Deletion of M2 Gene Open Reading Frames 1 and 2 of Human Metapneumovirus: Effects on RNA Synthesis, Attenuation, and Immunogenicity

Abstract

The M2 gene of human metapneumovirus (HMPV) contains two overlapping open reading frames (ORFs), M2-1 and M2-2. The expression of separate M2-1 and M2-2 proteins from these ORFs was confirmed, and recombinant HMPVs were recovered in which expression of M2-1 and M2-2 was ablated individually or together [r⌬M2-1, r⌬M2-2, and r⌬M2(1؉2)]. Each M2 mutant virus directed efficient multicycle growth in Vero cells. The ability to recover HMPV lacking M2-1 contrasts with human respiratory syncytial virus, for which M2-1 is an essential transcription factor. Expression of the downstream HMPV M2-2 ORF was not reduced when translation of the upstream M2-1 ORF was silenced, indicating that it is initiated separately. The r⌬M2-2 mutants exhibited a two-to fivefold increase in the accumulation of mRNA, normalized to the genome template, suggesting that M2-2 has a role in regulating RNA synthesis. Replication and immunogenicity were tested in hamsters. Animals infected intranasally with r⌬M2-1 or r⌬M2(1؉2) did not have recoverable virus in the lungs or nasal turbinates on days 3 or 5 postinfection and did not develop HMPV-neutralizing serum antibodies or resistance to HMPV challenge. Thus, M2-1 appears to be essential for significant virus replication in vivo. In animals infected with r⌬M2-2, virus was recovered from only 1 of 12 animals and only in the nasal turbinates on a single day. However, all of the animals developed a high titer of HMPV-neutralizing serum antibodies and were highly protected against challenge with wild-type HMPV. The HMPV r⌬M2-2 virus is a promising and highly attenuated HMPV vaccine candidate.Human metapneumovirus (HMPV) was first identified in 2001 in The Netherlands from infants and children with acute respiratory tract disease (38) and is now recognized to be worldwide in prevalence (22,41). HMPV resembles human respiratory syncytial virus (HRSV) with regard to disease signs and the ability to infect and cause disease in infants as well as in individuals of all ages (7,18,20,29,32,39,41). The contribution of HMPV to respiratory tract disease remains to be fully defined but appears to be sufficient to warrant the development of a vaccine, especially for the pediatric population. Reverse genetic systems were recently developed for HMPV, allowing the generation of infectious virus from cDNA and providing an important tool for characterizing HMPV biology and for designing live-attenuated HMPV vaccines (5, 25).HMPV has a negative-strand RNA genome of approximately 13 kb (4, 37). It has been classified, together with avian metapneumovirus, in the Metapneumovirus genus, Pneumovirus subfamily, Paramyxovirus family, of the order Mononegavirales. The Pneumovirus subfamily also contains the genus Pneumovirus, represented by HRSV. The Metapneumovirus gene order is N-P-M-F-M2-SH-G-L. By analogy to HRSV, the predicted HMPV proteins are the following: the nucleocapsid protein N, which encapsidates the RNA genome and, together with the phosphoprotein P and the RNA polymerase protein L, forms the ribonucleoprotein co...

Crystal structure of the essential transcription antiterminator M2-1 protein of human respiratory syncytial virus and implications of its phosphorylation

“…It seems unlikely that proteins sharing structural similarity would perform different functions in the relatively conserved mononegavirales RNA synthesis strategy. However, M2-1 already demonstrates precedent for this as M2-1 from human metapneumovirus is nonessential for mRNA transcription (22). Perhaps differences in M2-1 and VP30 function result from the binding partners that they mediate their function through.…”

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

“…160 This is consistent with studies that demonstrate that hMPV lacking the M2-1 gene could not replicate in hamsters. 161 However, is not essential for hMPV infectivity and growth in vitro as deletion of the ORF of the M2-1 protein is dispensable for viral replication in VERO cells. 161 In contrast, the hMPV M2-2 protein, defined as a terminator factor, since it inhibits viral replication and transcription, 162 interact with MAVS, 163 a protein that links the cytoplasmic viral sensors RIG-I and the melanoma differentiation associated protein 5 (MDA5) to the downstream TNF receptor-associated factors (TRAFs) and IkB kinases (IKKs), which are known to induce the type I IFN-pathway through the activation of IRF-3 and NFkB.…”

Rescue of Recombinant Marburg Virus from cDNA Is Dependent on Nucleocapsid Protein VP30

“…Interestingly, the necessity of the fourth nucleocapsid protein M2-1 for virus rescue is different among the pneumoviruses. While human respiratory syncytial virus M2-1 was found to be necessary for production of recombinant virus, recombinant human metapneumovirus lacking the M2-1 open reading frame was successfully recovered in cell culture (3,5). Concerning MARV, VP30 was not needed for replication and transcription of MARV minigenomes (17).…”