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SummaryIntrinsic restriction factors and viral nucleic acid sensors are important for the anti-viral response. Here, we show how upstream sensing of retroviral reverse transcripts integrates with the downstream effector APOBEC3, an IFN-induced cytidine deaminase that introduces lethal mutations during retroviral reverse transcription. Using a murine leukemia virus (MLV) variant with an unstable capsid that induces a strong IFNβ antiviral response, we identify three sensors, IFI203, DDX41, and cGAS, required for MLV nucleic acid recognition. These sensors then signal using the adaptor STING, leading to increased production of IFNβ and other targets downstream of the transcription factor IRF3. Using knockout and mutant mice, we show that APOBEC3 limits the levels of reverse transcripts that trigger cytosolic sensing, and that nucleic acid sensing in vivo increases expression of IFN-regulated restriction factors like APOBEC3 that in turn reduce viral load. These studies underscore the importance of the multiple layers of protection afforded by host factors.

Pathogenic retroviruses have evolved multiple means for evading host restriction factors such as apolipoprotein B editing complex (APOBEC3) proteins. Here, we show that murine leukemia virus (MLV) has a unique means of counteracting APOBEC3 and other cytosolic sensors of viral nucleic acid. Using virus isolated from infected WT and APOBEC3 KO mice, we demonstrate that the MLV glycosylated Gag protein (glyco-Gag) enhances viral core stability. Moreover, in vitro endogenous reverse transcription reactions of the glyco-Gag mutant virus were substantially inhibited compared with WT virus, but only in the presence of APOBEC3. Thus, glyco-Gag rendered the reverse transcription complex in the viral core resistant to APOBEC3. Glyco-Gag in the virion also rendered MLV resistant to other cytosolic sensors of viral reverse transcription products in newly infected cells. Strikingly, glyco-Gag mutant virus reverted to glyco-Gag–containing virus only in WT and not APOBEC3 KO mice, indicating that counteracting APOBEC3 is the major function of glyco-Gag. Thus, in contrast to the HIV viral infectivity factor protein, which prevents APOBEC3 packaging in the virion, the MLV glyco-Gag protein uses a unique mechanism to counteract the antiviral action of APOBEC3 in vivo—namely, protecting the reverse transcription complex in viral cores from APOBEC3. These data suggest that capsid integrity may play a critical role in virus resistance to intrinsic cellular antiviral resistance factors that act at the early stages of infection.

The type I interferon (IFN) response requires the coordinated activation of the latent transcription factors NF-kappaB, IRF-3 and ATF-2 which in turn activate transcription from the IFN-beta promoter. Here we have examined the type I interferon response in rhinovirus type 14-infected A549 cells, with particular emphasis on the status of the transcription factor IRF-3. Our results indicate that although rhinovirus type 14 (RV14) infection induces the activation of NF-kappaB and ATF-2, only very low levels of IFN-beta mRNA are detected. Analysis of ISG54 mRNA levels revealed very little induction of this IRF-3 responsive transcript and suggested that IRF-3 activation might be impaired. Examination of IRF-3 in RV14-infected cells demonstrated only low levels of phosphorylation, a lack of homodimer formation and an absence of nuclear accumulation indicating that this transcription factor is not activated. Inhibition of viral protein synthesis following infection resulted in an increase in IFN-beta mRNA levels indicating that viral gene products prevent induction of this pathway. Collectively, these results indicate that RV14 infection inhibits the host type I interferon response by interfering with IRF-3 activation.

Background The type I interferon (IFN) response is a critical component of the innate immune response to infection by RNA viruses and is initiated via recognition of viral nucleic acids by RIG-like receptors (RLR). Engagement of these receptors in the cytoplasm initiates a signal transduction pathway leading to activation of the transcription factors NF-κB, ATF-2 and IRF-3 that coordinately upregulate transcription of type I IFN genes, such as that encoding IFN-β. In this study the impact of poliovirus infection on the type I interferon response has been examined. Methods The type I IFN response was assessed by measuring IFN-β mRNA levels using qRT-PCR and normalizing to levels of β-actin mRNA. The status of host factors involved in activation of the type I IFN response was examined by immunoblot, immunofluorescence microcopy and qRT-PCR. Results The results show that poliovirus infection results in induction of very low levels of IFN-β mRNA despite clear activation of NF-κB and ATF-2. In contrast, analysis of IRF-3 revealed no transcriptional induction of an IRF-3-responsive promoter or homodimerization of IRF-3 indicating it is not activated in poliovirus-infected cells. Exposure of poliovirus-infected cells to poly(I:C) results in lower levels of IFN-β mRNA synthesis and IRF-3 activation compared to mock-infected cells. Analysis of MDA-5 and IPS-1 revealed that these components of the RLR pathway were largely intact at times when the type I IFN response was suppressed. Conclusions Collectively, these results demonstrate that poliovirus infection actively suppresses the host type I interferon response by blocking activation of IRF-3 and suggests that this is not mediated by cleavage of MDA-5 or IPS-1.

The type I interferon (IFN) response requires the coordinated activation of the latent transcription factors NF-κB, interferon regulatory factor 3 (IRF-3), and ATF-2, which in turn activate transcription from the IFN-β promoter. Synthesis and subsequent secretion of IFN-β activate the Jak/STAT signaling pathway, resulting in the transcriptional induction of the full spectrum of antiviral gene products. We utilized high-density microarrays to examine the transcriptional response to rhinovirus type 14 (RV14) infection in HeLa cells, with particular emphasis on the type I interferon response and production of IFN-β. We found that, although RV14 infection results in altered levels of a wide variety of host mRNAs, induction of IFN-β mRNA or activation of the Jak/STAT pathway is not seen. Prior work has shown, and our results have confirmed, that NF-κB and ATF-2 are activated following infection. Since many viruses are known to target IRF-3 to inhibit the induction of IFN-β mRNA, we analyzed the status of IRF-3 in infected cells. IRF-3 was translocated to the nucleus and phosphorylated in RV14-infected cells. Despite this apparent activation, very little homodimerization of IRF-3 was evident following infection. Similar results in A549 lung alveolar epithelial cells demonstrated the biological relevance of these findings to RV14 pathogenesis. In addition, prior infection of cells with RV14 prevented the induction of IFN-β mRNA following treatment with double-stranded RNA, indicating that RV14 encodes an activity that specifically inhibits this innate host defense pathway. Collectively, these results indicate that RV14 infection inhibits the host type I interferon response by interfering with IRF-3 activation.

Laboratories working with closely related viruses need simple and cost-effective ways to rapidly validate viral stocks, detect contamination and measure the abundance of viral RNA species. Using RT-PCR and specific primers an approach for the specific detection of rhinovirus type 14 (RV14) or poliovirus type 1 (PV1) is presented. It is demonstrated that viral sequences can be amplified directly from viral stocks or from infected cells. In addition, the utility of this protocol for the detection of low levels of contaminating PV1 in RV14 stocks is shown. Further, using quantitative real-time PCR It is shown that this approach can be used for the quantitative analysis of viral RNA and replication kinetics in infected cells. This method should be useful for laboratories working with PV and RV14 and could be adapted easily for use by laboratories working with other rhinovirus and enterovirus serotypes.

Obesity contributes to the development of insulin resistance, hyperglycaemia and metabolic syndrome (Kahn & Flier, 2000; Kahn et al. 2006). However, effects of obesity on the control of skeletal muscle mass and hypertrophy are not well understood. Muscle hypertrophy is marked by an increase in protein synthesis and addition of contractile filaments to generate muscle force (Glass, 2003). A vast array of signalling networks in response to external stimuli including insulin, insulin growth factor-1 (IGF-1), steroid hormones and also amino acids mediate hypertrophy of skeletal muscle (Glass, 2003). Though the external signals such as insulin and IGF-1 activate their specific signalling, they share common downstream signalling molecules such as PI3-K and Akt. IGF-1/insulin act on their cell surface specific receptors and initiate respective signalling by activation of PI3K followed downstream signalling via activation of Akt (Cheatham & Kahn, 1995; Stitt et al. 2004). The mechanism of distinct actions of these peptide hormones despite activating common signalling molecule: Akt is beginning to be understood. Reported studies suggest that different isoforms of Akt (Akt1, Akt2 and Akt3) confer signal specificity of insulin in glucose metabolism and IGF-1 in growth. Mutational analyses revealed that Akt1-deficient mice have retarded growth and perinatal mortality, where as Akt2 knockout mice have no growth defects, but have impaired glucose metabolism (Chen et al. 2001; Cho et al. 2001a,b;). On the other hand, Akt3 knockout mice had normal body weights and glucose metabolism, but reduced brain sizes (Garofalo et al. 2003). In general, IGF-1 and also insulin to a lesser extent mediate protein synthesis by activating Akt1. The activated Akt1 increases protein synthesis by activating mammalian target of rapamycin (mTOR). mTOR increases protein synthesis by phosphorylating S6 protein kinase (p-S6K), a positive regulator of protein synthesis. mTOR also inhibits the activity of eIF4E binding protein-1 (4E-BP1), a negative regulator of the protein initiation factor, eukaryotic translation initiation factor 4E (eIF-4E) (Proud & Denton, 1997; von Manteuffel et al. 1997). Glycogen synthase kinase 3β (GSK3β) is another substrate of Akt, whose repression is known to induce hypertrophy, and its activity is regulated negatively by the phosphorylation of serine 9 (p-GSK-3β) (Rommel et al. 2001). In a recent issue of The Journal of Physiology, Sitnick et al. (2009) investigated load-induced hypertrophy and underlying molecular mechanisms in response to high-fat diet. The authors investigated whether a high-fat diet (HFD) would alter the ability of skeletal muscle to respond to functional overload (FO). Further, they determined if underlying signalling mechanisms of hypertrophy are compromised in mice fed a high-fat diet. As expected, mice increased their body size in response to HFD. Measurements of fat (epididymal and retroperitoneal fat pads) and muscle (gastrocnemius and soleus) suggested that increases in body size were primarily due to increased fat composition in the body. The authors also determined that mice fed a high-fat diet were hyperinsulinemic, suggesting development of insulin resistance potentially mediated by increased intra-myocellular triglycerides in muscle. On all the tested days there was a significant increase in the mass of pantaris muscle in response to FO mice compared to control. After 14 and 30 days of FO the authors found a 10 and 16% reduction in the growth of the plantaris muscle in the HFD versus the low-fat diet (LFD) mice, suggesting a moderate decrease in the growth of plantaris muscle in response to HFD. The authors noticed that extended HFD nutrition (30 weeks) had a dramatic effect, showing no difference in the mass of plantarius muscle compared to control mice. These observations suggest that a HFD diet has a negative effect on the load-induced hypertrophy. The authors give interesting directions for the research into whether obesity may be a negative factor for load-induced muscular hypertrophy. On a path leading toward understanding the mechanism underlying this apparent relationship, the authors measured general protein translation and also status of the cell signalling molecules such as Akt, S6K1 and GSK3-β. The association of the ribosomal 40S and 60S subunits resulting in a 80S peak is an indicator of active translation process. The analyses demonstrate that the 80S peak in LFD mice was larger than in HFD mice suggesting that the translation process is greater in LFD mice compared to HFD. The authors further suggest that the activity of signalling molecules such as Akt and S6K1 were attenuated, but not for GSK3 β in FO/HFD mice compared FO/LFD mice. Though the analyses provided in the Sitnick et al. paper provide useful information about the status of the signalling molecules in hypertrophy, the data/analyses are not sufficient to provide accurate mechanistic insights. The potential link between obesity and hypertrophy at a mechanistic level may be delineated by further analyses of other important targets including IGF-1 expression and also using appropriate controls in the analyses. For example, the authors observed increased Akt phosphorylation in HFD control mice with apparent insulin resistance. This may be due to higher load on the plantaris muscle in the case of HFD mice due to an increased body mass. The measurement of IGF-1 levels would have been useful for interpreting information. Upon examining the Western blots of Akt presented in the paper, it is clear that total Akt levels increase in response to FO, irrespective of diet; however, the authors did not analyse if the total Akt levels show differences in response to FO in the two diets (LFD vs. HFD). The analyses are presented as a ratio of Akt Ser-373 to total Akt to indicate activity levels of Akt; but these analyses in the context of robust changes in total Akt levels will only provide limited information regarding Akt activity. The analyses would have been improved by probing for a housing-keeping protein such as GAPDH, and normalizing total Akt levels and also phosphorylated Akt levels to GAPDH and making comparisons between diets. The same suggestion applies to the quantification of the expression of S6K1, p-S6K1, GSK-3β and p-GSK-3β. All the molecules chosen in the study are not unique to either insulin or IGF-1, but common to both. It is not clear whether the HFD mice exhibited compromised hypertrophy due to attenuation of insulin signalling or IGF-1 signalling. In this context, future studies directed toward deciphering the specific proximal pathways such as IGF-1 serum levels/muscle expression of IGF-1, IGF-1/insulin receptor activation and also further study of specific Akt isoforms may begin to provide a molecular basis for the apparent relationship between obesity and hypertrophy. On the other hand, it has to be determined if obesity decreases activity of the mice. In summary, the recent paper by Stinick et al. suggests that diet plays a role in the hypertrophy of skeletal muscle, although the exact mechanisms for this process remain unclear. The authors provide a base upon which further investigations can build to elucidate the complex signalling mechanisms of diet and their influence on load-induced hypertrophy.