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Journal of Virology, June 2005, p. 6588-6597, Vol. 79, No. 11
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.11.6588-6597.2005

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

Ursula J. Buchholz,* Stéphane Biacchesi, Quynh N. Pham, Kim C. Tran, Lijuan Yang, Cindy L. Luongo, Mario H. Skiadopoulos, Brian R. Murphy, and Peter L. Collins

Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892-8007

Received 24 September 2004/ Accepted 2 February 2005

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{Delta}M2-1, r{Delta}M2-2, and r{Delta}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{Delta}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{Delta}M2-1 or r{Delta}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{Delta}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{Delta}M2-2 virus is a promising and highly attenuated HMPV vaccine candidate.

* Corresponding author: National Institutes of Health, NIAID, LID, Building 50, Room 6505, 50 South Dr., MSC 8007, Bethesda, MD 20892-8007. Phone: (301) 594-1533. Fax: (301) 496-8312. E-mail: ubuchholz{at}niaid.nih.gov.

Journal of Virology, June 2005, p. 6588-6597, Vol. 79, No. 11
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.11.6588-6597.2005




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