Advances in Tetravirus Research: New Insight Into the Infectious Virus Lifecycle and an Expanding Host Range.

Tetraviruses are a group of relatively unknown small RNA viruses with particles that display a characteristic T=4 capsid architecture. Tetraviruses are classified into three families, the Alphatetraviridae, Permutotetraviridae and Carmotetraviridae, according to the divergent characteristics of their respective viral replicases. Tetraviruses generally infect the larvae of lepidopteran insect species, many of which are important agricultural pests and, until recently, were thought to have an unusually narrow host range and tissue tropism. The development of experimental systems for studying the viral infectious life cycle in tissue culture has permitted the extension of the virus host range to mammalian cells and plants. This chapter will review recent advances in the understanding of the biology of tetraviruses, highlighting new information on the expression and functional characterisation of viral proteins and the development of biological systems for elucidating the molecular mechanisms of infection, viral replication and host range.

Providence virus: An animal virus that replicates in plants or a plant virus that infects and replicates in animal cells?

INTRODUCTION: Emerging viral diseases, most of which are zoonotic, pose a significant threat to global health. There is a critical need to identify potential new viral pathogens and the challenge is to identify the reservoirs from which these viruses might emerge. Deep sequencing of invertebrate transcriptomes has revealed a plethora of viruses, many of which represent novel lineages representing both plant and animal viruses and little is known about the potential threat that these viruses pose. METHODS: Providence virus, an insect virus, was used to establish a productive infection in Vigna unguiculata (cowpea) plants. Providence virus particles purified from these cowpea plants were used to infect two mammalian cell lines. FINDINGS: Here, we present evidence that Providence virus, a non-enveloped insect RNA virus, isolated from a lepidopteran midgut cell line can establish a productive infection in plants as well as in animal cells. The observation that Providence virus can readily infect both plants and mammalian cell culture lines demonstrates the ability of an insect RNA virus to establish productive infections across two kingdoms, in plants and invertebrate and vertebrate animal cell lines. CONCLUSIONS: The study highlights the potential of phytophagous insects as reservoirs for viral re-assortment and that plants should be considered as reservoirs for emerging viruses that may be potentially pathogenic to humans.

Expanding the host range of small insect RNA viruses: Providence virus (Carmotetraviridae) infects and replicates in a human tissue culture cell line.

Tetraviruses are small, positive (+ve)-sense ssRNA viruses that infect the midgut cells of lepidopteran larvae. Providence virus (PrV) is the only member of the family Carmotetraviridae (previously Tetraviridae). PrV particles exhibit the characteristic tetraviral T=4 icosahedral symmetry, but PrV is distinct from other tetraviruses with respect to genome organization and viral non-structural proteins. Currently, PrV is the only tetravirus known to infect and replicate in lepidopteran cell culture lines. In this report we demonstrate, using immunofluorescence microscopy, that PrV infects and replicates in a human tissue culture cell line (HeLa), producing infectious virus particles. We also provide evidence for PrV replication in vitro in insect, mammalian and plant cell-free systems. This study challenges the long-held view that tetraviruses have a narrow host range confined to one or a few lepidopteran species and highlights the need to consider the potential for apparently non-infectious viruses to be transferred to new hosts in the laboratory.

Complex pathways for regulation of pyrimidine metabolism by carbon catabolite repression and quorum sensing in Pseudomonas putida RU-KM3S.

Pseudomonads are metabolically versatile microbes that employ complex regulatory networks to control gene expression, particularly with respect to carbon and nitrogen metabolism. The aim of this study was to characterise the regulatory networks that control pyrimidine metabolism (hydantoin-hydrolysing activity) in Pseudomonas putida strain RU-KM3S, focussing on transcriptional activation of dihydropyrimidinase (Dhp) and ß-ureidopropionase (Bup), encoding dhp and bup, respectively. The two genes are arranged divergently on the chromosome and are separated by ORF1, encoding a putative transporter, which lies upstream of and in the same orientation as bup. The results from this study reveal that pyrimidine metabolism, as a function of Bup and Dhp activity in P. putida RU-KM3S, is controlled by a complex regulatory network including several global pathways in addition to induction by the substrate. Three major control pathways act at the level of transcriptional and include: (1) induction of transcriptional activation in the presence of hydantoin, (2) carbon catabolite repression mediated via a pathway independent of Crc and (3) quorum sensing that does not require a putative lux box located upstream of the dhp transcriptional start. Finally, the data suggest a minor role for the global regulators Anr, Vfr and Crc, likely through regulation of the activity of transcription factors interacting directly with the bup/ORF1-dhp promoter.

Subcellular localization and live-cell imaging of the Helicoverpa armigera stunt virus replicase in mammalian and Spodoptera frugiperda cells.

Whilst their structure has been well studied, there is little information on the replication biology of tetraviruses because of the lack of suitable tissue-culture cell lines that support virus replication. In this study, the potential site of Helicoverpa armigera stunt virus replication was investigated by transient expression of the replicase protein fused to enhanced green fluorescent protein (EGFP) in mammalian and insect cells. When EGFP was present at the C terminus of the protein, fluorescence was located in punctate cytoplasmic structures that were distinct from the peripheral Golgi, endoplasmic reticulum, early endosomes, lysosomes and mitochondria, but overlapped partially with late endosomes. In experiments where targeting to endosomal compartments was examined further by using Cascade Blue-dextran in live cells, no overlap between the replicase and active endocytic organelles was apparent. Analysis of the punctate structures using time-lapse imaging in live cells revealed that they undergo fusion, fission and 'kiss-and-run' events. Whilst the source of the membranes used to form the punctate structures remains unclear, we propose that the replicase sequesters membranes from the late endosomes and actively excludes host proteins, either by normal recycling processes or by a replicase-dependent mechanism that may result in the destabilization of the associated membranes and a release of luminal contents into the cytosol. This is the first study describing the localization of a tetravirus.

Regulation of hydantoin-hydrolyzing enzyme expression in Agrobacterium tumefaciens strain RU-AE01.

Optically pure D-: amino acids, like D-: hydroxyphenylglycine, are used in the semi-synthetic production of pharmaceuticals. They are synthesized industrially via the biocatalytic hydrolysis of p-hydroxyphenylhydantoin using enzymes derived from Agrobacterium tumefaciens strains. The reaction proceeds via a three-step pathway: (a) the ring-opening cleavage of the hydantoin ring by a D-: hydantoinase (encoded by hyuH), (b) conversion of the resultant D-: N-carbamylamino acid to the corresponding amino acid by a D-: N-carbamoylase (encoded by hyuC), and (c) chemical or enzymatic racemization of the un-reacted hydantoin substrate. While the structure and biochemical properties of these enzymes are well understood, little is known about their origin, their function, and their regulation in the native host. We investigated the mechanisms involved in the regulation of expression of the hydantoinase and N-carbamoylase enzyme activity in A. tumefaciens strain RU-AE01. We present evidence for a complex regulatory network that responds to the growth status of the cells, the presence of inducer, and nitrogen catabolite repression. Deletion analysis and site-directed mutagenesis were used to identify regulatory elements involved in transcriptional regulation of hyuH and hyuC expression. Finally, a comparison between the hyu gene clusters in several Agrobacterium strains provides insight into the function of D-: selective hydantoin-hydrolyzing enzyme systems in Agrobacterium species.

Induction of apoptosis in Saccharomyces cerevisiae results in the spontaneous maturation of tetravirus procapsids in vivo.

The Tetraviridae are a family of small, non-enveloped, insect RNA viruses consisting of one or two single-stranded, positive-sense genomic RNAs encapsidated in an icosahedral capsid with T=4 symmetry. Tetravirus procapsids undergo maturation when exposed to a low pH environment in vitro. While the structural biology of the conformational changes that mediate acid-dependent maturation is well understood, little is known about the significance of acid-dependent maturation in vivo. To address this question, the capsid-coding sequence of the tetravirus Helicoverpa armigera stunt virus was expressed in Saccharomyces cerevisiae cells. Virus-like particles were shown to assemble as procapsids that matured spontaneously in vivo as the cells began to age. Growth in the presence of hydrogen peroxide or acetic acid, which induced apoptosis or programmed cell death in the yeast cells, resulted in virus-like particle maturation. The results demonstrate that assembly-dependent maturation of tetravirus procapsids in vivo is linked to the onset of apoptosis in yeast cells. We propose that the reduction in pH required for tetraviral maturation may be the result of cytosolic acidification, which is associated with the early onset of programmed cell death in infected cells.