The nucleoprotein and the viral RNA of infectious salmon anemia virus (ISAV) are localized in the nucleolus of infected cells

Infectious salmon anemia virus (ISAV) has emerged as a virus of great concern to the aquaculture industry since it can lead to highly contagious and lethal infections in farm-raised salmon populations. While little is known about the transcription/replication cycle of ISAV, initial evidence suggests that it follows molecular mechanisms similar to those found in other orthomyxoviruses, which include the highly pathogenic influenza A (inf A) virus. During the life cycle of orthomyxoviruses, a panhandle structure is formed by the pairing of the conserved 5 and 3 ends of each genomic RNA. This structural motif serves both as a promoter of the viral RNA (vRNA)-dependent RNA polymerase and as a regulatory element in the transcription/replication cycle. As a first step toward characterizing the structure of the ISAV panhandle, here we have determined the secondary structures of the vRNA and the cRNA panhandles on the basis of solution nuclear magnetic resonance (NMR) and thermal melting data. The vRNA panhandle is distinguished by three noncanonical U ⅐ G pairs and one U ⅐ U pair in two stem helices that are linked by a highly stacked internal loop. For the cRNA panhandle, a contiguous stem helix with a protonated C ⅐ A pair near the terminus and tandem downstream U ⅐ U pairs was found. The observed noncanonical base pairs and base stacking features of the ISAV RNA panhandle motif provide the first insight into structural features that may govern recognition by the viral RNA polymerase.

Section: Discussionmentioning

confidence: 99%

Structural Characterization of the Viral and cRNA Panhandle Motifs from the Infectious Salmon Anemia Virus

Brinson

Szakal

Marino

2011

“…Furthermore, some studies consider nucleolar transit to be of relevance. For example, in the case of orthomyxoviruses there are studies suggesting that prior nucleolar localization is required for the nucleocapsid protein early in replication, which is followed by a combination of nucleoplasmic and nucleolar localizations (12,34). Second, what are the nature and significance of ␦Ag found in the nucleoplasm?…”

Section: Discussionmentioning

confidence: 99%

“…The present study includes examination of the nature and significance of the ␦Ag localization to the nucleolus. Other investigators have noted that for several viruses, specific and essential viral proteins, typically RNA-binding proteins, also locate to the nucleolus, and in some cases such localization only occurs early in infection, leading to the speculation that a "nucleolar transit" may be an essential step in genome replication (12,15). Consistent with but not proof of such a transit for ␦Ag is that during HDV genome replication, the majority of ␦Ag is no longer found in the nucleolus but in the nucleoplasm (4).…”

mentioning

confidence: 99%

Intracellular Localization of Hepatitis Delta Virus Proteins in the Presence and Absence of Viral RNA Accumulation

Han

Alves

Gudima

et al. 2009

Hepatitis delta virus (HDV) encodes one protein, hepatitis delta antigen (␦Ag), a 195-amino-acid RNA binding protein essential for the accumulation of HDV RNA-directed RNA transcripts. It has been accepted that ␦Ag localizes predominantly to the nucleolus in the absence of HDV genome replication while in the presence of replication, ␦Ag facilitates HDV RNA transport to the nucleoplasm and helps redirect host RNA polymerase II (Pol II) to achieve transcription and accumulation of processed HDV RNA species. This study used immunostaining and confocal microscopy to evaluate factors controlling the localization of ␦Ag in the presence and absence of replicating and nonreplicating HDV RNAs. When ␦Ag was expressed in the absence of full-length HDV RNAs, it colocalized with nucleolin, a predominant nucleolar protein. With time, or more quickly after induced cell stress, there was a redistribution of both ␦Ag and nucleolin to the nucleoplasm. Following expression of nonreplicating HDV RNAs, ␦Ag moved to the nucleoplasm, but nucleolin was unchanged. When ␦Ag was expressed along with replicating HDV RNA, it was found predominantly in the nucleoplasm along with Pol II. This localization was insensitive to inhibitors of HDV replication, suggesting that the majority of ␦Ag in the nucleoplasm reflects ribonucleoprotein accumulation rather than ongoing transcription. An additional approach was to reevaluate several forms of ␦Ag altered at specific locations considered to be essential for protein function. These studies provide evidence that ␦Ag does not interact directly with either Pol II or nucleolin and that forms of ␦Ag which support replication are also capable of prior nucleolar transit.Hepatitis delta virus (HDV) is a subviral agent with a singlestranded circular RNA genome. As reviewed recently (6, 14, 37) this 1,679-nucleotide RNA is replicated by RNA-directed RNA synthesis dependent upon redirection of the host RNA polymerase II (Pol II). Replication involves the production of the antigenome, an exact complement of the genome. The small delta antigen (␦Ag) is translated from a third RNA, one with the same antigenomic polarity but with a linear rather than circular conformation, of less than full-length, and possessing a 5Ј cap and a 3Ј poly(A). ␦Ag is a 195-amino-acid-long RNA binding protein that is essential in one or more ways for the accumulation of HDV RNA-directed RNA transcripts. There is a second form of ␦Ag that is 19 amino acids longer at the C terminus; this species arises due to specific RNA editing that occurs on the antigenomic RNA during replication. Unlike the small form, the large does not support HDV genome replication and is a dominant-negative inhibitor of the replication driven by the small form. However, large ␦Ag does have an essential function in that in the presence of HBV envelope proteins, it facilitates the assembly of HDV genomic RNA into new virus particles.Our previous immunostaining studies have shown that in the absence of HDV genome replication, small ␦Ag localizes predominantly to th...

“…Cells were then permeabilized for 1 h at room temperature with 0.1% Triton X-100 in 1ϫ PBS. The cells were fixed, and cell sections were blocked with 3% bovine serum albumin (BSA) containing 0.5% Tween 20 for 30 min at 37°C (25). A monoclonal antibody against the ISAV nucleoprotein (clone 2C2/H4; Grupo Bios, BiosChile) diluted 1:500 in 1ϫ PBS and 3% BSA (26) was incubated overnight at 4°C, detected by using a goat anti-mouse antibody conjugated with Alexa Fluor 568 (Life Technologies, USA) diluted 1:1,000 in 1ϫ PBS and 3% BSA, and incubated for 1 h at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Bona Fide Evidence for Natural Vertical Transmission of Infectious Salmon Anemia Virus in Freshwater Brood Stocks of Farmed Atlantic Salmon (Salmo salar) in Southern Chile

Marshall

Ramírez

Labra

et al. 2014

Infectious salmon anemia (ISA) is a severe disease that affects farmed Atlantic salmon (Salmo salar), causing outbreaks in seawater in most salmon-producing countries worldwide, with particular aggressiveness in southern Chile. The etiological agent of this disease is a virus belonging to the Orthomyxoviridae family, named infectious salmon anemia virus (ISAV). Although it has been suggested that this virus can be vertically transmitted, even in freshwater, there is a lack of compelling experimental evidence to confirm this. Here we demonstrate significant putative viral loads in the ovarian fluid as well as in the eggs of two brood stock female adult specimens that harbored the virus systemically but without clinical signs. The target virus corresponded to a highly polymorphic region 3 (HPR-3) variant, which is known to be virulent in seawater and responsible for recent and past outbreaks of this disease in Chile. Additionally, the virus recovered from the fluid as well as from the interior of the eggs was fully infective to a susceptible fish cell line. To our knowledge, this is the first robust evidence demonstrating mother-to-offspring vertical transmission of the infective virus on the one hand and the asymptomatic transmission of a virulent form of the virus in freshwater fish on the other hand. IMPORTANCEThe robustness of the data presented here will contribute to a better understanding of the biology of the virus but most importantly will constitute a key management tool in the control of an aggressive agent constantly threatening the sustainability of the global salmon industry.