Support Plasmids and Support Proteins Required for Recovery of Recombinant Respiratory Syncytial Virus

112

Human metapneumovirus (hMPV) is a newly discovered pathogen associated with respiratory tract illness, primarily in young children, immunocompromised individuals, and the elderly. The genomic sequence of the prototype hMPV isolate NL/1/00 without the terminal leader and trailer sequences has been reported previously. Here we describe the leader and trailer sequences of two hMPV isolates, NL/1/00 and NL/1/99, representing the two main genetic lineages of hMPV. Minigenome constructs in which the green fluorescent protein or chloramphenicol acetyltransferase genes are flanked by the viral genomic ends derived from both hMPV lineages and transcribed using a T7 RNA polymerase promoter-terminator cassette were generated. Cotransfection of minigenome constructs with plasmids expressing the polymerase complex components L, P, N, and M2.1 in 293T or baby hamster kidney cells resulted in expression of the reporter genes. When the minigenome was replaced by a sense or antisense full-length cDNA copy of the NL/1/00 or NL/1/99 viral genomes, recombinant virus was recovered from transfected cells. Viral titers up to 10 7.2 and 10 5.7 50% tissue culture infective dose/ml were achieved with the sense and antisense plasmids, respectively. The recombinant viruses replicated with kinetics similar to those of the parental viruses in Vero cells. This reverse genetics system provides an important new tool for applied and fundamental research.

Section: Discussionmentioning

confidence: 93%

Recovery of Human Metapneumovirus Genetic Lineages A and B from Cloned cDNA

Herfst

Graaf

Schickli³

et al. 2004

112

“…The HRSV M2-1 protein also has been shown to be an RNA-binding protein and binds to the N protein (9,15). Expression of the HRSV M2-1 protein, either from an added support plasmid or by promiscuous expression from the antigenome cDNA, appears to be essential for the recovery of recombinant HRSV from transfected cDNA, and it has not been possible to recover infectious HRSV in which the integrity of the M2-1 protein has been drastically disturbed by introduced mutations or by extensive deletion (10,12,28,34).…”

mentioning

confidence: 99%

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

Buchholz

Biacchesi

Pham

et al. 2005

Self Cite

128

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...

“…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).…”

mentioning

confidence: 99%

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

Enterlein

Volchkov

Weik

et al. 2006

Here we report recovery of infectious Marburg virus (MARV) from a full-length cDNA clone. Compared to the wild-type virus, recombinant MARV showed no difference in terms of morphology of virus particles, intracellular distribution in infected cells, and growth kinetics. The nucleocapsid protein VP30 of MARV and Ebola virus (EBOV) contains a Zn-binding motif which is important for the function of VP30 as a transcriptional activator in EBOV, whereas its role for MARV is unclear. It has been reported previously that MARV VP30 is able to support transcription in an EBOV-specific minigenome system. When the Zn-binding motif was destroyed, MARV VP30 was shown to be inactive in the EBOV system. While it was not possible to rescue recombinant MARV when the VP30 plasmid was omitted from transfection, MARV VP30 with a destroyed Zn-binding motif and EBOV VP30 were able to mediate virus recovery. In contrast, rescue of recombinant EBOV was not supported by EBOV VP30 containing a mutated Zn-binding domain.