV M protein have yet to be elucidated. To investigate the various functions of the M protein, we used a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) antisense agent designed to block translation of the M gene during infection. Treatment with the PPMO targeted against M mRNA (PPMO_M) led to dose-dependent decreases in M expression, and a 10 µM PPMO_M treatment applied at 1 hour post-infection (hpi) led to a >95% reduction in M protein at 24 hpi. Reductions in M expression had a significant impact on virus production and viral titers. Fluorescence in situ hybridization (FISH) probes used to detect vRNA showed that a reduction in M led to an increase in inclusion body (IB) count per cell, but a decrease in the average IB volume per cell at 12 and 24 hpi. HMPV-infected cells treated with PPMO_M exhibited lower levels of phosphoprotein (P) at the plasma membrane. Importantly, no change in viral ribonucleoprotein movement (vRNP) was observed when the M protein levels were reduced. These studies support a model where the M protein does not facilitate movement, but instead is critical for the transfer to and stable association of vRNPs with the assembly sites at the membrane.IMPORTANCEHuman metapneumovirus (HMPV) is a respiratory pathogen that can cause severe infections in humans, but there are no targeted treatment options for HMPV. To better characterize HMPV, a matrix (M) protein-targeted peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO_M) was designed to control M expression during infection. Inhibition of M expression resulted in infections with reduced viral filaments and less viral RNA at the cell periphery. A reduction in M protein expression did not disrupt the active transport of viral ribonucleoproteins (vRNPs) but did significantly decrease vRNP localization at membranes and increase the number of inclusion bodies within infected cells. Viral titers were significantly reduced when M protein expression was inhibited, and dose-dependent reductions in M protein directly correlated with viral particle production. This study uses a unique tool to characterize the HMPV M protein during infection.