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Practică medicală

New insights into molecular mechanisms replication and transcription of multiple genome segments of influenza A virus

Dr. Emil Ioniţă, Insitutul Cantacuzino

Rezumat:

Este cunoscut faptul ca virusul gripal A (IAV) contine in genomul sau un lant negativ segmentat de ARN, dar modul in care IAV reuseste sa-si echilibreze replicarea si transcrierea segmentelor sale multiple nu este cunoscut. Acest articol este o trecere in revista a datelor din literatura de specialitate asupra mecanismelor moleculare legate de competitia dintre segmentele multiple ale genomului IAV. Competitia este afectata de lungimea segmentului, de regiunea de codificare si de ( UTRs). Aceasta competitie este cel mai probabil activa in cursul infectiei virale timpurii, cand sunt disponibile cantitati limitate de polimeraze, iar acest lucru poate contribui la reglarea replicarii si transcriptiei segment specifice.

Cuvinte-cheie: virusul gripal A (IAV), polimeraza (IAV), segmente genomice, regiuni netraduse (UTRs).

Abstract:

It is known that Influenza A virus (IAV) contains a segmented negative-strand RNA genome, but how IAV balances the replication and transcription of its multiple genome segments is not understood.This article is a review of the literature data on molecular mechanisms regarding the competition between multiple genome segments of Influenza A virus(IAV). Competition is affected by segment length, coding region, and UTRs. This competition is probably most apparent early during infection, when limiting amounts of polymerases are present, and may contribute to the regulation of segment-specific replication and transcription.

Keywords: influenza virus A (IAV), (IAV) A polimerase, genome segments, untranslated regions (UTRs)

Introduction

Influenza A virus (IAV) of the family Orthomyxoviridae is an enveloped, negative-strand RNA virus. The IAV genome is composed of eight different vRNA segments that altogether encode up to 13 proteins [1],[2]. Each vRNA and cRNA possesses untranslated regions (UTRs) of varying length at the 3′ and 5′ ends. The first 12 and 13 nucleotides at the 3′ and 5′ UTRs of the vRNAs and cRNAs are highly conserved among different RNA segments. These highly conserved partly complementary UTRs, which form a “panhandle” or “corkscrew” conformation by alternative modes of base-pairing, constitute the promoter structure for RNA synthesis [3], [4].

The mechanism of replication and transcription varies greatly among viruses depending on the nature and structure of their viral genomes. Negative-strand RNA viruses replicate their viral genome via the synthesis of full length positive-strand complementary RNA (cRNA) molecules that in turn serve as templates for the synthesis of negative-strand virion RNA (vRNA) genomes. The negative-strand genomes also function as templates for the production of mRNAs [5], [6].

In non-segmented negative-strand RNA viruses, sequential transcription of successive genes results in a gradient of transcript abundance that steadily decreases towards the end of the template. Thus, the expression level of each gene is governed by the gene order [7]. This does, however, not apply to all negative-strand viruses as some of them acquired segmented genomes during their evolution. Each genome segment of these viruses is individually replicated and transcribed, necessitating careful regulation of these distinctive processes to generate sufficient vRNAs and proteins for the production of progeny virions [6].

Cells are transfected with either one or both reporter constructs (single or co-transfection). Luciferase expression is induced by expression of viral RNA polymerases and NP either by co-transfection of expression plasmids (transfection assay) or by virus infection (infection assay). The expression levels of the firefly and Gaussia luciferase reporter constructs are determined consecutively using a single tube, dual luciferase assay system. It has been determined the luciferase expression levels of the firefly (FNP) and Gaussia (GNP) luciferase reporter constructs when transfected alone or in combination by using the transfection assay.

The results indicate that expression of the firefly luciferase gene is negatively affected by co-transfection of the Gaussia luciferase reporter plasmid. Very similar results were obtained when an empty plasmid (pUC18) was included in the transfection mixture when only one reporter construct was transfected Thus, the observed differences in firefly luciferase expression do not result from a lower transfection efficiency of the firefly luciferase, but not of the Gaussia luciferase reporter construct, when an additional plasmid was included in the transfection mixture.

The results indicate that replication and transcription of the Gaussia and firefly luciferase genome segments are in competition with each other. If so the observed inhibitory effect of co-transfection of the Gaussia luciferase construct on the firefly luciferase expression level is expected to depend on the ratio of the transfected reporter constructs

The panhandle conformation antisense orientation between a PolI promoter and a ribozyme sequence.

The molecular mechanisms by which IAV replicates and transcribes its genome segments have generally been well studied. However, the way by which IAV regulates and balances the replication/transcription of its 8 genome segments is much less understood. In order to study and manipulate these processes, Widjaja I et al[8] have developed a dual reporter genome segment assay that enabled us to analyze whether the replication/transcription of one genome segment is affected by that of another.

Their data indicate that this is indeed the case as luciferase expression driven from a reporter genome segment was shown to be affected by the presence of other genome segments, both in the context of virus infection and in the presence of polymerase and NP proteins provided by transfection of the expression plasmids. Furthermore, their results indicate that genome segments are likely to compete with each other for the available viral proteins and that the balance between different genome segments is affected by reporter genome segment length, by the identity of 3′ and 5′ UTRs, and probably also by their coding regions.

These results indicate that replication/transcription of a genome segment can be negatively affected by the presence of another genome segment. This interference became less pronounced when the length of the smaller segment was extended, indicating that genome segment length plays a role in the competition between different segments. This “length effect” was also observed when natural genome segments were present in addition to the reporter construct, with the shortest segments, M and NS, giving the strongest inhibition of the reporter gene expression.

In agreement herewith, IAV defective interfering (DI) RNAs, which are formed by internal deletion of progenitor RNA segments, interfere with vRNA synthesis, probably because of the competitive advantage of the smaller DI RNA molecules (reviewed by Nayak [9]). In addition, our data indicate that the coding region of the vRNA segment may also be of importance, as the extended version of the Gaussia reporter segment was still able to outcompete its firefly luciferase counterpart, albeit less efficiently than its shorter version.

The segment UTRs are known to contain signals for transcription, replication and packaging of vRNP [10]. They have shown that the identity of the 3′ and 5′ UTRs also influences the competition between different segments.[8] Relatively minor differences were observed when reporter genome segments with different natural UTRs were compared in the competition assay. This result is in agreement with the observation that non-conserved regions of the UTRs contribute to some but limited extent to viral RNA replication, [12]. However, introducing three nucleotide changes in the 3′ UTR (G3A/U5C/C8U) of the NP segment, which is predicted to stabilize the UTR panhandle structure and is known to lead to increased reporter gene expression in infected cells, dramatically increased the competitive ability of the reporter segment, both when replication/transcription was driven by transfection of polymerase- and NP-encoding segments and when mediated by IAV infection.

Thus, while reporter segments carrying the natural NP UTRs or the mutant NPph UTRs were both efficiently expressed in the absence of competitor segments, large differences in luciferase expression were observed in favor of the luciferase segment carrying the panhandle-stabilizing mutations when other reporter segments were co-transfected or in IAV infected cells. In agreement herewith, recombinant viruses carrying two nucleotide changes (G3A/C8U) in the UTR of either the PB1 or PA segment displayed enhanced replication/transcription of the mutated segments in detriment of the wild-type UTR-bearing segments [13].

The most likely scenario suggested by their observations is that replication/transcription of one reporter segment interferes with that of another by sequestering UTR-binding proteins, probably polymerases, required for RNA synthesis. Several observations by Widjaja I et al[8] and others support this hypothesis: 1) increasing the amount of polymerase and NP proteins, but not of NP protein alone, alleviated the competition between different segments, 2) the polymerase proteins have been shown to bind to 5′ and 3′ UTRs of vRNAs, with most strong binding observed to the 5′ UTR, 3) introduction of mutations in the 3′ UTR (NPph) that stabilize the panhandle structure and are predicted to result in increased polymerase binding result in increased ability of the reporter segment to be replicated/transcribed in the presence of competitor segments ( 4) introduction of similar mutations in the 5′ UTR (NPphR) that are likely to interfere with polymerase binding [13], had a negative effect on the competitive ability of the reporter construct, and 5) panhandle-stabilizing mutations in the 3′ UTR (NPph), that increased the competitive ability of the reporter construct, partly compensated for replication-debilitating mutations in PB2 (R142A or E361A) but not in NP (M331K or F488G), suggesting a link between the interaction of the UTR with polymerase and the ability to compete with other segments.

Even so, the researchers experimental system[8] (i.e. the transfection assay) does not approach the complexity of the IAV infected cells with respect to number of vRNA segments and viral proteins present,their data suggest that IAV RNA segments compete with each other for available polymerases.It seems that this competition is expected to be most apparent early during infection, when only low amounts of polymerase are present. As the researchers concluded [8],it is clear that at this stage of the infection the low level of RNA polymerase plays a critical role in the regulation of segment-specific replication and/or transcription. At later times during infection competition between vRNA segments is expected to be alleviated by the increased levels of the polymerase subunits, thereby ensuring the efficient replication/transcription of all genome segments.

Conclusion

The balance between different reporter segments was most dramatically affected by the introduction of UTR panhandle-stabilizing mutations. Furthermore, only reporter genome segments carrying these mutations were able to efficiently compete with the natural genome segments in infected cells.

IAV genome segments compete for available polymerases. Competition is affected by segment length, coding region, and UTRs. This competition is probably most apparent early during infection, when limiting amounts of polymerases are present, and may contribute to the regulation of segment-specific replication and transcription.

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