Rift Valley Fever Virus and Autophagy: MyD88 and DRAM-1

Upon viral entry and release of the viral genome, viral RNA and DNA can be recognised by Toll-like Receptors (TLR), which once stimulated induce the expression of specific genes such as Interferon that are part of the antiviral response. TLR mediated signalling pathways predominantly signal through interferon regulatory factors (IRF) as well as Nuclear Factor-κB (NF-κB) and AP-1, eliciting the induction of the Interferon type-1 response and the expression of inflammatory cytokines as well as facilitating the presentation of viral antigens by MHC Class I and Class II molecules. In general, TLR-signalling is mediated via five adaptor proteins, MyD88, MyD88-adapter like (MAL), Toll/interleukin receptor (TIR) domain-containing adaptor protein inducing interferon β (TRIF), TRIF-related adaptor molecule (TRAM), and sterile α- and armadillo motif-containing protein (SARM). Following the activation of TLR, MyD88 us the key signalling protein for all TLRs (with the notable exception of TLR3 and subsets of TLR4).

TLR, PACT, MDA-5, and RIG-1 induced antiviral signalling by viral RNAs

As discussed in various posts before, autophagy is induced as a cellular survival response following starvation and as part of the ER stress response. In the context of viral replication, the autophagic machinery can be subverted by viral proteins to allow the formation of replication centers. Autophagy however is also part of the antiviral response as the degradation of viral proteins by the fusion of mature autophagosomes with the lysosome leads to the degradation of viral proteins and/or viral particles. Indeed, as discussed before, a number of viral proteins including Influenza Virus M2, coronaviral nsp-6 and KSHV K7 have the capability to inhibit autophagic flux, in particular the fusion of autophagosomes with lysosomes (e.g. by interacting with Beclin-1 directly or via Rubicon or by inhibition the formation of mature lysosomes via deactivating mTORC2) whilst inducing the formation of the phagophore by recruiting components of the autophagic machinery such as Beclin-1, LC3-C, or p62/SQSTM1 to the ER. Autophagy however is not only induced during the formation of the replication centers of RNA viruses, but also part of the antiviral response initiated by binding of viral RNA (genomic, antigenic, dsRNA intermediates) to TLRs. In this case, both MyD88 and TRIF have been shown to induce autophagy by at least two different mechanisms.

Akin to Bcl-2, purified MyD88 has been shown to bind Beclin-1 via the BH3-domain and activation of the TLR both by LPS and Poly(I:C) increases binding of Beclin-1to the TLR signalling complex and the formation of GFP-LC3 positive punctae in murine RAW 264.7 cells whilst decreasing binding of Beclin-1 to Bcl-2, thereby promoting the formation of the phagophore. The translocation of NF-κB into the nucleus not only induces the expression of cytokines, but also of DNA damage-regulated autophagy modulator-1 (DRAM-1) in Zebrafish infected with Mycobacterium tuberculosis and knockdown of DRAM-1 increases bacterial infection whereas the overexpression of DRAM-1 not only increases autophagic flux but decreases bacterial infection. Indeed, DRAM-1 overexpression increases RFP- positive punctae in cells expressing a tandem GFP-RFP-LC3 construct whilst decreasing the levels of p62/SQSTM-1 in a 3-MA sensitive manner. Recent data indicate that DRAM-1 induces the formation of the phagophore by binding p62/SQSTM-1 rather than Beclin-1.

TLR and MyD88 induction of autophagy via DRAM-1

                        Rift Valley Fever Virus and autophagy

Rift Valley Fever Virus (RVFV) is a segmented negative sense RNA virus belonging to the genus of Phleboviridae within the family of Bunyaviridae. As such, the RVFV genome consists of three RNA segments, one of which uses an ambisense coding strategy. The RNA dependent RNA Polymerase is encoded within the largest (L) segment, whereas the small  (S) segment encodes for the viral Nucleocapsid (N) protein as well as a non-structural protein (NSs) and the medium sized (M) segment encodes for the precursor of the viral glycoproteins (Gn) as well as for non-structural proteins. RVFV is transmitted via mosquito bites as well as droplet infection and contact with blood from infected animals while human-to-human to human transmission is rare. Infected patients can exhibit a wide range of symptoms, ranging from mild symptoms such as fever to ocular disease, encephalitis, and hemorrhagic fever.

Rift Valley Fever Virus: virion and genome

Following the release of the viral genome, the viral RNA has  been shown to activate RIG-1 mediated antiviral signalling without however activating MAVS and MAVS has been shown not to restrict RVFV replication in macrophages despite the induction of IFN-1. Since the RNA from both positive and negative strand RNA viruses not only activates RIG-1 or MDA-5 but also binds TLR, alternative pathways might include not only these but also induce (antiviral) autophagy via MyD88 as outlined above. Indeed, the infection of U2OS and murine embryonic fibroblasts (MEF) as well as Drosophila with an attenuated strain of RVFV induces both the formation of LC3-positive punctae as well as the degradation of p62/SQSTM-1 and the accumulation of mCherry positive punctae in U2O2 expressing a mCherry-GFP-LC3 tandem construct, indicating not only that the formation of autophagosomes is induced but also an increase in autophagic flux which is prevented in Atg5 -/- MEF. Furthermore, in ATG 5 -/- MEF as well as in U2OS cells transfected with siRNA targeting other components of the autophagic machinery (Beclin-1, Fip200, Atg5, Atg7, and Atg13) viral replication is significantly increased (albeit by varying levels), thus indicating that the induction of autophagy by RVFV is part of the antiviral response.

It should also be noted that both TRIF and TBK1 do not restrict RVFV replication, thus indicating that MyD88 plays a crucial role in inducing the antiviral autophagy response.

An additional component of the signal pathway involved in RVFV induced autophagy is TRAF-6 since in Traf6 mutant Drosophila -similar to mammalian cells- increased levels of viral RNAs can be detected as well as exhibiting impaired activation of autophagy. Since TRAF6 is known to activate autophagy by inhibiting the phosphorylation of Akt and thus the formation of the phagophore, a model was developed in which the binding of RVFV to TLR or alternatively viral RNA induces the recruitment of TRAF6 and thus inhibits the phosphorylation by Akt kinase and promoting the formation of the phagophore by activating Beclin-1. Indeed, loss of Traf-6 increases viral replication in Drosophila as well as in mammalian cell lines, thus highlighting the conservation of the antiviral signalling induced by RVFV across different host species.

The activation of MyD88 by RVFV is preceded by the binding of the virion to TLR which are located at the plasma membrane akin to LPS induced, MyD88 dependent, autophagy although the author of this post favours a model in which RVFV virions within the endosomal compartment activate endosomal TLR instead. In any case, the induction of autophagy by RVFV is independent of viral replication since UV inactivated virus not only reduces the phosphorylation of Akt as well  as decreasing p62/SQSTM-1 levels.

Model of RVFV mediated induction of autophagy: TRAF-6 decreases phosphorylated
Akt and thus induces the formation of the phagophore

RVFV and autophagy: induction of autophagy by endosome resident TLR?

A second mechanism leading to the induction of autophagy -in this case associated with viral replication- proposed by the author of this post might be dependent on the induction of the DNA damage response pathway (DDR). Following the infection of Human small airway lung epithelial cells (HSAEC), RVFV induces the phosphorylation of classic components of the DDR, namely Ataxia-Telangiectasia Mutated (ATM) (Ser-1981), Chk-2 (Thr-68), H2AX (Ser-139), and p53 (Ser-15) proteins, probably as result of the induction of Reactive Oxygen Species (ROS) by the viral NSs protein via downregulation of Superoxide Dismutase-1  (SOD1), which localises to the mitochondria and has been shown to induce NF-κB and activate p53. In this scenario, the formation of autophagosomes would be induced via DRAM-1 and independent of TLR signalling. Since the expression of NSs also induces the cleavage of Caspase-3 the formation of autophagosomes might be however prevented since Caspase-3 cleaves not only Beclin-1 but also Atg4D (preventing the lipidation of LC3-I). Experiments are therefore needed to investigate the intricate details of NSs on DRAM-1 induced autophagy.

RVFV NSs and autophagy: role of DRAM-1

Also, it remains to be seen if the expression of the viral glycoproteins located on the virion surface are sufficient for the induction of RVFV induced autophagy. Experiments using recombinant VLP in a VSV or Baculovirus backbone for instance as well as cells transfected with the plasmids allowing the expression of the glycoproteins as well treating cells with purified proteins should clarify this issue.

Finally, one of the questions I would like to see to be answered is, if in the case of KSHV the downregulation of MyD88 expression by the KSHV replication and transcription activator (RTA) protein interferes with the formation of autophagosomes as part of the antiviral signalling. Although not related to RVFV, experiments targeted at this question might give us some more insight into the importance of MyD88 initiated antiviral signalling. In more practical terms, the application of two autophagy inducers, Rapamycin and SMER28, have been demonstrated to reduce RVFV replication. The induction of autophagy might therefore offer novel treatment options for patients and infected livestock as well. In extensio, they might also be provide useful for treating patients infected with Coronaviruses such as MERS-CoV or SARS-CoV.





Further reading

Moy, R., Gold, B., Molleston, J., Schad, V., Yanger, K., Salzano, M., Yagi, Y., Fitzgerald, K., Stanger, B., Soldan, S., & Cherry, S. (2014). Antiviral Autophagy Restricts Rift Valley Fever Virus Infection and Is Conserved from Flies to Mammals Immunity, 40 (1), 51-65 DOI: 10.1016/j.immuni.2013.10.020 Shi CS, & Kehrl JH (2008). MyD88 and Trif target Beclin 1 to trigger autophagy in macrophages. The Journal of biological chemistry, 283 (48), 33175-82 PMID: 18772134 van der Vaart, M., Korbee, C., Lamers, G., Tengeler, A., Hosseini, R., Haks, M., Ottenhoff, T., Spaink, H., & Meijer, A. (2014). The DNA Damage-Regulated Autophagy Modulator DRAM1 Links Mycobacterial Recognition via TLR-MYD88 to Autophagic Defense Cell Host & Microbe, 16 (1) DOI: 10.1016/j.chom.2014.06.003 Mah LY, O'Prey J, Baudot AD, Hoekstra A, & Ryan KM (2012). DRAM-1 encodes multiple isoforms that regulate autophagy. Autophagy, 8 (1), 18-28 PMID: 22082963 Meijer AH, & van der Vaart M (2014). DRAM1 promotes the targeting of mycobacteria to selective autophagy. Autophagy PMID: 25484076 Wang RC, Wei Y, An Z, Zou Z, Xiao G, Bhagat G, White M, Reichelt J, & Levine B (2012). Akt-mediated regulation of autophagy and tumorigenesis through Beclin 1 phosphorylation. Science (New York, N.Y.), 338 (6109), 956-9 PMID: 23112296 Narayanan A, Amaya M, Voss K, Chung M, Benedict A, Sampey G, Kehn-Hall K, Luchini A, Liotta L, Bailey C, Kumar A, Bavari S, Hakami RM, & Kashanchi F (2014). Reactive oxygen species activate NFκB (p65) and p53 and induce apoptosis in RVFV infected liver cells. Virology, 449, 270-86 PMID: 24418562 Baer A, Austin D, Narayanan A, Popova T, Kainulainen M, Bailey C, Kashanchi F, Weber F, & Kehn-Hall K (2012). Induction of DNA damage signaling upon Rift Valley fever virus infection results in cell cycle arrest and increased viral replication. The Journal of biological chemistry, 287 (10), 7399-410 PMID: 22223653 Narayanan A, Popova T, Turell M, Kidd J, Chertow J, Popov SG, Bailey C, Kashanchi F, & Kehn-Hall K (2011). Alteration in superoxide dismutase 1 causes oxidative stress and p38 MAPK activation following RVFV infection. PloS one, 6 (5) PMID: 21655261 Liang Q, Chang B, Brulois KF, Castro K, Min CK, Rodgers MA, Shi M, Ge J, Feng P, Oh BH, & Jung JU (2013). Kaposi's sarcoma-associated herpesvirus K7 modulates Rubicon-mediated inhibition of autophagosome maturation. Journal of virology, 87 (22), 12499-503 PMID: 24027317Zhao Q, Liang D, Sun R, Jia B, Xia T, Xiao H, & Lan K (2014). Kaposi's Sarcoma-Associated Herpesvirus-Encoded RTA Impairs Innate Immunity via Ubiquitin-mediated Degradation of MyD88. Journal of virology PMID: 25320320 Mukhopadhyay S, Panda PK, Sinha N, Das DN, & Bhutia SK (2014). Autophagy and apoptosis: where do they meet? Apoptosis : an international journal on programmed cell death, 19 (4), 555-66 PMID: 24415198Liang C, Oh BH, & Jung JU (2014). Novel functions of viral anti-apoptotic factors. Nature reviews. Microbiology PMID: 25363821