This study outlines five key points that argue for a role for cellular mRNA decay enzymes in the SINV life cycle. First, SINV RNAs are clearly degraded with a measurable half-life during the course of an infection. Therefore, viral RNAs must interface with the cellular mRNA decay machinery during an infection. Second, the viral RNAs are refractory, particularly in cells, to deadenylation—the initial event in the major pathway of mRNA decay in the cell. This suggests that SINV has evolved a strategy to evade the major cellular decay enzymes, an adaptation that may significantly contribute to a productive infection. Third, a conserved feature of the alphavirus 3′ UTR, the RSEs, is part of a combinatorial set of regions in the 3′ UTR that mediates the repression of deadenylation. The conservation of this feature suggests that the ability to repress deadenylation could be a common feature of alphaviral 3′ UTRs. Fourth, the interaction between the viral 3′ UTR and a 38-kDa cellular protein is strongly associated with the observed block in deadenylation, suggesting a possible mechanism for the repression of poly(A) shortening which involves the usurping of cellular factors. Finally, the 3′ UTR from VEEV also represses deadenylation and interacts with a 38-kDa cellular protein, suggesting that a common strategy for inhibiting poly(A) tail shortening may be conserved among alphaviruses. Collectively, these data suggest that the influence of cellular mRNA decay enzymes must be taken into account if one is to fully appreciate the molecular biology of SINV, and likely other, viral infections.
Why would it be important for SINV to actively stabilize its transcripts, particularly the subgenomic mRNA that is so actively transcribed? The answer to this question likely involves the fact that levels of mRNAs are determined by both transcription and degradation rates. The fact that the subgenomic RNA is being transcribed throughout the infection is not sufficient to ensure its accumulation to physiological levels in the cell. Many cellular mRNAs are actively transcribed in the cell but accumulate only under specific conditions due to the fact that they are very actively degraded in a regulated manner (22
). Therefore, effective accumulation of viral transcripts requires that they possess a means to avoid rapid degradation by the cellular mRNA decay machinery. In addition, maintenance of a poly(A) tail is also necessary to ensure efficient translation of viral mRNAs.
Our ability to readily assess SINV mRNA decay revolved around two technologies, the use of a temperature-sensitive viral polymerase variant in vivo and the application of a cell-free in vitro system. Both of these experimental approaches are likely readily applicable to other viruses. Temperature-sensitive RNA polymerases have been described for numerous viruses (see, e.g., references 15
) and should be relatively easy to incorporate into recombinant constructs. This strategy allows one to specifically turn off viral RNA synthesis to allow the assessment of decay without any confounding secondary effects of drugs (26
). Our in vitro mRNA system from C6/36 cells (42
) is an adaptation of technology that we have previously developed and extensively exploited in HeLa cells (19
) and other eukaryotic systems (39
) to gain mechanistic insights into mRNA decay. Application of a combination of these approaches should provide a powerful tool to analyze the contribution of mRNA decay to viral infections.
In vivo and in vitro data clearly indicate that SINV RNAs have developed a means to maintain the integrity of their poly(A) tails and avoid deadenylation. This is particularly interesting because most mRNA decay initiates via this pathway (22
). In addition, transfected RNAs (which also are largely, if not exclusively, cytoplasmic) are also actively deadenylated (47
), further emphasizing the unique stability of SINV mRNAs. Despite being refractory to deadenylation, SINV RNAs are still clearly degraded (Fig. ). This implies the involvement of an alternative, deadenylation-independent pathway. There are several pathways of RNA decay in the cell that do not necessarily initiate with poly(A) tail shortening, including nonsense-mediated decay (1
), RNA interference (46
), and endonucleolytic decay (61
). An interesting question for future work will be to assess whether any of these known deadenylation-independent pathways, or perhaps a novel pathway, is responsible for the observed decay of viral mRNAs. Once the pathway(s) is identified, it may be possible to stimulate this pathway(s) of viral mRNA decay, creating a hostile environment for viral growth while having perhaps minimal effects on cellular gene expression.
Numerous deadenylase enzymes have been described in eukaryotic cells (22
), with CCR4, PAN2/3, and PARN among the major, most studied activities. One important question is whether the SINV 3′ UTR is capable of repressing the action of all deadenylases on viral transcripts or if it targets only specific enzymes. Since the 5′ cap-stimulated PARN enzyme (12
) appears to be the major deadenylase in C6/36 mosquito cell extracts (42
), our in vitro data would suggest that this enzyme is at least one target of the repressive effect of the 3′ UTR. Future studies using a combination of RNA interference knock-downs and in vitro reconstitution analyses will be required to assess the influence of the SINV 3′ UTR on the enzymatic activity of other deadenylases.
The results of our deletion analyses (Fig. ) suggest that multiple regions of the 3′ UTR influence the stability of SINV mRNAs. The mapping of a deadenylation repression element to RSE sequences provides the first biological function for this conserved feature of alphavirus 3′ UTRs. The core element may be repeated in its natural context in order to amplify repression through the additive effects of individual copies as has been seen in tethering experiments (2
). Combinatorial arrangements of mRNA stability elements have also been identified in cellular mRNAs (50
), suggesting that such an organization may be relatively common for regulated mRNAs. It will be interesting to see if the apparently unrelated RSEs of other alphaviruses such as VEEV possess a similar ability to repress deadenylation. The combination of multiple mRNA stabilizers in SINV may also contribute to the broad host range of the virus, as well as the relative overall effectiveness of a viral infection. In this regard, it is interesting that deletions of the 3′ UTR that remove the deadenylation-repression region described here were shown by Kuhn et al. to result in a defective virus that grows very poorly in mosquito C6/36 cells (33
). Defects in the ability of the 3′ UTR mutant virus to repress deadenylation could be at least partially responsible for these growth defects.
As shown in Fig. and , the interaction of a 38-kDa protein with the SINV and VEEV 3′ UTRs is strongly associated with a repression of deadenylation. The ability to combine protein binding/competition studies with functional assays as shown here demonstrates one of the advantages of the in vitro system used in these studies. Given its relative molecular size, this 38-kDa protein could be the Aedes
homolog of any number of a known cellular RNA stabilizers, including HuR (9
), TIA-1/TIAR (17
), or numerous others. Alternatively, the 38-kDa protein could also be a novel cellular protein that the virus has specifically usurped to remodel the messenger ribonucleoprotein composition of its 3′ UTR. It is important to point out that the UV cross-linking approach used in this study does not involve any purification steps. Identification of the 38-kDa protein, therefore, will require the application of affinity-based or conventional chromatographic methods.
In summary, the cellular mRNA decay machinery plays a very major role in host cell gene expression and quality control. The underlying hypothesis of this work is that viruses very likely have had to evolve a way to adapt to, and perhaps usurp, this powerful machinery. SINV and VEEV RNAs, through their 3′ UTRs, have evolved a novel way to avoid a major mRNA decay pathway in the cell and ensure efficient translation by maintenance of their poly(A) tails. Future questions include the identification of the mechanistic basis of the repression of deadenylation, the conservation of the ability to repress deadenylation among other alphaviruses, and the characterization of the apparently unusual, deadenylation-independent pathway of mRNA decay that acts upon these viral transcripts. Answers to these questions will allow the full impact of the cellular mRNA stability system on viral gene expression and growth to be assessed.