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Nobuhiro Suzuki, Said A. Ghabrial, Kook-Hyung Kim, Michael Pearson, Shin-Yi L. Marzano, Hajime Yaegashi, Jiatao Xie, Lihua Guo, Hideki Kondo, Igor Koloniuk and Bradley I. Hillman
A summary of this ICTV Report chapter has been published as an ICTV Virus Taxonomy Profile article in the Journal of General Virology, and should be cited when referencing this online chapter as follows:
Suzuki, N., Ghabrial, S.A., Kim, K., Pearson, M., Marzano, S.L., Yaegashi, H., Xie, J., Guo, L., Kondo, H., Koloniuk, I., Hillman, B.I., and ICTV Report Consortium. 2018, ICTV Virus Taxonomy Profile: Hypoviridae, Journal of General Virology, 99: 615–616.
The Hypoviridae, comprising one genus Hypovirus, is a family of capsidless viruses with positive-sense, single-stranded RNA genomes of 9.1–12.7 kb that possess either a single large open reading frame (ORF) or two ORFs. The ORFs appear to be translated from genomic RNA by non-canonical mechanisms, i.e., internal ribosome entry site-mediated and stop/restart translation. Hypoviruses have been detected in ascomycetous and basidiomycetous filamentous fungi, and are considered to be replicated in host Golgi-derived, lipid vesicles that contain their dsRNA as the replicative form. Some hypoviruses induce hypovirulence to host fungi, while others do not.
Table 1.Hypoviridae. Characteristics of the family Hypoviridae.
Cryphonectria hypovirus 1 strain EP713 (M57938), species Cryphonectria hypovirus 1, genus Hypovirus
Capsidless virus unable to form rigid particles
9.1–12.7 kb of linear, positive-sense, unsegmented RNA
Replication (synthesis of complementary RNA) and transcription (synthesis of genomic RNA) occur cytoplasmically in Golgi-derived membraneous vesicles
Directly from bi- or mono-cistronic genomic RNA containing a possible internal ribosomal entry site at the 5′-non-coding region
One genus including 4 species
No true virions are associated with hypoviruses. Pleomorphic vesicles 50–80 nm in diameter (Newhouse et al., 1983), devoid of any detectable viral structural proteins but containing replicative form dsRNA and polymerase activity (Fahima et al., 1994), are the only virus-associated particles that can be isolated from infected fungal tissue (Figure 1.Hypoviridae).
Figure 1.Hypoviridae. (Top) Thin section showing vesicles in fungal tissue; (bottom) thin section showing vesicle aggregate in fungal tissue surrounded by rough ER. Figure reproduced with permission from (Newhouse et al., 1983). The bar represents 100 nm.
The Mr of vesicles is unknown. They have a buoyant density in CsCl of approximately 1.27–1.30 g cm−3 and sediment through sucrose as a broad component of approximately 200S (Dodds 1980). Their pH stability is unknown. The vesicles can be purified in pH 5.0 buffer and resuspended in pH 7.0 buffer. The pH optimum for polymerase activity in vitro is 8.0; the optimum Mg2+ concentration for polymerase activity is 5 mM. Activity decreases dramatically at pH less than 7.0 or more than 9.0 (Fahima et al., 1993). The vesicles are unstable when heated, or dispersed in lipid solvents. The optimal temperature for polymerase activity is 30 °C; temperatures over 40 °C inactivate polymerase activity. Deoxycholate at concentrations of more than 0.5% inactivates polymerase activity (Fahima et al., 1993).
Vesicles contain a linear dsRNA genome of 9.1–12.7 kbp. The genome of Cryphonectria hypovirus 1 EP713, a member of the type species of the genus Hypovirus, is 12,712 bp. The coding (positive) strand contains a short 3′-poly(A) tail, of 20–30 nt when analyzed as a component of the dsRNA (Shapira et al., 1991b).
In the absence of an identifiable virion RNA, hypoviruses were originally classified, along with several other fungal viruses, as dsRNA viruses. However, the RdRP and RNA helicase of hypoviruses show sequence similarity and phylogenetic affinity to plant potyviruses, members of the expanded picorna-like supergroup with positive-sense RNA genomes (Koonin et al., 1991). In addition, the ability of positive strand ssRNA, but not dsRNA, to initiate infection is consistent with their current classification among the positive-sense RNA viruses. The predominant dsRNA form found in infected mycelia is regarded as a replicative intermediate or replicative form RNA. The accumulation of plus strand RNA, by contrast, is low due to the RNA silencing antiviral defense response of the fungal host, but greatly increases in fungal strains in which the corresponding Dicer (dcl2) or Argonaute gene (agl2) has been disrupted (Sun et al., 2009, Segers et al., 2007). Small interfering RNAs (siRNAs) spanning the entire genomic RNA are produced in a non-random fashion (Zhang et al., 2008). The presence of shorter-than-full-length, internally deleted, defective interfering (DI) and defective (D) replicative form dsRNA molecules is common among some members, and replicative form of satellite-like RNAs are present in other members (Shapira et al., 1991a, Yuan and Hillman 2001). The host RNA silencing pathway has been reported to promote DI RNA production (Nuss 2011). No function has been ascribed to any ancillary dsRNA. The 5′-terminus of the positive strand of dsRNA from Cryphonectria hypovirus 1-EP713 (CHV1-EP713) is blocked (Hiremath et al., 1986) but the nature of the blocking group is unknown. The 5′-terminus of the negative strand is unblocked. Both 5′-termini of dsRNA from Cryphonectria hypovirus 3-GH2 (CHV3-GH2) are unblocked (Tartaglia et al., 1986).
No structural proteins have been described for members of this family. Functions have been assigned to several nonstructural polypeptides encoded by members of the family. The 5′-proximal coding domain, ORF A, of CHV1-EP713 RNA encodes a papain-like protease, p29, and a highly basic protein, p40, derived, respectively, from the N-terminus and C-terminus of polyprotein p69, by a p29-mediated cleavage event (Choi et al., 1991a, Choi et al., 1991b) (Figure 2.Hypoviridae). A presumptive non-structural protein identified in vitro and in vivo, p29, has been shown by DNA-mediated transformation to contribute to suppression of host pigmentation, reduced sporulation, reduced laccase accumulation (Craven et al., 1993), to enhance vertical virus transmission (Suzuki et al., 2003), to serve as a suppressor of host RNA silencing (Zhang and Nuss 2008), and to promote RNA recombination of a co-infecting mycoreovirus (dsRNA virus) (Sun and Suzuki 2008). Protein p40 enhances viral RNA accumulation (Suzuki and Nuss 2002). RNA polymerase activity is associated with isolated vesicles from a CHV1-EP713 infected fungal isolate (Fahima et al., 1994, Fahima et al., 1993). The calculated mass of the CHV1 ORF B product, which contains a second papain-like protease, p48, and putative RNA polymerase and helicase domains, is approximately 362 kDa (3165 amino acids) based on its deduced aa sequence, but no protein of that mass has yet been detected from vesicles. Smaller virus-encoded proteins (~87 kDa) have been identified in the vesicle-associated polymerase complex, suggesting extensive processing of replication proteins and that ORF B processing occurs in vivo (Fahima et al., 1994). However, exact cleavage sites for these smaller proteins remain unknown. Other as-yet-unidentified viral and/or host proteases may contribute to the whole polyprotein processing. Note that 2A-like self-processing peptide motifs are detectable in some hypoviruses, although whether they are functional in infected fungal cells is an open question (Petrzik et al., 2016, Hisano et al., 2017). CHV3 and Cryphonectria hypovirus 4 (CHV4) each contains a putative UDP glycosyltransferase-encoding domain that is not present in CHV1 or Cryphonectria hypovirus 2 (CHV2), although the specific function of this domain has not been examined (Smart et al., 1999, Linder-Basso et al., 2005).
Figure 2.Hypoviridae. Genome organization of members of the family Hypoviridae. Arrows represent known or suspected sites of autoproteolysis. Cryphonectria hypovirus 4 has a genome organization similar to that of Cryphonectria hypovirus 3, but it is unknown whether the former undergoes autoproteolysis. The abbreviations pol, hel and ugt refer to the RNA-dependent RNA polymerase, RNA helicase, and UDP-glucose/sterol glucosyltransferase domains, respectively.
Host-derived lipids make up the vesicles that encapsulate the viral dsRNA (Newhouse et al., 1990).
Carbohydrates similar to those involved in fungal cell wall synthesis are associated with vesicles (Hansen et al., 1985).
A 5′-leader of approximately 300–500 nt, including several AUG triplets, precedes the AUG codon that initiates the first long ORF (Figure 2.Hypoviridae). Translational initiation for the first ORF on the genomic RNA is mediated by an internal ribosome entry site (IRES) residing in the 5′-non-coding region (NCR) and extending to the coding domain in the case of CHV1 (Chiba et al., 2018), although 5′-NCR sequences and predicted secondary structures are highly variable between hypovirus species (Mu et al., 2011). The viral coding region may be expressed from a single long ORF, or may be divided into two ORFs. If two ORFs are present (e.g., CHV1 and CHV2), the shorter, 5′-proximal ORF is designated ORF A. Its product may or may not be autocatalytically cleaved, depending on the virus. The UAA termination sequence at the end of ORF A is part of the pentanucleotide UAAUG in all members with two ORFs investigated to date. The AUG of the UAAUG pentanucleotide initiates the second long ORF, ORF B. In the case of hypoviruses with a two-ORF genome organization, the stop/restart translation mechanism is involved for the translation of their downstream ORFs. The pentanucleotide, UAAUG for CHV1 and CHV2 at the junction of the two ORFs (Figure 2.Hypoviridae), plays a critical role (Guo et al., 2009). The N-terminal product of ORF B contains a papain-like cysteine protease domain that is autocatalytically released from the growing polypeptide chain (e.g., p48 for CHV1-EP713 or p52 for CHV2-NB58) (Shapira and Nuss 1991, Hillman et al., 1994). No further processing in vitro has been demonstrated for the remaining 314 kDa polypeptide (2747 amino acids) from this ORF, although processed products have been detected in vivo (Fahima et al., 1994). Phylogenetic relatedness to members of the positive-sense, ssRNA family Potyviridae has been demonstrated by comparisons of protease, polymerase and helicase domains, although these domains are positioned differently in the two virus groups (Koonin et al., 1991). The 5′-proximal portions of hypovirus genomes are prone to recombination and show greater molecular variability than their 3′-proximal portions (Wang et al., 2013, Marzano et al., 2015, Du et al., 2017).
Synthesis of both positive- and negative strand viral RNA is believed to occur cytoplasmically in host-derived lipid vesicles that contain linear dsRNA, regarded as the replicative form of hypoviral genomic strand ssRNA. The polymerase associated with vesicles transcribes ssRNA molecules in vitro that correspond in size to full-length dsRNA. Approximately 80% of the polymerase products in vitro are of positive-sense polarity (Fahima et al., 1993). Except for CHV2 p50, hypoviral proteins are synthesized as part of a polyprotein. The polyprotein is autocatalytically cleaved by viral proteases such as CHV1 p29 and p48, and CHV2 p52. CHV1 p29 enhances the replication in cis and in trans by suppressing antiviral RNA silencing (Segers et al., 2007), regardless of whether expressed from the viral genome or transgenically. The other CHV1 ORF A-coded protein p40, a basic protein, also enhances the replication in cis only (Suzuki and Nuss 2002). However, the ORF A coding domain, except the first 22 codons, is dispensable for CHV1 viability (Suzuki et al., 2000). The p48 protein encoded by CHV1 ORF B is required for initiation but not maintenance of viral RNA replication (Jensen and Nuss 2014).
No antibody has ever been raised from virus-associated particle preparations. Antibodies directed against CHV1-EP713-encoded p29 were used to demonstrate association with the trans-Golgi network membranes. Antibodies directed against the conserved RNA polymerase domain of ORF B, expressed in bacteria, were used to identify an 87 kDa protein in a CHV1-EP713 infected fungal strain (Fahima et al., 1994).
All members infect the chestnut blight fungus, Cryphonectria parasitica. Classified members result in reduced virulence (hypovirulence) on chestnut trees and altered fungal morphology in culture, but many unclassified family members have little or no discernible effect on the fungal host. Some unclassified hypovirus-related viruses infect other filamentous fungi, e.g., Sclerotinia sclerotiorum (Marzano et al., 2015, Khalifa and Pearson 2014, Hu et al., 2014, Xie et al., 2011), Valsa ceratosperma (Yaegashi et al., 2012), Phomopsis longicolla (Koloniuk et al., 2014), and Fusarium spp. (Wang et al., 2013, Li et al., 2017, Li et al., 2015, Osaki et al., 2016), Macrophomina phaseolina (Marzano et al., 2016), and Agaricus bisporus (Deakin et al., 2017). Infection of fungal mycelium is known only through fusion, or anastomosis, of infected hyphae with uninfected hyphae. The frequency of transmission through asexual spores (conidia) varies from a few to close to 100%. Transmission through sexual spores (ascospores) is not known to occur. Transmission via cell-free extracts has not been demonstrated but transfection of protoplasts with full-length synthetic transcripts derived from cDNA clones has been successful for hypoviruses CHV1-EP713, CHV1-Euro7, CHV1-EP721, and an unclassified hypovirus-related virus Sclerotinia sclerotiorum hypovirus 2-Lactuca (SsHV2-L) (Marzano et al., 2015, Chen et al., 1994, Lin et al., 2007, Chen and Nuss 1999). Classified members have been identified throughout chestnut-growing areas of Europe, North America and Asia (Bryner et al., 2012, Peever et al., 1998). dsRNA-containing vesicles have been associated with abnormal Golgi apparatus in freeze-substituted thin sections (Newhouse et al., 1990). No nuclear or mitochondrial associations, nor virus-associated inclusions, have been noted.
Hypo: from hypovirulence.
Many strains and isolates have been identified by RNA hybridization, RFLP, or nucleotide sequence analysis. Among the four species, subgroupings of CHV1 with CHV2 and CHV3 with CHV4 are apparent (Hillman and Suzuki 2004). The creation of additional genera accommodating unclassified hypovirus-related viruses of the family (Related, Unclassified Viruses; Figure 3.Hypoviridae) has been proposed in which “Alphahypovirus” would include CHV1, CHV2 and unclassified hypoviruses, and “Betahypovirus” would include CHV3, CHV4 and unclassified hypoviruses, respectively (Khalifa and Pearson 2014, Yaegashi et al., 2012). Beihai hypo-like virus and Beihei sipunculid worm virus 6 may represent members of an additional genus (Figure 3.Hypoviridae).
Figure 3.Hypoviridae. Molecular phylogenetic analyses of hypoviruses and related viruses. Amino acid sequences of the polyproteins containing RNA-dependent RNA polymerase and RNA helicase domains were aligned using MAFFT version 7 (Katoh et al., 2017), and then the alignment was filtered using Gblocks 0.91b (Talavera and Castresana 2007). A maximum likelihood phylogenetic tree was generated using PhyML 3.0 (Guindon et al., 2010) with the best-fit model LG + G + I. Pink shading indicates viruses in genus Hypovirus, either assigned to species (red labels) or unclassified, related viruses (black labels); blue shading indicates unclassified fusariviruses. The tree is rooted to the fusariviruses as outgroup. Numbers at the nodes indicate bootstrap support over 70% (100 replicates). This phylogenetic tree and corresponding sequence alignment are available to download from the Resources page.
The possession of an undivided positive-sense ssRNA genome, expressed via long polyprotein precursors, demonstrates an affinity with viruses of the picornavirus supergroup (Koonin et al., 1991). Deduced aa sequences of polymerase, helicase and protease motifs of members of the family Hypoviridae suggest that their closest relatives are the members of the genus Bymovirus in the family Potyviridae (Koonin et al., 1991). Sequence similarity and phylogenetic affinity is also noted between hypoviruses and several unclassified fusariviruses represented by Fusarium graminearum dsRNA mycovirus 1 strain DK21 (Kwon et al., 2007, Zhang et al., 2014) (Figure 3.Hypoviridae). However, fusariviruses have a different genome organization and relative position of the polymerase and helicase domains, and so are not included in the family Hypoviridae.
Since only one genus (Hypovirus) is currently recognized in the family Hypoviridae, the family description above corresponds to the genus description. For clarity, the additional information that can be found on the genus page is also presented below.
Viruses belonging to different species are differentiated by genetic organization or genome expression, as well as by major differences in nucleic acid sequence identity. Thus, Cryphonectria hypovirus 1 (CHV1) differs from Cryphonectria hypovirus 2 (CHV2) in the presence or absence, respectively, of a papain-like proteinase in ORF A. CHV1 and CHV2 isolates share less than 60% overall aa sequence identity with each other. Cryphonectria hypovirus 3 (CHV3) and Cryphonectria hypovirus 4 (CHV4) each contain a single ORF, but isolates of the two species share less than 50% overall sequence identity. In addition, infection by CHV1 isolates results in a white or near-white phenotype in the fungus; CHV2 infection results in an orange-brown phenotype; CHV3 and CHV4 isolates have little effect on fungal pigment. Infection by members of any of the four species may reduce fungal virulence.
Sclerotinia sclerotiorum hypovirus 1-SZ-150
Sclerotinia sclerotiorum hypovirus 2-5472
Sclerotinia sclerotiorum hypovirus 2-SX247
Sclerotinia sclerotiorum hypovirus 2-Lactuca
Valsa ceratosperma hypovirus 1-MVC86
Phomopsis longicolla hypovirus 1-ME711
Fusarium graminearum hypovirus 1-HN10
Fusarium graminearum hypovirus 2-JS16
Fusarium langsethiae hypovirus 1-AH32
Macrophomina phaseolina hypovirus 1- Mp2003b
Agaricus bisporus virus 2-C19-C23-C1
Wuhan insect virus 14-WHZM10168
Beihai hypo-like virus 1-BHZC36965
Beihai sipunculid worm virus 6-BHNXC41400
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