The Role of the Flavivirus Replicase in Viral Diversity and Adaptation (original) (raw)
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Journal of General Virology, 2007
West Nile virus (WNV) has successfully spread throughout the USA, Canada, Mexico, the Caribbean and parts of Central and South America since its 1999 introduction into North America. Despite infecting a broad range of both mosquito and avian species, the virus remains highly genetically conserved. This lack of evolutionary change over space and time is common with many arboviruses and is frequently attributed to the adaptive constraints resulting from the virus cycling between vertebrate hosts and invertebrate vectors. WNV, like most RNA viruses studied thus far, has been shown in nature to exist as a highly genetically diverse population of genotypes. Few studies have directly evaluated the role of these mutant spectra in viral fitness and adaptation. Using clonal analysis and reverse genetics experiments, this study evaluated genotype diversity and the importance of consensus change in producing the adaptive phenotype of WNV following sequential mosquito cell passage. The results indicated that increases in the replicative ability of WNV in mosquito cells correlate with increases in the size of the mutant spectrum, and that consensus change is not solely responsible for alterations in viral fitness and adaptation of WNV. These data provide evidence of the importance of quasispecies dynamics in the adaptation of a flavivirus to new and changing environments and hosts, with little evidence of significant genetic change.
RNA Structure Duplications and Flavivirus Host Adaptation
Trends in Microbiology, 2016
Flaviviruses include a highly diverse group of arboviruses with a global distribution and a high human disease burden. Most flaviviruses cycle between insects and vertebrate hosts; thus, they are obligated to use different cellular machineries for their replication and mount different mechanisms to evade specific antiviral responses. In addition to coding for viral proteins, the viral genome contains signals in RNA structures that govern the amplification of viral components and participate in triggering or evading antiviral responses. In this review, we focused on new information about host-specific functions of RNA structures present in the 3' untranslated region (3' UTR) of flavivirus genomes. Models and conservation patterns of RNA elements of distinct flavivirus ecological groups are revised. An intriguing feature of the 3' UTR of insect-borne flavivirus genomes is the conservation of complex RNA structure duplications. Here, we discuss new hypotheses of how these RNA elements specialize for replication in vertebrate and invertebrate hosts, and present new ideas associating the significance of RNA structure duplication, small subgenomic flavivirus RNA formation and host adaptation.
Virus Evolution, 2021
Flaviviruses display diverse epidemiological and ecological features. Tick-borne and mosquito-borne flaviviruses (TBFV and MBFV, respectively) are important human pathogens that alternate replication in invertebrate vectors and vertebrate hosts. The Flavivirus genus also includes insect-specific viruses (ISFVs) and viruses with unknown invertebrate hosts. It is generally accepted that viruses that alternate between taxonomically different hosts evolve slowly and that the evolution of MBFVs and TBFVs is dominated by strong constraints, with limited episodes of positive selection. We exploited the availability of flavivirus genomes to test these hypotheses and to compare their rates and patterns of evolution. We estimated the substitution rates of CFAV and CxFV (two ISFVs) and, by taking into account the time-frame of measurement, compared them with those of other flaviviruses. Results indicated that CFAV and CxFV display relatively different substitution rates. However, these data, t...
NS4/5 mutations enhance flavivirus Bamaga virus infectivity and pathogenicity in vitro and in vivo
PLOS Neglected Tropical Diseases
Flaviviruses such as yellow fever, dengue or Zika viruses are responsible for significant human and veterinary diseases worldwide. These viruses contain an RNA genome, prone to mutations, which enhances their potential to emerge as pathogens. Bamaga virus (BgV) is a mosquito-borne flavivirus in the yellow fever virus group that we have previously shown to be host-restricted in vertebrates and horizontally transmissible by Culex mosquitoes. Here, we aimed to characterise BgV host-restriction and to investigate the mechanisms involved. We showed that BgV could not replicate in a wide range of vertebrate cell lines and animal species. We determined that the mechanisms involved in BgV host-restriction were independent of the type-1 interferon response and RNAse L activity. Using a BgV infectious clone and two chimeric viruses generated as hybrids between BgV and West Nile virus, we demonstrated that BgV host-restriction occurred post-cell entry. Notably, BgV host-restriction was shown to be temperature-dependent, as BgV replicated in all vertebrate cell lines at 34˚C but only in a subset at 37˚C. Serial passaging of BgV in Vero cells resulted in adaptive mutants capable of efficient replication at 37˚C. The identified mutations resulted in amino acid substitutions in NS4A-S124F, NS4B-N244K and NS5-G2C, all occurring close to a viral protease cleavage site (NS4A/2K and NS4B/NS5). These mutations were reverse engineered into infectious clones of BgV, which revealed that NS4B-N244K and NS5-G2C were sufficient to restore BgV replication in vertebrate cells at 37˚C, while NS4A-S124F further increased replication efficiency. When these mutant viruses were injected into immunocompetent mice, alongside BgV and West Nile virus chimeras, infection and neurovirulence were enhanced as determined by clinical scores, seroconversion, microneutralisation, viremia, histopathology and immunohistochemistry, confirming the involvement of these residues in the attenuation of BgV. Our studies identify a new mechanism of host-restriction and attenuation of a mosquito-borne flavivirus.
The role of viral persistence in flavivirus biology
Pathogens and Disease, 2014
In nature, vector-borne flaviviruses are persistently cycled between either the tick or mosquito vector and small mammals such as rodents, skunks, and swine. These viruses account for considerable human morbidity and mortality worldwide. Increasing and substantial evidence of viral persistence in humans, which includes the isolation of RNA by RT-PCR and infectious virus by culture, continues to be reported. Viral persistence can also be established in vitro in various human, animal, arachnid, and insect cell lines in culture. Although some research has focused on the potential roles of defective virus particles, evasion of the immune response through the manipulation of autophagy and/or apoptosis, the precise mechanism of flavivirus persistence is still not well understood. We propose additional research for further understanding of how viral persistence is established in different systems. Avenues for additional studies include determining whether the multifunctional flavivirus protein NS5 has a role in viral persistence, the development of relevant animal models of viral persistence, and investigating the host responses that allow vector-borne flavivirus replication without detrimental effects on infected cells. Such studies might shed more light on the viral-host relationships and could be used to unravel the mechanisms for establishment of persistence.
Genome biology and evolution, 2013
Protein structure is commonly regarded to be conserved and to dictate function. Most proteins rely on conformational flexibility to some degree. Are regions that convey conformational flexibility conserved over evolutionary time? Can changes in conformational flexibility alter protein function? Here, the evolutionary dynamics of structurally ordered and disordered (flexible) regions are investigated genome-wide in flaviviruses, revealing that the amount and location of structural disorder fluctuates highly among related proteins. Some regions are prone to shift between structured and flexible states. Increased evolutionary dynamics of structural disorder is observed for some lineages but not in others. Lineage-specific transitions of this kind could alter the conformational ensemble accessible to the same protein in different species, causing a functional change, even if the predominant function remains conserved. Thus, rapid evolutionary dynamics of structural disorder is a potenti...
PloS one, 2014
RNA secondary structures in the 3'untranslated regions (3'UTR) of the viruses of the family Flaviviridae, previously identified as essential (promoters) or beneficial (enhancers) for replication, have been analysed. Duplicated enhancer elements are revealed as a global feature in the evolution of the 3'UTR of distantly related viruses within the genera Flavivirus and Pestivirus. For the flaviviruses, duplicated structures occur in the 3'UTR of all four distantly related ecological virus subgroups (tick-borne, mosquito-borne, no known vector and insect-specific flaviviruses (ISFV). RNA structural differences distinguish tick-borne flaviviruses with discrete pathogenetic characteristics. For Aedes- and Culex-associated ISFV, secondary RNA structures with different conformations display numerous short ssRNA direct repeats, exposed as loops and bulges. Long quadruplicate regions comprise almost the entire 3'UTR of Culex-associated ISFV. Extended duplicated sequence a...
Flavivirus NS1: a multifaceted enigmatic viral protein
Virology journal, 2016
Flaviviruses are emerging arthropod-borne viruses representing an immense global health problem. The prominent viruses of this group include dengue virus, yellow fever virus, Japanese encephalitis virus, West Nile virus tick borne encephalitis virus and Zika Virus. These are endemic in many parts of the world. They are responsible for the illness ranging from mild flu like symptoms to severe hemorrhagic, neurologic and cognitive manifestations leading to death. NS1 is a highly conserved non-structural protein among flaviviruses, which exist in diverse forms. The intracellular dimer form of NS1 plays role in genome replication, whereas, the secreted hexamer plays role in immune evasion. The secreted NS1 has been identified as a potential diagnostic marker for early detection of the infections caused by flaviviruses. In addition to the diagnostic marker, the importance of NS1 has been reported in the development of therapeutics. NS1 based subunit vaccines are at various stages of deve...
Host influence in the genomic composition of flaviviruses: A multivariate approach
Flaviviruses present substantial differences in their host range and transmissibility. We studied the evolution of base composition, dinucleotide biases, codon usage and amino acid frequencies in the genus Flavivirus within a phylogenetic framework by principal components analysis. There is a mutual interplay between the evolutionary history of flaviviruses and their respective vectors and/or hosts. Hosts associated to distinct phylogenetic groups may be driving flaviviruses at different pace and through various sequence landscapes, as can be seen for viruses associated with Aedes or Culex spp., although phyloge-netic inertia cannot be ruled out. In some cases, viruses face even opposite forces. For instance, in tick-borne flaviviruses, while vertebrate hosts exert pressure to deplete their CpG, tick vectors drive them to exhibit GC-rich codons. Within a vertebrate environment, natural selection appears to be acting on the viral genome to overcome the immune system. On the other side, within an arthropod environment, mutational biases seem to be the dominant forces.
Adaptor protein complexes-1 and 3 are involved at distinct stages of flavivirus life-cycle
Scientific Reports, 2013
Intracellular protein trafficking pathways are hijacked by viruses at various stages of viral life-cycle. Heterotetrameric adaptor protein complexes (APs) mediate vesicular trafficking at distinct intracellular sites and are essential for maintaining the organellar homeostasis. In the present study, we studied the effect of AP-1 and AP-3 deficiency on flavivirus infection in cells functionally lacking these proteins. We show that AP-1 and AP-3 participate in flavivirus life-cycle at distinct stages. AP-3-deficient cells showed delay in initiation of Japanese encephalitis virus and dengue virus RNA replication, which resulted in reduction of infectious virus production. AP-3 was found to colocalize with RNA replication compartments in infected wild-type cells. AP-1 deficiency affected later stages of dengue virus infection where increased intracellular accumulation of infectious virus was observed. Therefore, our results propose a novel role for AP-1 and AP-3 at distinct stages of infection of some of the RNA viruses. M embers of the genus Flavivirus within the family Flaviviridae comprise of several medically important pathogens including the Japanese encephalitis virus (JEV), dengue virus (DENV), yellow fever virus (YFV) and West Nile virus (WNV) which cause significant morbidity and mortality in humans, animals and birds 1. Flaviviruses are enveloped viruses with a positive-sense, single-strand RNA genome of approximately 11 kb that is translated as a single polyprotein precursor of ,3300 amino acids in length and proteolytically cleaved into 10 viral proteins: three structural (capsid, pre-membrane/membrane (prM-M), and envelope) and seven non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins 1. Flaviviruses enter their host cells through a process of receptor-mediated endocytosis followed by subsequent fusion within the endosomal compartment to release the viral genome into the cytoplasm for translation and replication 2. Replication of the viral RNA genome occurs on virus-induced host cell membranes. Such structures may serve as a scaffold for anchoring the viral replication complexes, which consist of viral RNA, viral proteins, and host cell factors. Virus assembly occurs within the endoplasmic reticulum-derived membrane compartments and non-infectious virions traverse through the Golgi stack to reach the trans-Golgi network (TGN) where furin-mediated cleavage between prM-M leads to conformational changes rendering the virion infectious. Infectious virus is subsequently released from cells via the secretory pathway 1,3. Adaptor complexes (AP-1 through 5) are heterotetrameric protein complexes comprising one each of the two large sub-units c, a, d, e and f and b1-5 and one medium sub-unit m1-5 and one small sub-unit s1-5 respectively. APs are involved in distinct intracellular vesicular transport pathways which play a vital role in maintaining cellular homeostasis 4-6. AP-1 is involved in the trafficking of cargo molecules in the biosynthetic pathway from the trans-Golgi network (TGN) to endosomes and back. AP-2 which is one of the most extensively-studied adaptor complex has been shown to be involved in the endocytic pathway at the plasma membrane. AP-3 is reported to function in the transport of selected proteins in the endo-lysosomal pathway. AP-4 is involved in sorting of proteins destined to basolateral surface in polarized cells. AP-5 is the newest member of the family discovered recently and has been proposed to function at the late endosomes 6. Despite the prominent role played by APs in intracellular trafficking pathways, their involvement in flavivirus life-cycle has not been characterized. A number of earlier studies investigating the internalization of flaviviruses have shown the involvement of clathrin-dependent and lipid raft-dependent pathways for virus entry but the role of APs in stages post-entry has not been investigated 2. A genome-wide RNA interference screen for identifying cellular proteins associated with WNV infection identified AP-1 m1 subunit and AP-3 s2 subunit as some of the host factors required for both WNV and DENV infection 7. Similarly, another study identified AP1M1 as one of the genes required for