Programmed Ribosomal Frameshift Alters Expression of West Nile Virus Genes and Facilitates Virus Replication in Birds and Mosquitoes

Abstract

West Nile virus (WNV) is a human pathogen of significant medical importance with close to 40,000 cases of encephalitis and more than 1,600 deaths reported in the US alone since its first emergence in New York in 1999. Previous studies identified a motif in the beginning of non-structural gene NS2A of encephalitic flaviviruses including WNV which induces programmed −1 ribosomal frameshift (PRF) resulting in production of an additional NS protein NS1′. We have previously demonstrated that mutant WNV with abolished PRF was attenuated in mice. Here we have extended our previous observations by showing that PRF does not appear to have a significant role in virus replication, virion formation, and viral spread in several cell lines in vitro. However, we have also shown that PRF induces an over production of structural proteins over non-structural proteins in virus-infected cells and that mutation abolishing PRF is present in ∼11% of the wild type virus population. In vivo experiments in house sparrows using wild type and PRF mutant of New York 99 strain of WNV viruses showed some attenuation for the PRF mutant virus. Moreover, PRF mutant of Kunjin strain of WNV showed significant decrease compared to wild type virus infection in dissemination of the virus from the midgut through the haemocoel, and ultimately the capacity of infected mosquitoes to transmit virus. Thus our results demonstrate an important role for PRF in regulating expression of viral genes and consequently virus replication in avian and mosquito hosts.

Last 20 aa of the West Nile virus NS1′ protein are responsible for its retention in cells and the formation of unique heat-stable dimers

“…This is in contrast to virus grown in mammalian or insect culture, which has shown that viral replication in vitro is not different between viruses lacking PRF/NS19 and those encoding PRF and producing NS19 (Melian et al, 2010;Ye et al, 2012). Our other recent study also found no difference in replication in avian DF-1 cells between WT and PRF-deficient mutant (A30A9) WNV KUN viruses (Melian et al, 2014). Combined together, these results indicate that the role of PRF/NS19 in viral replication may be virus species and perhaps host species specific.…”

“…We have also shown that a viral mutant (Stop Mutant) producing a truncated form of NS19, but not deficient in ribosomal frameshift, replicates with the same efficiency in cells of different origin and has a relatively similar virulence in mice to the WT WNV KUN . In combination with previous work (Melian et al, 2010(Melian et al, , 2014, this suggests that the PRF itself (and not the NS19 protein) is important for viral pathogenesis in the mammalian system. Further work in the mosquito and avian hosts using Stop Mutant producing truncated NS19 will determine whether fulllength NS19 is indeed required for viral replication/ transmission in this vector/host system.…”

“…It seems highly unlikely that such a conserved mechanism as 21 PRF producing a stable NS19 protein has evolved in a distinct group of viruses without a significant impact on viral growth/transmission properties in at least one of the vector or host systems. Relatively modest attenuation in mice of PRF-and NS19-deficient WNV KUN virus (A30A9) (Melian et al, 2014) and our data showing insignificant attenuation of Stop Mutant WNV KUN in mice suggest that mammalian hosts are unlikely to be the primary driver for the evolution of this mechanism. As mammals are only incidental hosts for these viruses, and these viruses predominantly cycle through the Culex mosquito vector and avian hosts, it is more likely that this is where the PRF mechanism evolved initially, and where it is likely to have a significant impact on virus replication and/or transmission.…”

“…As mammals are only incidental hosts for these viruses, and these viruses predominantly cycle through the Culex mosquito vector and avian hosts, it is more likely that this is where the PRF mechanism evolved initially, and where it is likely to have a significant impact on virus replication and/or transmission. Recent work with WNV KUN and JEV viruses conducted in the mosquito and avian systems supports this hypothesis, with a difference in pathogenicity in birds, virus growth in avian cell culture and transmission in Culex mosquitoes observed between viruses encoding PRF and expressing NS19 and those lacking PRF and NS19 (Melian et al, 2014;Takamatsu et al, 2014).…”

“…Due to the intricate relationship between the ribosomal frameshift and the production of NS19, it is difficult to determine whether attenuation of NS19-lacking viruses is a result of the loss of the NS19 protein or the frameshift itself (Melian et al, 2010;Ye et al, 2012). It is possible that the frameshift mechanism evolved to primarily control the ratio of structural to non-structural proteins (Melian et al, 2014) and its byproduct NS19 is generated to increase the relative level of functioning NS1. The work presented here showed that truncation of NS19 did not detrimentally affect virus growth in mammalian and insect cell culture or WNV KUN pathogenicity in mice after peripheral inoculation (Fig.…”