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Circular satellite RNAs
Virus classification Edit this classification
Informal group: Subviral agents
Informal group: Satellite nucleic acids
Informal group: Circular satellite RNAs

Virusoids are circular single-stranded RNA(s) dependent on viruses for replication and encapsidation.[1] The genome of virusoids consists of several hundred (200–400) nucleotides and does not code for any proteins.

Virusoids are essentially viroids that have been encapsulated by a helper virus coat protein. They are thus similar to viroids in their means of replication (rolling circle replication) and in their lack of genes, but they differ in that viroids do not possess a protein coat. Both virusoids and a few viroids encode a hammerhead ribozyme.

Virusoids, while being studied in virology, are subviral particles rather than viruses. Since they depend on helper viruses, they are classified as satellites. Virusoids are listed in virological taxonomy as Satellites/Satellite nucleic acids/Subgroup 3: Circular satellite RNA(s).[2]

Definition

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Depending on whether a lax or strict definition is used, the term virusoid may also include Hepatitis D virus (HDV). Like plant virusoids, HDV is circular, single-stranded, and supported by a helper virus (Hepatitis B virus) to form virions; however, the virions possess a much larger genome size (~1700 nt) and encode a protein.[3][4] They also show no sequence similarity with the plant virusoid group.

History

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The first virusoid was discovered in Nicotiana velutina plants infected with Velvet tobacco mottle virus R2 (VTMOV).[5][6] These RNAs have also been referred to as viroid-like RNAs that can infect commercially important agricultural crops and are non–self-replicating single stranded RNAs.[7] RNA replication of virusoids is similar to that of viroids but, unlike viroids, virusoids require specific "helper" viruses.

Replication

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The circular structure of virusoid RNA molecules is ideal for rolling circle replication, in which multiple copies of the genome are generated in an efficient manner from a single replication initiation event.[8] Another advantage to circular RNAs as replication intermediates is that they are inaccessible and resistant to exonucleases. Additionally, their high GC content and high degree of self-complementarity make them very stable against endonucleases. Circular RNAs impose constraints on RNA folding by which secondary structures that are favored for replication differ from those assumed during ribozyme-mediated self-cleavage.

Plant satellite RNAs and virusoids depend on their respective helper viruses for replication, while the helper viruses themselves are dependent upon plants to provide some of the components required for replication.[9] Therefore, a complex interaction involving all three major players including satellites, helper viruses and host plants is essential for satellite / virusoid replication.

A hammerhead ribozyme, not from a virusoid (PDB: 2GOZ​)

satLTSV replication has been shown to occur through the symmetric rolling circle mechanism,[10] wherein the satLTSV self-cleaves both (+) and (-) strands. Both the (+) and (-) strands of satLTSV were found to be equally infectious.[11] Nevertheless, since only the (+) strand is packaged in the LTSV particles, the origin of assembly sequence (OAS) / secondary structure is assumed to be present on the (+) strand only.

Gellatly et al., 2011 demonstrated that the entire satLTSV molecule possesses sequence and structural significance wherein any mutations (insertions / deletions) causing disruption in the overall rod-like structure of the virusoid molecule are lethal to its infectivity.[11] Foreign nucleotides introduced into the molecule will only be tolerated if they preserve the overall cruciform structure of the satLTSV. Furthermore, the introduced foreign sequences are eliminated in successive generations to ultimately reproduce the wild-type satLTSV.

Therefore, in satLTSV RNA, the entire sequence seems to be essential for replication. This contrasts with satRNA of TBSV or the defective-interfering RNAs,[12] in which only a small portion of their respective sequences / secondary structures were found to be sufficient for replication.

Role of ribozyme structures in the self-cleavage and replication of virusoids

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Virusoids structurally resemble the viroids as they possess native secondary structures that form double-stranded rod-like molecules with short terminal branches.[13][14] They also contain hammerhead ribozymes that are involved in autocatalytic cleavage of satRNA multimers during rolling circle replication.[1] It was proposed that the hammerhead ribozyme structure of satLTSV is formed only transiently, similar to that observed by Song & Miller (2004) with satRPV (Cereal yellow dwarf polerovirus serotype RPV) RNA.[15] This hammerhead structure contains a short stem III that is stabilized by only two base-paired nucleotides. This unstable conformation thus suggests that a double hammerhead mode of cleavage takes place. These structures are similar to those reported for CarSV and newt ribozymes,[16][17] which implies an ancient relationship between these divergent RNAs. The observation by Collins et al., 1998 that the dimer of the satRYMV RNA is more efficiently self-cleaved than the monomer is consistent with the double hammerhead mode of cleavage. The self-cleavage of the satRYMV in the (+) strand and not in the (-) strand implies that the satRYMV replicates through an asymmetric mode of rolling circle replication, similar to other sobemoviral satellites with the exception of satLTSV.[18]

Evolutionary origin

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A group I intron (PDB: 1grz​)

Considering properties such as their diminutive size, circular structure and the presence of hammerhead ribozymes, viroids may have had an ancient evolutionary origin distinct from that of the viruses. Likewise, the lack of any sequence similarity between the satellite RNAs and their host viruses, host plants and insect vectors implies that these satellite RNAs have had a spontaneous origin. Alternatively, the siRNAs and microRNAs generated during viral infections may have been amplified by helper virus replicases, whereby these molecules assembled to form satellite RNAs.

Virusoids and viroids have been compared to circular introns due to their size similarity. It has been proposed that virusoids and viroids originated from introns.[19][20] Comparisons have been made between the (-) strand of viroids and the U1 small nuclear ribonucleoprotein particle (snRNPs), implicating that viroids could be escaped introns.[19][20][21][22] Dickson (1981) also observed such homologies within both the (+) and (-) strands of viroids and virusoids.[23] In particular, virusoids and viroids exhibit several structural and sequence homologies to the group I introns such as the self-splicing intron of Tetrahymena thermophila.

A phylogeny based on a manually-adjusted alignment in 2001 suggests that virusoids may form a clade of their own as a sister group to Avsunviroidae, which also possess hammerhead ribozymes. However, the said alignment is not available, making the results hard to reproduce.[24]

Virusoids and other circular RNAs are ancient molecules that are being explored with renewed interest.[25][26] Circular RNAs have been shown to possess a number of functions, ranging from modulation of gene expression, interactions with RNA binding proteins (RBPs) acting as miRNA sponges and have been linked to a number of human diseases, including aging and cancer.[27][28]

Developments

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Abouhaidar et al., 2014 demonstrated the only example of protein translation and messenger RNA activity in the Rice yellow mottle virus small circular satellite RNA (scRYMV).[29][30] This group suggested that the scRYMV be designated as a virusoid satelliteRNA that could serve as a model system for both translation and replication.

The most promising application of these subviral agents is to make specific vectors that can be used for the future development of biological control agents for plant viral diseases. The vector system could be applied for the overexpression and silencing of foreign genes. The unique example of a foreign expression vector is Bamboo mosaic virus satellite RNA (satBaMV),[31] which possesses an open reading frame that encodes a 20-kDa P20 protein. It was observed that when this nonessential ORF region was replaced with a foreign gene, expression of the foreign gene was enhanced or overexpressed.[31] In the case of gene silencing, various satellite RNA-based vectors can be used for sequence-specific inactivation. Satellite Tobacco Mosaic Virus (STMV) was the first subviral agent to be developed as a satellite virus-induced silencing system (SVISS).[32]

References

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Virusoid

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A virusoid is a subviral infectious agent composed of a small, circular, single-stranded , typically 220–450 in length, that typically lacks protein-coding capacity, though some, such as the satellite RNA of rice yellow mottle , encode small proteins, and requires a helper for both replication and transmission to host cells. Unlike viroids, which are autonomous pathogens that replicate using host , virusoids are RNAs dependent on the replicase provided by their associated helper , such as members of the sobemovirus or luteovirus families, and are often encapsidated within the helper virus's protein coat./06%3A_Acellular_Pathogens/6.04%3A_Viroids_Virusoids_and_Prions) First identified in 1981 by J. W. Randles and colleagues in infected with velvet mottle , virusoids primarily infect and can modulate the symptoms of the diseases caused by their helper viruses, sometimes attenuating or exacerbating effects on crops like subterranean clover or . Virusoids replicate via a rolling-circle mechanism similar to viroids, producing multimeric RNA intermediates that are processed by self-cleaving ribozymes into unit-length monomers, but this process relies entirely on the helper virus's machinery rather than host enzymes. Their RNA genomes exhibit rod-like secondary structures with high base-pairing, though less stable than those of viroids due to fewer extra-stable hairpins, enabling efficient packaging and transmission within the helper virion. Notable examples include the virusoid associated with subterranean clover mottle virus, which causes mild symptoms in legumes, and the satellite RNA of lucerne transient streak virus, which can influence viral pathogenesis in forage crops./06%3A_Acellular_Pathogens/6.04%3A_Viroids_Virusoids_and_Prions) Although primarily studied in plant virology, virusoids highlight the diversity of -based pathogens and their evolutionary links to viroids and satellite viruses, contributing to broader understanding of RNA folding, replication strategies, and subviral dependencies in infectious diseases. Their discovery expanded the known spectrum of acellular pathogens, revealing how small RNAs can parasitize cycles, typically without encoding their own proteins, though exceptions exist.

Overview and Characteristics

Definition and Key Features

Virusoids are small, circular, single-stranded molecules, typically ranging from 220 to 388 in length, that function as infectious RNAs but lack autonomous replication capability, instead relying on helper viruses for both replication and packaging into virions. These RNAs are covalently closed, forming a stable circular structure without free ends, which enhances their resistance to exonucleases and contributes to their in host plants. Unlike autonomous pathogens, virusoids do not encode any proteins and exert their effects through RNA-based mechanisms, such as modulating host responses or interfering with helper virus replication. A defining feature of virusoids is their encapsidation within the proteins of helper viruses, which facilitates their transmission and protection during infection, setting them apart from non-encapsidated agents like viroids. This packaging occurs in association with specific viruses, particularly sobemoviruses, where the virusoid is incorporated into the viral particles alongside the helper . Virusoids exhibit a high guanine-cytosine (, often exceeding 50%, which promotes extensive base-pairing and results in a compact, rod-like secondary that confers thermodynamic stability. This rod-like conformation, with 66-73% of involved in base-pairing, is crucial for maintaining the integrity of the during replication and infection cycles. Representative examples of virusoid genomes include those associated with sobemoviruses, such as the 366-nucleotide virusoid of velvet tobacco mottle virus (VTMoV) and the 324-nucleotide virusoid of lucerne transient streak virus (LTSV), both of which demonstrate the typical size range of 220-388 nucleotides and dependency on their respective helper viruses. These structures highlight the virusoid's role as parasitic elements that exploit viral machinery without contributing coding capacity, emphasizing their evolutionary adaptation as minimalistic replicators in plant pathosystems.

Distinctions from Viroids and Other Satellite RNAs

Virusoids differ from viroids primarily in their encapsidation and replication dependencies. While viroids are naked, circular single-stranded RNAs that replicate autonomously using host RNA polymerases, virusoids are encapsidated within the coat proteins of helper viruses and require the helper virus's for replication. This encapsidation provides virusoids with enhanced stability and transmissibility, contrasting the unprotected nature of viroids, which rely solely on host cellular machinery in the nucleus or chloroplasts. In comparison to linear satellite RNAs, virusoids are distinguished by their circular structure and possession of self-cleaving , such as hammerhead motifs, which facilitate into monomeric units without encoding any proteins. Linear satellite RNAs, often non-circular and varying in size, depend on helper viruses for both replication and packaging but lack these autonomous cleavage mechanisms, making them more susceptible to degradation. This circularity and ribozyme activity confer greater structural integrity to virusoids, enabling efficient rolling-circle replication mediated by the helper virus. Virusoids also contrast with satellite viruses, which encode their own proteins despite relying on helper viruses for replication functions. In contrast, virusoids completely lack coding capacity for structural proteins and are fully dependent on the helper virus for both replication and encapsidation, positioning them as non-autonomous satellite RNAs rather than minimal viruses. The classification of virus (HDV) presents some overlap and debate with virusoids. HDV, a ~1700 circular RNA, exhibits viroid-like features including activity and host usage but is larger, encodes a delta antigen for partial autonomy, and requires for envelopment, leading to its occasional description as a virusoid-like satellite agent.
FeatureVirusoidsViroidsLinear Satellite RNAsSatellite Viruses
Size (nt)220–388246–401200–1500800–1500
Circular, non-coding ssRNACircular, non-coding ssRNALinear, often non-coding ssRNALinear, coding ssRNA
Replication EnzymesHelper virus RdRpHost Helper virus RdRpHelper virus RdRp
EncapsidationBy helper virus coat proteinNone (naked)By helper virusOwn protein
Host Range (e.g., with sobemoviruses) (e.g., , ) (e.g., with cucumoviruses) (e.g., with necrotic viruses)

History and Discovery

Initial Identification

The initial discovery of virusoids occurred in when J. W. Randles and colleagues identified an unusual viroid-like in plants of Nicotiana velutina infected with the R2 strain of velvet tobacco mottle (VTMoV). This was detected during purification of VTMoV particles from infected tissue, revealing a previously unknown subviral component alongside the 's primary genomic RNAs. Characterization of the RNA involved extraction from viral capsids followed by separation via , which estimated its molecular weight at approximately 1.2 × 10⁵ daltons, corresponding to about 366 . confirmed the presence of the RNA within isometric viral particles approximately 30 nm in diameter and further supported its covalently closed circular structure, a feature shared with but rare among viral RNAs. Initially, the circular form led to confusion with , as it exhibited similar resistance and thermal denaturation profiles; however, it was distinctly encapsidated within VTMoV virions, unlike naked viroid RNAs. This encapsidation marked it as a novel subviral agent, termed a "viroid-like RNA," and was detailed in a seminal 1981 publication in . Early infectivity studies demonstrated that the viroid-like RNA could not replicate independently and required co-inoculation with VTMoV genomic ( 1) for systemic in host plants such as Nicotiana clevelandii. This dependency highlighted its nature, reliant on the helper for replication and transmission, further distinguishing it from autonomous viroids. The specificity of this interaction was evident, as the RNA failed to replicate with unrelated viroids or viral RNAs, underscoring the unique association with VTMoV.

Major Milestones and Subsequent Discoveries

In the , following the initial identification of virusoids, researchers reported their presence in several sobemoviruses, including transient streak virus (LTSV) and velvet mottle virus (VTMoV), where circular satellite RNAs were characterized as encapsidated, viroid-like entities dependent on helper for replication. These findings expanded the known diversity of virusoids within the Sobemovirus genus, with subsequent isolations in subterranean clover mottle virus (SCMV) confirming their structural similarities to viroids, such as circularity and lack of coding capacity. Although Southern mosaic virus (SBMV), the of sobemoviruses, supports replication of heterologous virusoids like those from LTSV, native virusoids were not directly associated with it during this period. During the 1990s, a key advancement came with the recognition of hammerhead ribozymes in virusoid RNAs, enabling self-processing and linking their replication to autonomous cleavage mechanisms. A 1990 study on VTMoV virusoids demonstrated that plus-strand RNAs formed hammerhead structures facilitating site-specific self-cleavage, a process essential for generating monomeric units during rolling-circle replication. This discovery paralleled earlier observations in 1987, where both polarities of VTMoV and nodiflorum mottle virus (SNMV) virusoids exhibited self-cleavage activity, solidifying the functional analogy to viroids. Later in the decade, a virusoid was identified in rice yellow mottle virus (RYMV), the smallest known at 220 , further highlighting the structural conservation of hammerhead motifs across sobemovirus-associated RNAs. The 2000s saw virusoids contextualized within broader evolutionary frameworks, including comparisons to animal pathogens. Hepatitis delta virus (HDV), with its circular RNA genome and ribozyme-mediated processing, emerged as an animal-derived analog to plant virusoids, sharing viroid-like replication strategies despite requiring hepatitis B virus as a helper. A 2001 phylogenetic analysis reassessed relationships among viroids, virusoids, and HDV, revealing shared rod-like secondary structures and suggesting ancient common origins through sequence covariation in functional domains. Post-2014 research has been relatively sparse for virusoids specifically but has advanced understanding of RNA dynamics, including folding and evolution. A 2016 review by the American Phytopathological Society emphasized how virusoids modulate helper virus pathogenicity, often attenuating symptoms in sobemovirus infections through interference with replication or host responses. Recent studies from 2022–2024 have explored RNA folding pathways, noting that viroid-like agents, including virusoids, achieve kinetic control over secondary structures to evade host and facilitate replication. These works highlight ongoing evolutionary pressures on circular RNAs, with virusoid-specific investigations underscoring their stability in diverse sobemovirus hosts.

Molecular Structure

Genomic Composition

Virusoids are composed of single-stranded, positive-sense RNA molecules that form a covalently closed circular genome, distinguishing them from linear satellite RNAs. This circular configuration arises without the need for their own protein capsids and enables replication that depends on helper viruses. The genome adopts a rod-like secondary structure due to extensive intramolecular base-pairing, resulting in a highly compact fold that spans much of the RNA length. Genome sizes exhibit variability, with virusoids typically ranging from 220 to 450 nucleotides. Sequence features typically include elevated GC content (around 55–70%), which stabilizes the secondary structure, generally minimal open reading frames with limited or no protein-coding capacity, and conserved motifs enabling recognition by the helper virus's RNA-dependent RNA polymerase for replication. However, recent studies have identified exceptions, such as a 220-nucleotide circular RNA associated with rice yellow mottle virus that encodes small proteins via novel translation mechanisms. These elements ensure the virusoid's dependence on the helper for propagation without encoding their own replicative machinery. The and rod-like folding confer resistance to host exonucleases, promoting genomic in infected cells. Electron microscopy visualizes these structures as slender rods approximately 50 nm long, reflecting their base-paired conformation, while reverse transcription PCR (RT-PCR) followed by sequencing provides detailed nucleotide-level characterization. Some virusoid genomes incorporate elements for self-processing during replication.

Ribozyme Structures and Functions

Virusoids frequently incorporate the motif, a conserved catalytic sequence of approximately 50 that enables site-specific self-cleavage without requiring protein enzymes. This motif is embedded within the genome and facilitates the processing of replication intermediates. The of the hammerhead in virusoids consists of a formed by three helical stems (I, II, and III) connected by a conserved catalytic core of 15 . Stem I pairs with the substrate region upstream of the cleavage site, stem II forms the catalytic domain, and stem III provides stability; the overall fold adopts a Y-shaped tertiary stabilized by non-canonical base pairs. Catalysis is magnesium-dependent, with Mg²⁺ ions coordinating the 2'-hydroxyl nucleophile and facilitating the reaction that generates a 2',3'-cyclic and a 5'-hydroxyl terminus. In virusoids, the primary function of the hammerhead is to cleave multimeric replication intermediates produced via a rolling-circle mechanism into unit-length monomeric RNAs, enabling circularization and subsequent or further replication. Self-cleavage occurs specifically at the N+17/GUX triplet (where N is any and X is A, C, or U), ensuring precise processing of the plus-strand . This ribozyme activity integrates into the broader replication cycle by processing linear multimers immediately after transcription. Prominent examples include the virusoids associated with velvet tobacco mottle virus (VTMoV) and the satellite RNA of southern bean mosaic virus (satSBMV), both of which harbor hammerhead ribozymes in their plus strands. These motifs exhibit evolutionary conservation across viral isolates, with the core sequence and helical stems maintaining functional integrity despite sequence variations in flanking regions. Experimental evidence for activity derives from cleavage assays using synthetic transcripts of virusoid RNAs, which demonstrate rapid self-processing under physiological conditions ( 7.0–8.0, 37–50°C, and 5–10 mM Mg²⁺). For instance, dimeric transcripts of the sTRSV virusoid (a model for hammerhead-containing satellites) undergo complete cleavage to monomers during or shortly after transcription, confirming the motif's autonomy and efficiency. Similar assays with VTMoV and satSBMV transcripts validate the conservation and magnesium requirement, with cleavage rates approaching 1 min⁻¹ at neutral .

Replication and Life Cycle

Mechanism of Replication

Virusoids replicate via a rolling circle mechanism that relies on the (RdRp) provided by their helper . The process initiates with the circular plus-strand serving as a template, where the helper RdRp synthesizes a complementary negative-strand , resulting in the production of tandem multimeric negative strands. These linear multimers can extend to several unit lengths, enabling efficient amplification of the . The negative-strand multimers then act as templates for the synthesis of plus-strand RNAs by the same RdRp, generating complementary tandem plus-strand multimers. These multimeric intermediates are processed through ribozyme-mediated self-cleavage, typically by hammerhead ribozymes present in the plus strands, which precisely cleave the concatemers at specific sites to produce monomeric linear RNAs with defined termini. Subsequent ligation, facilitated by host or helper virus-associated enzymes, circularizes these monomers to yield mature infectious circles. Replication involves a negative-sense intermediate, with the plus-strand functioning as the primary infectious form that predominates in infected cells. Efficiency of the process is supported by promoter-like sequences within the virusoid that resemble the 3' terminal regions of the helper genome, aiding in RdRp recruitment and initiation of transcription. In virusoids, such as those associated with sobemoviruses, this mechanism ensures robust replication dependent on the helper's enzymatic machinery. In vivo evidence for this replication strategy comes from analyses of infected plant tissues, where Northern blot hybridization has revealed the accumulation of monomeric circular forms alongside dimeric and higher-order multimeric linear RNAs of both polarities, confirming the presence of rolling circle intermediates during active infection. For instance, in rice plants co-infected with rice yellow mottle virus and its associated virusoid, such blots detect these species, underscoring the dynamic processing and amplification steps.

Role of Helper Viruses

Virusoids are obligate parasites of their associated helper viruses, relying on them for essential functions that enable their replication, encapsidation, and dissemination within host cells and organisms. Specifically, virusoids lack the genetic capacity to encode their own (RdRp), necessitating the provision of this enzyme from the helper virus to facilitate their rolling-circle replication mechanism. Additionally, virusoids depend on the helper virus's coat proteins for encapsidation, which protects the genome and allows for efficient cell-to-cell movement and systemic spread. In plant systems, virusoids are commonly associated with sobemoviruses such as rice yellow mottle virus (RYMV), where the helper virus supplies both the RdRp for replication and structural proteins for packaging the 220-nucleotide circular satellite . Although traditionally considered non-coding, the RYMV-associated virusoid encodes a 16 kDa protein via shunting, representing an exception to the typical lack of protein-coding capacity. These associations highlight the virusoids' inability to independently infect hosts or propagate. Transmission of virusoids occurs through co-packaging within the helper virus's virions, allowing mechanical inoculation or vector-mediated delivery to new host tissues or without separate dissemination mechanisms. This symbiotic relationship can influence the helper virus's ; virusoids may attenuate symptoms by competing for replication resources, reducing helper virus , or enhance severity by altering host responses, depending on the specific virusoid-helper pair and genotype. Experimental studies have demonstrated the critical dependence on helpers: inoculation of purified virusoid RNA alone results in no detectable replication or accumulation in host cells, whereas co-inoculation with the corresponding helper virus leads to robust RNA synthesis and packaging, confirming the absence of autonomous infectivity.

Examples and Diversity

Virusoids in Plant Viruses

Virusoids associated with plant viruses represent a distinct class of subviral agents that are encapsidated by their helper viruses and rely on them for replication, transmission, and movement within the host. These entities are typically small, circular, single-stranded RNAs lacking coding capacity, ranging from 220 to approximately 390 nucleotides in length, and often feature self-cleaving ribozyme structures such as hammerhead motifs that process multimeric replication intermediates into unit-length monomers. In plants, virusoids are predominantly linked to members of the genus Sobemovirus (family Solemoviridae), where they can attenuate or exacerbate disease symptoms, influencing agricultural productivity in key crop families. One of the earliest identified oids is the viroid-like associated with velvet mottle (VTMoV), a sobemovirus first isolated from wild Nicotiana velutina in arid regions of in 1981. This circular satellite , known as vVTMoV, consists of 366 and is encapsidated within VTMoV particles alongside the viral genomic . In experimental infections of (), the presence of vVTMoV contributes to mild symptoms characterized by chlorotic mottling of leaves, though the virusoid itself does not independently cause disease. The VTMoV helper provides the (RdRp) essential for vVTMoV replication via a rolling-circle mechanism. Another notable example is the satellite RNA of southern bean mosaic virus (SBMV), a sobemovirus that infects legumes such as common bean (Phaseolus vulgaris). The satSBMV, a circular RNA of approximately 391 nucleotides, is encapsidated by SBMV and modulates the severity of mosaic and mottle symptoms in infected beans, often attenuating disease expression to milder forms compared to infections with the helper virus alone. This interaction highlights the regulatory role of virusoids in host-virus dynamics within the Fabaceae family, a major agricultural group. The satellite RNA associated with rice yellow mottle virus (RYMV), prevalent in African rice (Oryza sativa) crops, exemplifies the smallest known plant virusoid at 220 . This covalently closed (satRYMV) contains hammerhead ribozymes in both polarities, enabling site-specific self-cleavage essential for its replication and processing. Found in RYMV-infected plants in , satRYMV is encapsidated by the helper virus and can reduce symptom severity, such as yellow mottling and stunting, thereby influencing yield losses in staple crops. The of transient streak virus (LTSV), which affects (Medicago sativa) and other , is a small of 322 that depends on LTSV—a with sobemovirus affinities—for replication and spread. This alters streak symptoms in infected hosts, typically intensifying transient chlorotic streaking on leaves and stems, and is encapsidated in isometric particles similar to those of its helper. Discovered in Australian plants, it underscores the impact of virusoids on forage crop health. Overall, plant virusoids are distributed primarily among viruses infecting the (e.g., RYMV in ) and (e.g., SBMV in beans, LTSV in ) families, posing challenges to global through altered disease dynamics in and production systems. These associations emphasize the parasitic nature of virusoids, which exploit sobemovirus RdRp for replication while potentially mitigating or enhancing host damage.

Virusoids in Animal Viruses

The hepatitis delta virus (HDV) represents the primary example of a virusoid-like agent in animal hosts, characterized by its single-stranded, circular genome of approximately 1679–1700 in length. Unlike typical plant virusoids, HDV requires the (HBV) for provision of envelope proteins to facilitate its packaging and transmission, establishing it as a dependent on a helper virus. This genome exhibits a rod-like structure due to extensive base pairing, enabling efficient replication within infected hepatocytes. A distinctive feature of HDV is its self-cleaving , which is structurally and mechanistically distinct from the hammerhead ribozyme found in many virusoids. The HDV , present in both genomic and antigenomic , catalyzes site-specific cleavage at a cytidylate-adenylate bond, facilitating processing of multimeric RNA intermediates into unit-length monomers during replication. This ribozyme activity was first demonstrated for the antigenomic RNA, highlighting its role in the virus's rolling-circle replication mechanism without reliance on host or viral proteins for cleavage. In terms of , HDV can establish infection through either with HBV, where both viruses are acquired simultaneously, or of an individual already chronically infected with HBV, leading to accelerated liver damage. often results in more severe outcomes, including in up to 20% of cases and a significantly higher risk of progression to or compared to HBV alone. , while typically self-limiting, can still cause acute with elevated mortality in certain populations. HDV's uniqueness among virusoid-like entities lies in its larger genome size relative to non-coding plant virusoids and its encoding of the delta antigen (HDAg), a multifunctional protein essential for replication and assembly, which blurs the distinction between true virusoids and viroid-like satellites. The small (S-HDAg) and large (L-HDAg) isoforms of this regulate RNA synthesis and interact with HBV envelope proteins for virion production, respectively. Recent studies have identified HDV-like circular RNAs in diverse animal , including , birds, amphibians, and , which can propagate using helper viruses other than HBV, such as unrelated flaviviruses, indicating a broader ecological distribution of these viroid-like agents as of 2023.

Evolutionary Origins

Proposed Origins

One prominent hypothesis posits that virusoids originated from self-splicing group I , retaining key features such as circularity and activity that enable autonomous processing and replication. This idea stems from observed sequence and structural similarities between virusoids, viroids, and group I introns, including a conserved 16-nucleotide and complementary elements arranged in a comparable 5'-to-3' order. For instance, the exhibits homology in its D stem and pathogenicity regions with the group I intron, suggesting virusoids may represent "escaped" introns from organellar genomes that adapted to a viral lifestyle. Another proposed origin links virusoids to viroid-like evolution from primordial RNAs in the pre-cellular , where self-replicating RNA molecules predominated before the emergence of DNA-based life. Phylogenetic analyses of viroids and viroid-like satellite RNAs, including virusoids, support a monophyletic ancestry consistent with these entities as "living fossils" of such an ancient RNA replicator phase. This timeline aligns with the hypothesized era, approximately 3.5 to 4.0 billion years ago, during which simple RNA structures could have evolved catalytic capabilities without protein assistance. A third suggests virusoids arose through acquisition from host genomes, akin to transposon-like elements co-opted by viruses, based on structural parallels such as rod-like conformations with extensive base-pairing, inverted repeats, and flanking direct repeats. These features mirror those of transposable elements and retroviral proviruses, implying that deletions in host-derived sequences could have generated compact, mobile circles dependent on viral helpers for propagation. Supporting evidence for these independent origins includes the lack of significant between virusoids and their helper viruses, indicating that virusoids did not derive directly from viral genomes but rather evolved separately before associating with them for replication and transmission. This autonomy in sequence, coupled with conserved structures for self-cleavage, underscores virusoids' distinct evolutionary path.

Phylogenetic and Evolutionary Relationships

Virusoids are classified by the International Committee on Taxonomy of Viruses (ICTV) as a subset of satellite RNAs, specifically those that form covalently closed circular molecules, distinguishing them from linear satellite RNAs that require helper viruses for replication and packaging. This grouping emphasizes their dependence on helper viruses, primarily plant sobemoviruses, such as those associated with velvet tobacco mottle virus (VTMoV) and southern mosaic virus (SCPMV, formerly the cowpea strain of SBMV), while highlighting structural similarities to viroids in their circularity and lack of protein-coding capacity. Subgroups within satellite RNAs include these circular virusoids, reflecting their viroid-like features but with distinct replication dependencies. Sequence analyses reveal low overall conservation among virusoids, with divergence driven by non-coding regions, though cores—often hammerhead motifs—show higher similarity essential for self-cleavage during replication. A 2001 phylogenetic reassessment using manually adjusted multiple alignments demonstrated clustering of VTMoV-associated virusoids with certain groups, while SCPMV-associated forms grouped separately among viroid-like satellite RNAs, supported by bootstrap values and likelihood mapping that accounted for insertions, deletions, and rearrangements. This low global similarity (typically <50% identity) underscores the challenges in reconstructing deep evolutionary histories, yet local structural alignments confirm functional constraints on catalytic domains. Virusoids share circular genomes with viroids, suggesting a possible common ancestral RNA element adapted for autonomous replication in viroids versus helper-virus dependence in virusoids, though they utilize distinct RNA polymerases—host Pol II for viroids versus viral RdRp for virusoids. Phylogenetic reconstructions support a monophyletic origin for these subviral agents, with virusoids branching as a derived group within viroid-like RNAs, potentially arising from recombination events between viroid progenitors and viral elements. In contrast, the hepatitis delta virus (HDV) ribozyme, a self-cleaving motif in an animal satellite-like RNA, exhibits no sequence or structural homology to the hammerhead ribozymes prevalent in plant virusoids, indicating convergent evolution toward similar phosphodiester bond cleavage functions despite independent origins. This divergence highlights parallel selective pressures for RNA circularization and autocatalytic processing across eukaryotic hosts. Recent metatranscriptome mining has expanded the known diversity of viroid-like agents, including virusoid satellites, revealing modular evolutionary patterns where ribozyme domains and non-coding loops recombine independently, fostering rapid adaptation and host range expansion in plant-associated circular RNAs. A 2024 study identified novel circular RNA agents with mosaic architectures, showing virusoid satellites cluster with viroid subgroups based on shared modular elements like hammerhead variants, while exhibiting higher sequence divergence in flanking regions (>70% variability), supporting a model of evolution through template switching during replication.

Biological Impact and Applications

Pathogenicity and Host Interactions

Virusoids, as non-coding satellite RNAs, generally exhibit limited direct pathogenicity but modulate severity in their hosts through interactions with helper viruses and host cellular machinery. In hosts, virusoids often attenuate the symptoms induced by the helper virus by competing for replication resources, such as host polymerases and viral replicases, thereby reducing and associated tissue damage. For instance, certain satellite RNAs of sobemoviruses can lessen severe in infected , promoting milder symptoms. Similarly, some virusoids exacerbate symptoms; the virusoid of Velvet mottle virus (VTMoV) intensifies chlorotic lesions in leaves compared to infections without the virusoid. Satellite viruses analogous to virusoids, such as hepatitis delta virus (HDV) dependent on , can exacerbate in humans, leading to acute with , , and potential hepatic failure. HDV's pathogenicity arises from interference with host and immune evasion, resulting in more severe chronic than HBV alone. Host range for virusoids is restricted to plants, where systemic spread occurs cell-to-cell via plasmodesmata facilitated by the helper virus movement protein, and long-distance through the . HDV spreads systemically in humans via the bloodstream, using HBV envelope proteins for entry. Virusoid-host interactions primarily involve RNA-based mechanisms rather than protein effectors, as these agents lack coding capacity. A key process is the induction of , where virusoid-derived small interfering RNAs (siRNAs) target and silence host genes involved in defense or development, contributing to symptom expression such as altered growth or pigmentation. This silencing can suppress host antiviral responses or compete with helper virus suppressors of , indirectly modulating pathogenicity without direct enzymatic activity. Overall, these interactions highlight virusoids' role in fine-tuning disease outcomes through resource competition and regulatory effects.

Research Developments and Biotechnological Uses

Research on virusoids has advanced significantly since the , particularly in understanding their folding and replication for . Circular satellite RNAs, including virusoids, have been explored as stable scaffolds for RNA-based tools due to their viroid-like structures. A 2024 review in Viruses details how thermodynamically controlled folding of virusoid and satellite RNAs enables replication without protein coding, informing designs for synthetic circular RNAs. These principles have aided development of circular guide RNAs for , enhancing stability and reducing off-target effects in plant antiviral systems, similar to Cas13-mediated interference. In plant , virusoid-inspired satellite RNAs have been adapted for virus-induced (VIGS). For example, modified circular satellite RNAs of sobemoviruses serve as vectors to target endogenous genes in monocots and dicots, competing with helper viruses to suppress without severe symptoms. As of 2025, advances in lipid nanoparticle (LNP) formulations have improved delivery of circular RNAs, including virusoid-derived elements, achieving sustained expression by protecting against degradation—techniques shown in optimized LNPs for circRNA vaccines. These efforts support scalable production for agricultural resistance, such as against sobemovirus infections. Recent 2025 studies further elucidate virusoid silencing, showing chloroplast-replicating -like agents induce siRNAs that regulate host responses, bridging and virusoid mechanisms for enhanced antiviral strategies. Despite advances, challenges include virusoid instability in non-plant systems due to reliance on specific polymerases, limiting persistence outside . Ongoing preclinical work on synthetic ribozymes and siRNAs modeled on virusoid structures incorporates stabilizing modifications to improve delivery and reduce immune recognition, though clinical is constrained by .

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