Determination of the henipavirus phosphoprotein gene mRNA editing frequencies and detection of the C, V and W proteins of Nipah virus in virus-infected cells

Abstract

The henipaviruses, Nipah virus (NiV) and Hendra virus (HeV), are highly pathogenic zoonotic paramyxoviruses. Like many other paramyxoviruses, henipaviruses employ a process of cotranscriptional mRNA editing during transcription of the phosphoprotein (P) gene to generate additional mRNAs encoding the V and W proteins. The C protein is translated from the P mRNA, but in an alternate reading frame. Sequence analysis of multiple, cloned mRNAs showed that the mRNA editing frequencies of the P genes of the henipaviruses are higher than those reported for other paramyxoviruses. Antisera to synthetic peptides from the P, V, W and C proteins of NiV were generated to study their expression in infected cells. All proteins were detected in both infected cells and purified virions. In infected cells, the W protein was detected in the nucleus while P, V and C were found in the cytoplasm.Nipah virus (NiV) and Hendra virus (HeV) are paramyxoviruses in the genus Henipavirus, the subfamily Paramyxovirinae, within the family Paramyxoviridae. HeV causes febrile respiratory illness in humans and animals; of all the febrile illnesses caused by HeV in Australia from 1994 to 2007, there were 17 equine deaths and two human fatalities out of three cases (Hanna et al., 2006;Hooper & Williamson, 2000; Murray et al., 1995a, b). The first human NiV infections were detected during an outbreak of severe febrile encephalitis in peninsular Malaysia and Singapore from autumn 1998 to spring 1999 (Chua et al., 2000). NiV has subsequently been established as the cause of fatal human encephalitis in Bangladesh in 2001, 2003, 2007(Banerjee, 2007Hsu et al., 2004) as well as in India in (Chadha et al., 2006.Fruit-eating bats of the genus Pteropus are a natural reservoir for NiV and HeV (Chua et al., 2002;Halpin et al., 2000;Yob et al., 2001). Humans are infected by exposure to infected fruit bats or material contaminated by infected bats (Hsu et al., 2004), but are also infected via intermediate hosts such as pigs (Amal et al., 2000;Chew et al., 2000;Paton et al., 1999) or by direct human-tohuman contact (Gurley et al., 2007). No vaccines or specific antiviral drugs are currently available for NiV. Although patients from the Malaysian outbreak who received ribavirin showed a lower mortality rate (Chong et al., 2001), ribavirin was unable to protect NiV-infected hamsters from fatal disease (Georges-Courbot et al., 2006).The genomes of NiV and HeV are 18 246 and 18 234 nt, respectively, making them significantly larger than most other paramyxoviruses, with the exceptions of rodentborne Beilong and J viruses (Jack et al., 2005;Li et al., 2006). The N, P and L proteins are required and sufficient for mini-genome replication, similar to other members of the subfamily Paramyxovirinae (Halpin et al., 2004). The P genes of the subfamily Paramyxovirinae, which are 2 704 nt for NiV and 2 698 nt for HeV ( Supplementary Fig. S1, available in JGV Online), contain several open reading frames (ORFs) in addition to the P ORF. Like most other paramyxovirinae, henipavir...

Nipah Virus C and W Proteins Contribute to Respiratory Disease in Ferrets

“…The C protein utilizes alternative start codons shortly after the P start codon; the C open reading frame (ORF) is out of frame with P, and therefore, the C protein shares no homology with P (26). The V and W proteins are produced through mRNA editing by the viral polymerase (27,28), resulting in P, W, and V proteins that share an N-terminal domain but contain unique C-terminal domains.…”

Kinetic analysis of RNA editing of Newcastle disease virus P gene in the early period of infection

“…Th e only diff erence among the P gene-derived transcripts was the number of G residues inserted. Th erefore, the typical nucleic acid detection techniques, such as PCR and real-time PCR, cannot be used for the quantitation, whereas the genetic sequencing is the only way to diff erentiate these transcripts, which has limited the research on RNA editing (Galinski et al, 1992;Ghosh et al, 1996;Hausmann et al, 1996;Kolakofsky et al, 2005;Kulkarni et al, 2009;Lo et al, 2009Lo et al, , 2012. Up to date, little was known about NDV regulation of RNA-editing frequency.…”

Novel Nipah Virus Immune-Antagonism Strategy Revealed by Experimental and Computational Study

“…The goal of the modeling was to use the available data and the results of the present study to re-create the profile of responses that occur during infection by a wild-type virus and thereby to elucidate the overall immuneantagonist strategy used by NiV. Therefore, in order to gain insight into the immune response during actual NiV infection, we proceeded to simulate the effects of all three immune antagonists expressed in a temporal pattern derived from published experiments with wild-type virus (32,34). The experimental data on viral antagonist expression was obtained with To account for the additional time necessary for protein translation, simulations for wild-type NiV were delayed by 1 h. Relative levels of expression of NiV P, V, and W over time are shown in Fig.…”

“…Research on NiV is difficult due to the highly pathogenic properties that have limited studies on the full virus to biosafety level 4 facilities (32,34,40,47). The paramyxovirus NDV has been established as a useful vector with which to study the effects of immune antagonists from human viruses (20,49).…”