the family reoviridae accommodates a wide range of members that infect protists , fungi , plants , invertebrates , and vertebrates , and are characterized by 912 segmented double - stranded rna ( dsrna ) genomes , multi - layered virion structures , and particle - associated enzymes for rna synthesis .
the family now consists of 15 genera divided into two subfamilies spinareovirinae (  turreted  or spiked reoviruses ) and sedoreovirinae ( non - spiked reoviruses ) ( attoui et al . , 2012 ) .
among the 15 genera is a relatively recently established genus mycoreovirus containing three members ( mycoreovirus 13 , myrv1 - 3 ) that were isolated in two phytopathogenic fungi , specifically , the chestnut blight fungus ( myrv1 and myrv2 ) and the white root rot fungus ( myrv3 ) by the groups of drs .
bradley i. hillman , william macdonald , and naoyuki matsumoto ( enebak et al . , 1994 ; hillman and suzuki , 2004 ; hillman et al . , 2004
myrv1 and myrv2 have 11-segmented genomes ( figure 1 ; s1s11 , termed with an increasing order of mobility in sds - polyacrylamide gel electrophoresis ) , while myrv3 has a 12-segmented genome ( s1s12 ) .
the mycoreovirus belongs to the spinareovirinae subfamily and its members retain conserved ntp binding motif and di - histidine motif specifically conserved in the subfamily ( suzuki et al .
, 2004 ; supyani et al . , 2007 ; spear et al . , 2012 ) .
each segment has a large single orf corresponding between 42 and 98% of its entire size .
light gray boxes refer to orfs and the sizes of their protein products are shown . the locations and
rdrp , guanylyltransferase , and ntp binding protein are assigned to s1 , s3 , and s6 . vp4 and
the plus - sense strand of each dsrna segment possesses the strictly conserved 5 pentanucleotide ( 5-gauca ) , single orf and 3 heptanucleotide ( gcaguca-3 ) .
myrv1 has a double - shelled particle structure of approximately 80  nm in diameter .
. both could involve large extents of genome segment alterations , but they are different in the generation mechanism .
reassortment events , involving exchange of genome segments between two viruses , occur at varying rates during genome packaging in coinfected cells .
genome rearrangements , defined as  alterations of considerable tracts of sequence within single genome segments often in the form of deletions and extensions  ( taniguchi and urasawa , 1995 ) , are a common phenomenon in all major genera of the family reoviridae ( table 1 ) .
they occur under natural conditions ( murao et al . , 1996 ; schnepf et al . , 2008 ) and laboratory conditions , for example , by serial passage of rotaviruses at high multiplicities of infection ( mois ; hundley et al
, 1985 ; kojima et al . , 2000 ) and exclusive maintenance in one of their two hosts ( plant , vector insect ) in the case of plant reoviruses ( nuss , 1984 ) .
rearrangements can be regarded as non - homologous , intramolecular rna recombination and exclude intermolecular rna recombinations that are documented frequently in plus - sense ( + ) , single - stranded rna ( ssrna ) viruses but are infrequently reported for reoviruses ( taniguchi and urasawa , 1995 ; desselberger , 1996 ) .
rearrangements are hypothesized to happen by copy choice ( template switch ) mechanism during rna synthesis mediated by the rna - dependent rna polymerase ( rdrp ) complex , as is rna recombination in ssrna viruses .
nsp , non - structural protein ; del , deletion ; dup+ , duplication with the original orf extended ; dup , duplication with the original orf unaltered ; inter , intergenic recombination ; wt , wild - type virus ; lab , laboratory ; nat , natural . *
we overview genome alterations , with a focus on unusual genome rearrangements observed in mycoreoviruses and discuss differences between other reovirus rearrangements in type of genome segment alterations and possible mechanisms underlying their occurrence .
readers are referred to excellent review articles on rna recombination ( lai , 1992 ; nagy and simon , 1997 ; simon - loriere and holmes , 2011 ; sztuba - solinska et al . , 2011 ) .
three types of very unusual genome alterations were reported to occur in mycoreoviruses that differ from those reported for other reoviruses .
the first example is complete loss of one of the segment s8 reported for myrv3 , a very rare event for a reovirus .
an entire set of genome segments are believed to be incorporated and packaged into single core particles during replication ( plus - sense strand synthesis ) .
therefore , all reovirus strains with rearranged segments still contain a set of all genome segments whether intact or altered . to our knowledge ,
( 2004 ) is the only example of reovirus that lacks a genome segment and is still viable .
these observations allowed the authors to conclude that myrv3 s8 is dispensable for maintenance under laboratory conditions .
the second type of mutations found in myrv1 is unique among reovirus examples in their dependence on a distinct virus or a viral protein .
specifically , myrv1 rearrangements are observed at extremely high rates in the fungal host either coinfected with the prototype hypovirus cryphonectria hypovirus 1/ep713 ( chv1-ep713 ) or expressing a multifunctional protein p29 encoded by chv1-ep713 ( sun and suzuki , 2008 ) .
after repeated subculturing of those fungal strains for one to two months , several dozen percent of resulting myrv1 isolates carry genome rearrangements . from doubly infected fungal mycelia , a myrv1 variant ,
myrv1/s10ss is isolated from 22.4% of subcultures , which harbors an altered segment s10ss , a deleted form of s10 , in place of an intact s10 .
s10ss lacks approximately 75% of the internal orf region while retaining the 5 and 3 terminal regions ( sun and suzuki , 2008 ) . in transformant fungal strains with the chv1 p29 coding domain ,
a variety of rearrangements are generated in addition to s10ss that include s1l , s2l , s3l , and s6l ( figure 2 ) .
in contrast to s10ss , these rearranged segments involve orf extensions , but are distinct from many previously reported animal reovirus examples .
extensions have been reported mostly from rotaviruses and those entail all head - to - tail tandem partial duplication occurring downstream of the termination codon of the authentic orf . thus , unaltered protein products are being synthesized from their cognate transcripts in infected cells . however , altered myrv1 genome segments , i.e. , s1l , s2l , s3l , and s6l are all with duplicated orfs in - frame with their preceding orfs , resulting in extension of orfs by 1.41.9 fold .
importantly , the products of the expected sizes are detectable in mycelia infected with myrv1 variants although s1l - encoded product remains to be detected ( sun and suzuki , 2008 ; tanaka et al . , 2011 ) .
these extended segments s1l , s2l , s3l , and s6l are frequently concomitant with s10ss although its significance is unclear .
s1 , s3 , and s6-encoded proteins carry sequence motifs characteristic of rdrp ( hillman et al . , 2004 ) , guanylyltransferase ( supyani et al . , 2007 ) , and ntp binding proteins ( suzuki et al . , 2004 ) .
it should be noted that these sequence motifs are duplicated in the expected protein products .
the maximum size expansion of an orthoreovirus was predicted to be 10% of the entire size ( approximately 2.0  kb ) ( roner and steele , 2007 ) .
congruent with this notion , myrv1/s1l  +  s10ss variant , with the largest extension per segment ( approximately 2.3  kb ) , has a genome - based extension of 1.8  kb after deduction of the deleted s10 sequence in s10ss .
these extensions correspond to approximately an 8% increase on a genome - size ( 23,433  bp ) basis .
there are two types of myrv1 rearrangements reported : extensions ( s1l , s2l , s3l , and s6l ) and deletions ( s4ss and s10ss ) that could happen by single events .
s1l , s2l , and s3l result from in - frame orf fusion but s4ss and s10ss do not always involve in - frame orf deletions .
genetic organizations of the normal ( top ) and rearranged forms ( bottom ) of genome segments is shown .
for extended segments s1l ( a ) , s2l ( b ) , s3l ( c ) , and s6l ( d ) , three fragments colored differently ( blue , red , and green ) are separated by break points .
the red portions refer to duplicated sequence while red color gradation is to indicate orientation ( head - to - tail for all rearrangements ) . in internal deletion mutations s4ss and s10ss ( e , f )
, only 1122% ( for s4ss ) and 1043% ( for s10ss ) of the intact orfs are retained .
note that s4ss and s10ss each have at least 4 ( s4ss1s4ss4 ) and 2 variants ( s10ss1 and 2 ) and one each of them ( s4ss1 and s10ss1 ) are shown in ( e ) and ( f ) , respectively .
solid lines denote the 5 and 3 utrs , while blue and green boxes refer to the n and c terminal portions of orf retained after each rearrangement event .
nucleotide positions for the start and stop codons , and rearrangement breakpoints are shown above the diagrams of each normal segments .
nucleotide and aa sequences adjacent to the altered sites on normal and rearranged segments are also shown below the diagrams of each altered segment .
neither sequence heterogeneity nor mismatch was detected at the junction sites for s1l , s2l , and s3l , while mismatches are found at or close to breakpoints of s4ss and s10ss ( sun and suzuki , 2008 ; eusebio - cope et al .
myrv1 s4 ( eusebio - cope et al . , 2010 ) and s10 ( suzuki , unpublished results ) , albeit very infrequently , are rearranged in a chv1 p29 independent fashion .
myrv1/s4ss variants carry deletion mutations on s4 ( s4ss1 , s4ss2 , s4ss3 , and s4ss4 ) lacking approximately 80% of the s4 orf but retaining its ability to be transcribed and replicated .
the genome segments including s4ss , s4ss1s4ss4 are different from each other in deletion endpoint .
a reassortant variant of myrv1 that contains substantial deletions in both s4 and s10 ( myrv1/s4ss1  +  s10ss2 ) was generated in fungal colonies coinfected with myrv1/s4ss1 and myrv1/s10ss2 .
surprisingly , myrv1/s4ss  +  s10ss shows comparable replication levels to the wild - type myrv1 .
this is the first example of a reovirus that is replication - competent without two viral proteins .
unlike members of the genera orthoreovirus ( kobayashi et al . , 2007 ) , orbivirus ( boyce et al . , 2008 ; matsuo et al . ,
2010 ) , and rotavirus ( komoto et al . , 2006 ) , no reverse genetics system has been developed for mycoreoviruses .
however , the myrv1 rearrangements ( shown in figure 2 ) provide insights into the functional roles of the genome segments and their protein products .
common to all these altered rearrangements , they are competent to be packaged and transcribed given the comparable accumulation levels of their transcripts in infected cells .
these results show that signals for packaging , transcription , and replication reside within the remaining genome sequence .
the viability of myrv1/s4ss  +  s10ss indicates that neither myrv1 non - structural protein encoded by s4 ( vp4 ) nor s10 ( vp10 ) is required for virus replication .
2010 ) reported that vp4 contributes to mycelial growth and efficient vertical transmission through conidia .
vp10 contributes inversely to the reduction of aerial mycelial growth induced by the virus ( sun and suzuki , 2008 ) .
recombination events altering protein sequences are often deleterious to its function and purified from the population ( simon - loriere and holmes , 2011 ; sztuba - solinska et al . , 2011 ) .
in fact , most extensions reported previously for reoviruses occur downstream of the termination codon and thus result in no change of protein sequences ( kojima et al . , 1996 ) .
in this sense , the fact that several myrv1 variants with extension rearrangements are viable is surprising .
interpretation of functional roles for extended segments ( s1l , s2l , s3l , and s6l ) in virus replication is complex due to coexistence of the extended and normal unaltered genome segments in fungal colonies infected by myrv1 variants .
therefore , heterocomplementation of rearranged segments by the normal segments can not be ruled out . in this regard
( 2011 ) , that fungal colonies infected with myrv1/s1l  +  s10ss2 , myrv1/s2l  +  s10ss2 , and myrv1/s3l  +  s10ss2 are similarly restored in aerial mycelial growth to some extent ( figure 3 ) .
the restoration levels of aerial mycelial growth increase as the ratio of transcript levels of rearranged s1l to normal segment s1 rises ( tanaka et al . , 2011 )
fungal colonies were grown on pda for 8 days under the bench top conditions approximately 24c . c. parasitica strain ep155 was infected with wild - type myrv1 ( myrv1 ) , myrv1/s10ss2 ( myrv1/s10ss ) , myrv1/s1l + s10ss2 ( myrv1/s1l + s10ss ) , myrv1/s2l + s10ss2 ( myrv1/s2l + s10ss ) , or myrv1/s3l + s10ss2 ( myrv1/s3l + s10ss ) .
as in other reoviruses , rearrangements are considered to contribute to evolution and molecular diversity of mycoreoviruses , while mycoreoviruses with such rearrangements including the myrv3 s8 loss are found under laboratory conditions , but not in the natural environment .
myrv1 variants with large deletions may have selective advantages in replication or packaging over wild - type myrv1 .
myrv1 variants with large extensions require chv1 p29 for their maintenance , suggesting no selective advantage of these variants in the absence of p29 .
maintenance of animal viruses in cultured host cells frequently induces mutations that confer selective advantages under laboratory conditions and lead to virulence attenuation in host animals . in this regard , myrv1 with genome rearrangements are different because they retain the ability to induce symptoms , while distinct from those induced by wild - type myrv1 ( figure 3 ) , including the reduction of host virulence to plants .
rather , myrv1 is reminiscent of plant reoviruses which undergo various mutations when they are maintained exclusively in one of their hosts , plant and insect vectors in laboratory conditions .
examples include those of would tumor virus ( wtv ) and rice dwarf virus ( rdv ) that are vectored by different species of leafhoppers ( table 1 ) .
in fact , transmission defective isolates of wtv emerge after being maintained for a long period of time in cultured insect cells that carry internal deletions on segments encoding structural and non - structural proteins ( nuss , 1984 ; anzola et al . , 1987 ) . in the case of rdv
maintained only in plants , point mutations rather than internal deletions accumulate resulting in little expression of protein products encoded by the mutated segments .
. however , one single passage in cultured insect vector cell lines resulted in inverse population structures ( pu et al . , 2011 ) .
two major mechanisms are proposed for it , specifically , replication independent breakage - rejoining recombination , and replication dependent rdrp - mediated recombination ( template switch or copy choice ) .
the non - replicative , breaking - joining mechanism entails dsrna or ssrna fragmentation and re - ligation for which several variations are proposed such as self - recombination requiring no proteins , and rnase - catalyzed cleavage and ligation ( chetverin , 1999 ) . the second model , replicase - mediated mechanism is more widely accepted ( lai , 1992 ; nagy and simon , 1997 ; sztuba - solinska et al . , 2011 ) .
in this model , the nascent rna and rdrp complex jumps after proceeding to a certain point from one template to another ( intergenic or intermolecular recombination ) or to another point of the same template ( intragenic or intramolecular recombination ) .
template switches could occur in homologous and non - homologous ( illegitimate ) manners such that their crossover regions are of similar and unrelated sequences respectively .
non - homologous recombination is sometimes considered to be facilitated by sequences features of template and acceptor strands , such as direct , inverted repeats and/or secondary structures .
a number of viral rna and protein elements as well as host factors are involved in rna recombinations in different single - stranded , plus - sense rna plant virus / host systems ( serviene et al . , 2005 ; sztuba - solinska et al . ,
recently , rna silencing was shown to contribute to the generation of defective - interfering ( di ) rnas of the prototype hypovirus ( chv1 ; zhang and nuss , 2008 ) , associated with the picornavirus superfamily ( koonin et al . , 1991 ) , that infects the chestnut blight fungus and confers hypovirulence .
rearrangements reported for members of the family reoviridae could be accounted for by an intramolecular template switch , in which the replicase complex jumps backward ( for extension ) or forward ( for deletions ) on the same template molecule .
for example , intragenic rearrangements of human rotavirus segment s11 are hypothesized to be generated in the process of plus - sense strand synthesis ( transcription ) mediated by short direct repeats of 46 nucleotides ( kojima et al .
( 2001 ) inferred that duplications of human rotavirus genes 7 and 11 were presumed to occur by template switch mediated by an inverted repeat - involving secondary structure during the replication step ( gault et al .
a rare example is intergenic recombination with a crossover of two rotavirus segments encoding nsp2 and nsp5,6 ( cao et al . , 2008 ;
recent structural studies led to 3d modeling of rdrps from two reoviruses , human orthoreovirus and rotavirus .
they are commonly characterized by 4-window structures which allow entry of substrates ( rear window ) and templates ( left window ) , and exit of template rna ( front window ) and nascent rna ( bottom window ; tao et al . , 2002 ; lu et al . , 2008
based on this rdrp structural prediction , a loop model was proposed to explain how reovirus extension rearrangements occur ( matthijnssens et al . , 2006 ) . according to this model ,
extension rearrangements occur during the transcription step via intramolecular template switch from the transcribed region of the minus - strand to upstream regions of the same molecule re - entering from the left channel .
it remains unknown how the mechanism of intergenic recombination or large deletion rearrangements fits into the rdrp 3d model .
addressing how p29 is involved in the enhanced emergence of myrv1 rearrangements is an interesting challenge .
hypovirus rna are replicated in golgi - derived vesicles of 80  nm whose production is enhanced in the cytoplasm upon viral infection ( fahima et al . , 1994 ; hillman and suzuki , 2004 ; jacob - wilk et al . , 2006 ) .
reovirus rnas are synthesized in viroplasms or virus factories formed in the cytoplasm of infected cells by viral and host components ( patton et al . , 2006 ; cheung et al . , 2010 ) .
chv1 p29 is a multifunctional protein acting as a symptom determinant ( choi and nuss , 1992 ; craven et al . , 1993 ; suzuki et al . , 1999 , 2003 ) , protease ( choi et al
, 1991a , b ) , an rna silencing suppressor ( segers et al . , 2006 ) , and an enhancer of heterologous and homologous viruses ( suzuki et al . , 2003 ; sun et al . , 2006 )
. some functional domains of p29 are mapped such as the n terminal aa 124 essential for virus viability , adjacent 2574 for the symptom determinant and the c terminal half for the proteolytic catalytic domain ( suzuki and nuss , 2002 ; hillman et al . , 2004 ) .
firstly , p29 may interact the rna synthesis machinery to perturb transcription ( plus - sense rna synthesis ) and/or replication ( minus - sense rna synthesis ) , leading to enhanced rate of template switch of rdrp - mediated via rna sequence features such as inverted or direct repeats .
another possibility is that p29 alters the physiological state of infected cells , which enhances selection of pre - existing mutant viruses with rearranged segments by unknown means .
, it remains unknown whether chv1 p29 reside in myrv1 core particles viewed as a nano - scale rna synthesis factory ( guglielmi et al . , 2010 ) to perturb myrv1 rna synthesis .
sun and suzuki ( 2008 ) showed that chv1 p29 interacts with myrv1 vp9 , considered to be a non - structural protein , in vitro and in vivo , providing a clue to the mechanism .
as in cells infected with other reoviruses , viroplasm is produced in myrv1-infected cells ( sun and suzuki , unpublished data ) .
one or two viral non - structural proteins are required as matrix proteins for production of reoviral viroplasms which are believed to be the site of virus replication where core particle assembly and rna synthesis occur .
consistent with this notion , knocking - down of the genes responsible for functional viroplasm formation is detrimental for virus replication ( campagna et al .
as discussed above , a myrv1 variant shows comparable replication competency without vp4 and vp10 .
therefore , vp4 and vp10 are unlikely to be matrix proteins of viroplasms given the essentiality of viroplasm .
one of key points to be addressed is what the possible role of vp9 in myrv1 replication is .
it is of interest to infer that vp9 is a viroplasm matrix protein that is remodeled upon interaction with p29 .
moreover , to explore whether chv1 p29 is incorporated into myrv1 core particles is a prerequisite .
the other possibility may be associated with the reason why almost a half of the myrv1 segments , whether encoding structural or non - structural proteins , are generated at high rates .
chv1 p29 , as a symptom determinant , slightly enhances mycelial growth and suppresses asexual sporulation and orange pigmentation , which involve complex regulatory pathways .
additionally , p29 is an rna silencing suppressor which may potentially be able to perturb the cellular physiological state .
the p29 protein might be involved in selection and maintenance of myrv1 variants with rearrangements .
apparent  reversion  of s1l , s2l , s3l , and s6l to respective normal segments ( sun and suzuki , 2008 ; tanaka et al . ,
2011 ) in the absence of p29 suggests a role for the protein in the maintenance of the extended segments .
three unusual types of genome alterations are found in the genus mycoreovirus ; a s8-deficient myrv3 mutant , a myrv1 variant with two rearranged segments ( s4ss and s10ss ) each lacking three fourths of the orf , and myrv1 variants with genome rearrangements ( s1l , s2l , s3l , s6l , and s10ss ) induced by a multifunctional protein p29 of an unrelated virus , chv1 . while s8-lacking myrv3 strains have properties indistinguishable from wild - type myrv3 , most myrv1 variants with rearranged segments induced different symptoms in the chestnut blight fungus than the wild - type virus .
therefore , these genome segment alterations are useful for gaining insights into functional roles of genome segments of mycoreoviruses for which a reverse genetics system is unavailable like many other reoviridae members .
the chv1 p29-dependent rearrangements of myrv1 are a novel type of intriguing virus / virus interactions , and accordingly there are a few important unanswered questions . what is the biological significance of the interactions between the two viruses ?
what function of chv1 p29 is related to rearrangements ? does p29 contribute to generate de novo genome rearrangements or to select pre - existing rearrangements by altering the cellular state , while it is poorly defined at present ?
does chv1 p29 induce genome rearrangements on other reoviruses such as myrv2 and myrv3 ? experiments to address these issues are underway .
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .
 the family reoviridae is one of the largest virus families with genomes composed of 912 double - stranded rna segments . 
 it includes members infecting organisms from protists to humans . 
 it is well known that reovirus genomes are prone to various types of genome alterations including intragenic rearrangement and reassortment under laboratory and natural conditions . 
 recently distinct genetic alterations were reported for members of the genus mycoreovirus , mycoreovirus 1 ( myrv1 ) , and myrv3 with 11 ( s1s11 ) and 12 genome segments ( s1s12 ) , respectively . while myrv3 s8 is lost during subculturing of infected host fungal strains , myrv1 rearrangements undergo alterations spontaneously and inducibly . 
 the inducible myrv1 rearrangements are different from any other previous examples of reovirus rearrangements in their dependence on an unrelated virus factor , a multifunctional protein , p29 , encoded by a distinct virus cryphonectria parasitica hypovirus 1 ( chv1 ) . 
 a total of 5 myrv1 variants with genome rearranged segments ( s1s3 , s6 and s10 ) are generated in the background of a single viral strain in the presence of chv1 p29 supplied either transgenically or by coinfection . 
 myrv1 s4 and s10 are rearranged , albeit very infrequently , in a chv1 p29 independent fashion . 
 a variant of myrv1 with substantial deletions in both s4 and s10 , generated through a combined reassortment and rearrangement approach , shows comparable replication levels to the wild - type myrv1 . in vivo and in vitro interactions of chv1 p29 and myrv1 vp9 
 are implicated in the induction of myrv1 rearrangements . 
 however , the mechanism underlying p29-mediated rearrangements remains largely unknown . 
 myrv1 s4 rearrangements spontaneously occurred independently of chv1 p29 . in the absence of reverse genetics systems for mycoreoviruses , molecular and biological characterization of these myrv1 and myrv3 variants 
 contribute to functional analyses of the protein products encoded by those rearranged segments . 