What type of genome is carried by ambisense viruses

Here we consider a novel gene identified in viral genomes in opposite direction, as positive in influenza and negative in coronaviruses, suggesting an ambisense genome strategy for both virus families.

Noteworthy, the identified novel genes colocolized in the same RNA regions of viral genomes, where the previously known opposite genes are encoded, a so-called ambisense stacking architecture of genes in virus genome. It seems likely, that ambisense gene stacking in influenza and coronavirus families significantly increases genetic potential and virus diversity to extend virus-host adaptation pathways in nature.

These data imply that ambisense viruses may have a multivirion mechanism, like "a dark side of the Moon", allowing production of the heterogeneous population of virions expressed through positive and negative sense genome strategies.

Core Tip: A novel genes identified in viral genomes in opposite direction, as positive in influenza and negative in coronaviruses, are considered.

The identified novel genes colocolized in the same RNA regions of viral genomes, where the previously known opposite genes are encoded, a so-called ambisense stacking architecture of genes in virus genome. Orthomyxo- and coronaviruses are two families of enveloped viruses containing single stranded linear RNA genomes. Orthomyxovirus family includes seven genera: Alphainfluenzavirus, Betainfluenzavirus, Deltainfluenzavirus, Gammainfluenzavirus, Isavirus, Thogotovirus, and Quaranjavirus.

These viruses infect wide range of hosts including mammals, birds, rodents, fish, ticks and mosquitoes. Orthomyxoviridae viruses contain six to eight segments of negative-sense single stranded RNA with a total genome length of Kb[ 1 ].

Coronaviridae is divided into the four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Deltacoronavirus. Alpha- and betacoronaviruses infect mammals, while gamma- and deltacoronaviruses primarily infect birds.

The size of genomic positive sense RNA of coronaviruses ranges from 26 to 32 kilobases, one of the largest genome among RNA viruses[ 2 ]. Here we mainly consider alphainfluenza viruses and betacoronaviruses as a typical members in both families. Genome of influenza A viruses is composed of 8 segments of single-stranded RNAs with mol.

Each segment encodes one or several unique polypeptides through the canonical negative sense genome strategy Table 1. It means that genome RNA of negative sense polarity is transcribed by the virus polymerase to produce positive sense mRNAs, which recognized by ribosomes to translate individual viral proteins Figure 1.

In addition to the negative sense genes, influenza A virus genome segments were found to contain long open reading frames ORFs, genes in opposite positive sense orientation. There are three groups of data showing in vivo expression potential of these negative stranded genes. Later, it was also supported that influenza A virus NSP8 could be efficiently expressed from either a plasmid or a recombinant vaccinia virus in mammalian cells and the synthetized NSP8 was localized in the perinuclear endoplasmic reticulum ER and post-ER cellular compartments[ 12 ]; and 3 There are data that mice infected with influenza virus produce CTL response specific to epitopes presented in the influenza NSP8 protein[ 12 - 14 ].

These findings also demonstrate that translation of sequences locating on the negative RNA strand of a single-stranded RNA genome of influenza A virus can develop in vivo and can initiate antiviral CTL response and immunosurveillance. The mature product of the NSP8 gene has not been yet identified in biological systems such virus-infected cells and animals. The failure to detect NEG8 protein could be due to a number of factors other than the complete absence of translation from genomic RNA.

It would not be surprising if negative polarity genes are only expressed physiologically under special circumstances in vivo determining host cell tropism of influenza viruses. Recently, similar ambisense polarity has been revealed in coronaviruses genomes[ 15 ]. The coronavirus genome RNA contains two groups of genes expressing proteins through the positive sense strategy. In addition to the positive sense genes, we have identified numerous long open reading frames in negative sense orientation Table 2 ; Figure 2B.

Like in the case of the ambisense genes of flu viruses, coronavirus negative sense genes have all elements characteristic of the mRNA molecules which are recognized by host ribosomes: classical AUG or alternative CUG[ 17 ] start codons, termination codons, IRES, and Kozak-like sequences at the start area[ 18 , 19 ]. However, unlike to influenza A viruses, coronavirus ambisense polarity has opposite configuration: a positive sense genome strategy and a negative sense orientation of the novel negative sense genes, so called a negative sense genes or negative gene proteins NGPs.

The identification of coronavirus negative-polarity genes implies two possible mechanisms of their expression and synthesis of the corresponding mRNAs and proteins. To realize pathway I coronavirus genome contains poly A sequence positions nt functioning as a viral polymerase binding site and transcrip tion initiation signal Figure 2B. The function and role of the newly discovered ambipolar viral genes have not yet been determined. The possible functional significance of the novel ambisense genes is not yet generally clear.

The read-through process requires at least two elements. Second, the nucleotide context surrounding the termination codon and in particular the two downstream codons appear important for readthrough of TMV RNA in vivo Skuzeski et al. Ribosomal frameshifting is a strategy frequently employed by various organisms to produce more than one protein from overlapping reading frames.

It may occur in either direction. For a number of retroviruses, heptanucleotide signals are involved as "slippery sequences" in the frameshift. In addition to the specific sequence signal a second type of information bears relevance to frameshifting Hatfield and Oroszlan, : stem-loop structures immediately downstream of the "slippery sequences" have significant influence on the efficiency of the frameshift event. The viral genome of PLRV consists of 5. An intergenic region located in the centre of the RNA genome separates a 5' cluster of genes ORFs 1, 2a and 2b , which are divergent among the luteoviruses sequenced so far, from a highly conserved gene block ORFs 3, 4 and 5 in the 3' half.

The signal responsible for efficient frameshift in PLRV is composed of the slippery sequence UUUAAAU followed by a sequence that has the potential to adopt two alternative folding patterns, either a structure involving a pseudoknot, or a simple stem-loop structure. Kujawa et al. A mechanism whereby the 5' cistron was bypassed by ribosomes has been suggested for Agronowski , cited by Hull , for the expression of the beet yellows closterovirus K cistron, which is downstream of a 6.

The latter cistron has no internal AUG codons in any of the reading frames, from where the expression of the K cistron could be initiated.

Recently, Grieco et al. Three other mechanisms will be briefly mentioned here to explain the expression strategies used by caulimoflower mosaic virus CaMV , a dsDNA virus that, to express the different cistrons, transcribes their genome into two RNA species, the polycistronic 35S and the monocistronic 19S Rothnie et al.

Studies carried out to analyse the translation of the CaMV in protoplasts suggested a mechanism by which ribosomes enter at the cap site as normal and begin scanning. But at some point near the 5' end of the leader, they are somehow transferred to a region at the 3' end of the leader, without scanning linearly through the central portion of the leader. This process has been termed "ribosome shunt" and the sites between which it occurs have been defined Futterer et al.

The shunt mechanism has been observed to operate between two separate RNA molecules, thus reinforcing the fact that the ribosomes are transferred directly from one part of the leader to another without scanning the sequence in between Rothnie et al. TAV is a complex protein that appears to be involved in many aspects of the virus life cycle De Tapia et al. It has been shown to be part of abundant vacuolated inclusion bodies as well as determinant of host specificity and a factor influencing symptom development in infected plants Broglio, ; De Tapia et al.

Translational trans-activation has been demonstrated for CaMV and figwort mosaic virus Bonneville et al. Like the "ribosome shunt", trans-activation seems to act on ribosomes that have begun scanning of the RNA at the 5' end.

The process of trans-activation seems to allow ribosomes that have translated one ORF to remain competent to translate further downstream ORFS, or to become initiation competent once more Rothnie et al.

It was previously though that there was no obligate role for splicing in either plant or animal pararetroviruses. However, the finding of splicing in rice tungro bacilliform virus was the first case to break this rule Futterer, More recently, Kiss-Laszlo et al.

Virus mutants in which the splice acceptor site in ORF II is inactivated are not infectious, indicating that splicing plays as essential role in the CaMV life cycle. These different strategies described above can be used exclusively, or, as a combination of strategies for a particular virus. Adkins, S. Virology Ahlquist, P. Current Opinion in Genetics and Development 2: Journal of Molecular Biology, , Allison, R. Journal of Virology, Banerjee, A.

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There may also be a requirement for host cell proteins. Their genomes are translated shortly after penetration into the host cell to produce the RdRp and other viral proteins required for synthesis of additional viral RNAs. Positive-strand RNA viruses often use large complexes of cellular membranes for genome replication. They actively modify host cell membranes to construct viral replication scaffolds. For each of these groups of viruses, the first synthetic event after genome penetration is transcription.

This is accomplished by viral proteins including the RdRp that enter cell with the genome. RdRp is the key player for all of these processes Fig.

Ribbon diagram of flavivirus RdRp. Fingers, palm, and thumb subdomains are colored in blue, green, and red, respectively. Motifs A, C, E, F, the G-loop, and the priming loop are colored in orange, yellow, gray, magenta, cyan, and purple, respectively. N-ter and C-ter indicate the termini of the RdRp domain.

RdRps of RNA viruses probably arose from a common ancestor. The RdRp, in association with other proteins required for viral genome synthesis is often called the replicase complex. The biochemical requirements for genome synthesis may or may not be identical to those required for synthesis of mRNAs.

If the two processes differ, the term transcription complex is sometimes used to describe the particular set of proteins required for viral mRNA synthesis. Upon penetration into the host cell, ribosomes assemble on the genome to synthesize viral proteins. During the replication cycle of positive-strand RNA viruses, among the first proteins to be synthesized are those needed to synthesize additional genomes and mRNAs. A functional definition of a positive-strand virus is that purified or chemically synthesized genomes are infectious Fig.

Schematic representation of replication of positive-strand RNA virus genomes. The genome of a positive-strand RNA virus is an mRNA that is translated, upon entry into the cells, to produce proteins needed for transcription and genome replication for example, RdRp.

After initial rounds of translation, the genome serves as the template for synthesis of copy RNA. RdRp is a nonstructural protein, meaning that it is not found within the assembled virion.

Instead it is translated directly from the infecting genome shortly after penetration. RdRp and other viral proteins needed for viral RNA synthesis are encoded as a polyprotein that is cleaved by virally encoded proteases.

In the case of the picornaviruses and the flaviviruses, all viral proteins structural and nonstructural are synthesized as part of a single long polyprotein. Other positive-strand RNA viruses i.

For each of these groups of viruses, the first synthetic event after genome penetration is transcription Fig. This is accomplished by viral proteins including viral RdRp that enter cell with the genome.

They associate with the genome through interactions with RNA-binding nucleocapsid N or capsid proteins. Therefore, naked purified away from protein genomic RNA is not infectious, cannot be translated, and will eventually be degraded if transcription is blocked.

Before genome replication can proceed, viral mRNAs must be transcribed and translated. If purified virions are gently lysed under appropriate buffer conditions, with the addition of NTPs, mRNAs will be transcribed in the test tube. However, genome RNA will not be synthesized under these conditions Table Schematic representation of replication of genomes of minus-strand RNA viruses. Upon entry into the cell, the active transcription complex synthesizes mRNAs.

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