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Low temperature-dependent salmonid alphavirus glycoprotein processing and recombinant virus-like particle formation.

ABSTRACT: Pancreas disease (PD) and sleeping disease (SD) are important viral scourges in aquaculture of Atlantic salmon and rainbow trout. The etiological agent of PD and SD is salmonid alphavirus (SAV), an unusual member of the Togaviridae (genus Alphavirus). SAV replicates at lower temperatures in fish. Outbreaks of SAV are associated with large economic losses of ~17 to 50 million $/year. Current control strategies rely on vaccination with inactivated virus formulations that are cumbersome to obtain and have intrinsic safety risks. In this research we were able to obtain non-infectious virus-like particles (VLPs) of SAV via expression of recombinant baculoviruses encoding SAV capsid protein and two major immunodominant viral glycoproteins, E1 and E2 in Spodoptera frugiperda Sf9 insect cells. However, this was only achieved when a temperature shift from 27°C to lower temperatures was applied. At 27°C, precursor E2 (PE2) was misfolded and not processed by host furin into mature E2. Hence, E2 was detected neither on the surface of infected cells nor as VLPs in the culture fluid. However, when temperatures during protein expression were lowered, PE2 was processed into mature E2 in a temperature-dependent manner and VLPs were abundantly produced. So, temperature shift-down during synthesis is a prerequisite for correct SAV glycoprotein processing and recombinant VLP production.

SUBMITTER: Metz SW

PROVIDER: S-EPMC3185042 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Low temperature-dependent salmonid alphavirus glycoprotein processing and recombinant virus-like particle formation.

Metz Stefan W SW Feenstra Femke F Villoing Stephane S van Hulten Marielle C MC van Lent Jan W JW Koumans Joseph J Vlak Just M JM Pijlman Gorben P GP

PloS one 20111003 10

Pancreas disease (PD) and sleeping disease (SD) are important viral scourges in aquaculture of Atlantic salmon and rainbow trout. The etiological agent of PD and SD is salmonid alphavirus (SAV), an unusual member of the Togaviridae (genus Alphavirus). SAV replicates at lower temperatures in fish. Outbreaks of SAV are associated with large economic losses of ~17 to 50 million $/year. Current control strategies rely on vaccination with inactivated virus formulations that are cumbersome to obtain a ...[more]

PMID: 21991361

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Project description:BackgroundPancreas disease (PD) is a viral fish disease which in recent years has significantly affected Norwegian salmonid aquaculture. In Norway, the aetiological agent salmonid alphavirus (SAV) has been found to be represented by the subtype 3 only. SAV subtype 3 has in previous analyses been found to show a lower genetic divergence than the subtypes found to cause PD in Ireland and Scotland. The aim of this study was to evaluate the nucleotide (nt) and amino acid divergence and the phylogenetic relationship of 33 recent SAV subtype 3 sequences. The samples from which the sequences were obtained originated from both PD endemic and non-endemic regions in an attempt to investigate agent origin/spread. Multiple samples throughout the seawater production phase from several salmonid populations were included to investigate genetic variation during an outbreak. The analyses were mainly based on partial sequences from the E2 gene. For some samples, additional partial 6 K and nsP3 gene sequences were available.ResultsThe nucleotide divergence for all gene fragments ranged from total identity (0.0% divergence) to 0.45% (1103 nt fragment of E2), 1.11% (451 nt fragment of E2), 0.94% (6 K) and 0.28% (nsP3). This low nucleotide divergence corresponded well to previous reports on SAV 3 sequences; however the observed divergence for the short E2 fragment was higher than that previously reported. When compared to SAVH20/03 (AY604235), amino acid substitutions were detected in all assessed gene fragments however the in vivo significance of these on for example disease outbreak mortality could not be concluded on. The phylogenetic tree based on the 451 nt E2 fragment showed that the sequences divided into two clusters with low genetic divergence, representing only a single SAV subtype.ConclusionsThe analysed sequences represented two clusters of a single SAV subtype; however some of the observed sequence divergence was higher than that previously reported by other researchers. Larger scale, full length sequence analyses should be instigated to allow further phylogenetic and molecular epidemiology investigations of SAV subtype 3.

Project description:Alphaviruses are small enveloped RNA viruses that bud from the host cell plasma membrane. Alphavirus particles have a highly organized structure, with a nucleocapsid core containing the RNA genome surrounded by the capsid protein, and a viral envelope containing 80 spikes, each a trimer of heterodimers of the E1 and E2 glycoproteins. The capsid protein and envelope proteins are both arranged in organized lattices that are linked via the interaction of the E2 cytoplasmic tail/endodomain with the capsid protein. We previously characterized the role of two highly conserved histidine residues, H348 and H352, located in an external, juxtamembrane region of the E2 protein termed the D-loop. Alanine substitutions of H348 and H352 inhibit virus growth by impairing late steps in the assembly/budding of virus particles at the plasma membrane. To investigate this budding defect, we selected for revertants of the E2-H348/352A double mutant. We identified eleven second-site revertants with improved virus growth and mutations in the capsid, E2 and E1 proteins. Multiple isolates contained the mutation E2-T402K in the E2 endodomain or E1-T317I in the E1 ectodomain. Both of these mutations were shown to partially restore H348/352A growth and virus assembly/budding, while neither rescued the decreased thermostability of H348/352A. Within the alphavirus particle, these mutations are positioned to affect the E2-capsid interaction or the E1-mediated intertrimer interactions at the 5-fold axis of symmetry. Together, our results support a model in which the E2 D-loop promotes the formation of the glycoprotein lattice and its interactions with the internal capsid protein lattice.IMPORTANCE Alphaviruses include important human pathogens such as Chikungunya and the encephalitic alphaviruses. There are currently no licensed alphavirus vaccines or effective antiviral therapies, and more molecular information on virus particle structure and function is needed. Here, we highlight the important role of the E2 juxtamembrane D-loop in mediating virus budding and particle production. Our results demonstrated that this E2 region affects both the formation of the external glycoprotein lattice and its interactions with the internal capsid protein shell.

Project description:MicroRNAs (miRNAs) control multiple biological processes including the innate immune responses by negative post-transcriptional regulation of gene expression. As there were no studies on the role(s) of miRNAs in viral diseases in Atlantic salmon, we aimed to identify miRNAs responding to salmonid alphavirus (SAV) infection. Their expression were studied at different time points post infection with SAV isolates associated with different mortalities. Furthermore, the genome sequences of the identified miRNAs were analysed to reveal putative cis-regulatory elements, and, finally, their putative target genes were predicted.Twenty differentially expressed miRNAs (DE miRNAs) were identified. The expression of the majority of these increased post infection with maximum levels reached after the viral load were stabilized or decreasing. On the other hand, some miRNAs (e.g. the miRNA-21 family) showed decreased expression at the early time points post infection. There were significant differences in the temporal expression of individual miRNA associated with different SAV isolates. Target gene prediction in SAV responsive immune network genes showed that seventeen of the DE miRNAs could target 24 genes (e.g. IRF3, IRF7). Applying the Atlantic salmon transcriptome as input 28 more immune network genes were revealed as putative targets (e.g. IRF5, IRF4). The majority of the predicted target genes promote inflammatory response. The upstream sequences of the miRNA genes revealed a high density of cis-regulatory sequences known as binding sites for immune network transcription factors (TFs). A high expression in the late phase could therefore be due to increased transcription promoted by immune response activated TFs. Based on the in silico target predictions, we discuss their putative roles as early promotors or late inhibitors of inflammation. We propose that the differences in expressions associated with different SAV isolates could contribute to their differences in mortality rates.This study represents the first steps in exploring miRNAs important in viral-host interaction in Atlantic salmon. We identified several miRNAs responding to SAV infection. Some likely to prohibit harmful inflammation while other may promote an early immune response. Their predicted functions need to be validated and further studied in functional assays to fully understand their roles in immune homeostasis.

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Low temperature-dependent salmonid alphavirus glycoprotein processing and recombinant virus-like particle formation.

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Low temperature-dependent salmonid alphavirus glycoprotein processing and recombinant virus-like particle formation.

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