Virology tidbits: November 2014

Wednesday, 26 November 2014

Chikungunya Virus nsP2 and the ER stress response

Both cellular and viral proteins translocate into the Endoplasmic Reticulum to be post-translationally modified by ER resident glycosylases and folded by ER president chaperones prior being transported to other cellular organelles and/or being exported to the plasma membrane for release. The accumulation of proteins of misfolded or unfolded proteins within the ER results in the induction of the ER stress response -also known as Unfolded Protein Response (UPR)- as described elsewhere. In general, this process involves the increased expression of genes encoding for chaperones as well as the reduction of both transcription and translation as well as the induction of autophagy; prolonged ER stress however can lead to the induction of apoptosis via intrinsic pathways involving the depolarisation of the mitochondrial membrane. UPR is initiated by the dissociation of Ca²⁺ dissociated heavy chain binding protein (BiP)/Glucose regulated Protein 78 (GRP78) from dsRNA RNA dependent protein kinase (PERK), thus allowing PERK to phosphorylate the alpha subunit of the eukaryotic translation factor-2 (eIF2α), which in turn inhibits translation. Phosphorylated eIF2α also selectively increases the translation of Activating Transcription Factor- 4 (ATF4). As outlined before, ATF4 increases the expression of CHOP/GADD153, thus in turn not only upregulates apoptosis but also autophagy via upregulation of Beclin-1. CHOP expression is however also induced by the two other branches of the UPR, namely the IRE1- and ATF6α.

The ER stress response: three branches which are intersected

Chikungunya virus (CHIKV) is the causative agent of an arthropod (mosquito) transmitted disease which is characterised by a high fever, rash, joint pain, and arthritis which was reported in 1952 in Tanzania but has spread since to Europe, Asia, Oceania and more recently to America, including the Caribbean and North America.

Chikungunya infections in the Americas Dec 2013 to Nov 2014 (as of Nov 26th 2014)
(Data provided by PAHO www.paho.org)

Being a member of the Alphaviridae genus of the family Togaviridae, CHIKV has a positive strand ssRNA genome of 11.6 kb in size that encodes for both the structural and non-structural (nsP) proteins. In accordance with other prototype members of the Alphaviridae, the 5’ two thirds encodes for the four viral nsP (nsP 1-4) whereas the structural proteins are encoded within a subgenomic m 26S RNA, which in turn derives from a precursor 42S RNA. Both the non-structural (nsP) and structural proteins are expressed as polyproteins and cleaved by cellular and viral proteases. In the case of the polyprotein containing the structural proteins, following the (autocatalytic) cleavage of the Capsid protein, the remaining polyprotein translocates into the ER host proteases cleave the protein into the precursor E2 and E1 proteins both of which are N-glycosylated before being transported to TGN-46 positive structures -the viral replication center- in close proximity to the Golgi via NDP52 positive autophagy-like vesicles.

Prototype Alphavirus particle and genome

CHIKV proteins and autophagy: NDP52 positive particles
are transported to the viral RTC (model)

CHIKV and the ER stress response: structural proteins

Since the polyprotein containing the structural proteins (excluding the Capsid protein) translocates into the ER where it is modified, expression of the CHIKV glycoproteins -namely E3E26KE1- by itself outside the context of viral replication might be sufficient to induce the ER stress response. Indeed, expression of the polyprotein in Vero cells results in the upregulation of both ATF4 and BiP/GRP78 analogue to the expression of Sindbis Virus (SINV) E2 and E1 proteins, as well as enhancing tunicamycin induced expression of ATF4 and BiP/GRP78.

If however the expression of CHIKV E3E26KE1 under these conditions also results in the induction of ATF6, IRE1, and subsequent activation of Caspase -4/-12 as well as autophagy has not been determined, but seems likely. It should be noted that in the case of SINV E2 and E1 proteins, BiP associates with both E2 and E1 thus activating ATF6 and IRE1 mediated pathways.

CHIKV and the ER stress response: non-structural protein 2 (nsP2)

In Vero cells transfected with a plasmid expressing nsP2 and treated with tunicamycin, at 16 h post transfection, tunicamycin mediated induction of both BiP and ATF4 reporter genes are inhibited. Since this inhibition is dependent on the ability of nsP2 to localise to the nucleus or to induce shut-off of the host cells transcription by degradation of the catalytic subunit of RNA Polymerase II, the expression of CHIKV nsP2 might inhibit the ER stress response by inhibiting UPR associated increase in selective translation induced by PERK phosphorylated eIF2α.

CHIKV and ER stress: induction of the UPR by viral envelope proteins

Since the expression of the viral envelope proteins induces the ER stress response whilst the expression of the viral nsP2 protein inhibits the same, the final aspect we have to discuss is if the infection of cells in vitro and of mice in vivo with CHIKV induces the ER stress response. Following the infection of Vero cells with CHIKV the induction of UPR reporter genes was measured at 6 and 16 hrs p.i., at a timepoint where the induction of the ER stress response induces autophagy as well as oxidative stress but when the formation of the viral replication centers does not take place. Compared to cells treated with tunicamycin (positive control), CHIKV infected Vero cells do not upregulate the expression of GRP94, ATF4, or CHOP reporter genes despite increased phosphorylation of eIF2α as early as 8 hrs p.i. . Taken together with the results obtained from Vero cells transfected with nsP2 this indicates that nsP2 is at least partly responsible for inhibiting eIF2α mediated increase of reporter gene expression despite the expression of E2 and the E3E2 precursor.

Because SINV has been reported to induce the ATF6 and IRE-1 mediated ER stress response, it might be possible that CHIKV infection mediates the accumulation of the spliced version of XBP1 and thus activates CHOP. Indeed, CHIKV infection of Vero cells as well as primary murine splenic macrophages and murine embryonic fibroblasts, partially activates XBP1 but to a lesser extent than in cells treated with tunicamycin. Despite only partially inhibition of XBP1 splicing, XBP1 protein cannot be detected in the nucleus of infected cells, indicating that sXBP1 is rendered inactive. Additionally, spliced XBP1 is absent from tissue derived from mice infected with CHIKV at 6 d p.i. despite displaying signs of CHKV specific inflammation.

CHIKV non-structural and structural proteins and the UPR

In the context of viral infected cells the inhibition of the ER stress response by the expression of CHIKV proteins might be part of counteracting an antiviral response induced by the expression of the envelope proteins. The induction of the ER stress response not only induces autophagy and apoptosis -both antiviral responses in themselves- but also triggers inflammatory pathways, including p38/MAP Kinase, JNK kinase, and the NF-κB mediated induction of TNF-α or IL-6. In the case of CHIKV, this is corroborated by findings that both CHKIV nsP2 and SINV nsP2 are potent inhibitors of STAT1 phosphorylation as mutations in CHIKV nsP2 (P718S) and Sindbis virus (SINV) nsP2 (P726S) render replicons sensitive to IFN induced signalling. The importance of nsP2 is also highlighted by the observation that in CHIKV infected cells, nsP2 (in addition to nsP3 and nsP4) is stabilised by heat shock protein 90 (Hsp90) and that inhibition of Hsp90 not only decreases viral replication and inflammation but also nsP2 levels. Since in cells not expressing nsP2, nsP3, or nsP4 Hsp90 stabilises IRE-1 and is thus required for the induction of the UPR, it might be possible that the interaction with CHIKV nsP's contributes to the inhibition of IRE-1 mediated signalling pathways, including splicing of XBP1.

CHIKV proteins, UPR, and the immune response

Tuesday, 18 November 2014

HIV-1 and autophagy: Gag, Nef, and Tat

Human Immunodeficiency Virus (HIV) is a lentivirus and the causative agent of Acquired Immunodeficiency Syndrome (AIDS), infecting cells of the human immune system including CD4+ T lymphocytes, dendritic cells and macrophages as well as cells of the nervous system, in particular microglia. The infection of these cells can lead apoptosis or to latently infected cells, the latter constituting a viral reservoir.

HIV genome (linearised)

HIV-1 Gag, Nef and autophagy: inhibition of autophagy by Beclin-1 binding?

Patients infected with a virus that harbours a deletion of the nef gene either do not progress to full blown AIDS or -if so- with a considerable delay compared to patients infected with “standard” HIV as well as lower virus loads, suggesting that the expression of Nef is necessary for maintaining high viral loads and AIDS pathogenicity. In addition, deletions of nef within the genome of the simian equivalent of HIV, SIV, have similar effects in Rhesus monkeys. In general, Nef is believed to contribute to the pathogenesis of AIDS by interfering with the antiviral immune response on multiple levels, including the downregulation of cell surface molecules (MHC-Class I, Tetraspanins, and CD4) thus preventing the recognition of infected cells by CD8⁺ lymphocytes and promoting the dissemination of infectious virions respectively, thus partially overlapping with the functions of other accessory proteins such as Vpu, Vif, and Vpr/Vpx (in HIV-2). In addition, Nef from non-pathogenic SIV has been shown to downregulate the expression of TCR-CD3 thus preventing the activation of bystander T lymphocytes - similar to HIV-1 Δ Nef but not wt HIV-1. In all instances however are indirect effects of Nef expression rather than direct functions.

HIV-1 Nef: domains (top) and membrane topology (bottom)

As part of the innate antiviral response against HIV-1 in macrophages, HIV-1 is degraded via autophagy in a Nef dependent manner as cells infected with a HIV-1 Δ Nef exhibit increased viral titers, suggesting that Nef inhibits either the formation of mature autophagosomes or alternatively the degradation of mature autophagosomes by inhibiting the fusion with the lysosome, akin to the coronaviral PLP2 or nsp-6. Experimental evidence suggests that Nef does bind Beclin-1 either directly or indirectly thus blocking the maturation of the autophagosome but interestingly not the induction of autophagy. Indeed, Nef co-localises with both Atg7 and Atg12 in addition to Beclin-1 and 2xFYVE-GFP ( a marker for membranes containing phosphatidylinositol 3-phosphate (PI3P) ) probably by binding Beclin-1, indicating that the formation of phagosomes is not impaired. So how is the fusion with lysosomes inhibited? As mentioned in a previous post, Beclin-1 is also required for the fusion of the lysosome with the autophagosome via a complex anchored at the lysosomal membrane, a process which can be inhibited by Rubicon binding to Beclin-1. In the case of HIV-1 Nef, binding of Nef to the complex therefore might either prevent UVRAG from binding Beclin-1 or the Beclin-1/UVRAG complex from being formed at the lysosome (personal opinion). Given that Nef not interferes with the formation of autophagic vesicles, the binding of Nef per se seems not sufficient to impair the function of Beclin-1 during the formation of the phagosome suggesting that Nef either does not bind to ER localised Beclin-1 or is prevented from doing so by a different HIV-1 derived protein. Unfortunately, the author of this post is not aware of any study expressing Nef by itself in the context of investigating autophagy, but past results obtained by the author of this post indicate that in SupT1 cells Nef does exhibit a punctate distribution pattern in the cytoplasm.

Localisation of Nef in SupT1 cells stably transfected with a inducible plasmid treated with 4-HT (middle and bottom)

or untreated (top) and co-transfected with HIV-1 Vpr (top and bottom) Arrows indicate punctae in Nef expressing cells

One possibility is that in infected cells another viral protein is inducing autophagy which is subsequently inhibited by Nef. Indeed, the same study, which showed that Nef inhibits autophagy, also demonstrated that the viral Gag/p17 protein co-localises and co-purifies with LC3-II in extracts of monocyte derived macrophages infected with a pseudotyped VSV -HIV construct. Interestingly, the accumulation of autophagic markers is dependent on Nef -but is the formation of the phagophore? If so, then the question remains how does Nef impair the degradation of the autophagosome? The answer might be, that Nef interacts with a (newly identified) negative regulator of autophagy, Golgi-associated plant pathogenesis-related protein 1 (GAPR-1). Transfection of HeLa cells with a siRNA targeting GAPR-1 increases the number of Beclin-1/WIPI2 positive punctae, and thus early autophagosomes, whereas in control cells Beclin-1 is mainly localised to the Golgi. GAPR-1 therefore tethers Beclin-1 to the Golgi and renders Beclin-1 inactive. Binding of Golgi resident Beclin-1 -but not ER resident Beclin-1- by Nef might lead to the formation of Beclin-1/Nef/GAPR-1 complex, thus preventing Golgi resident Beclin-1 from being forming a complex with UVRAG. I should note that this represents a model which needs to be tested, but may be supported by findings that the expression of a D174A/D175A mutant of Nef which does not bind Beclin-1 still inhibits autophagy despite that a synthetic peptide which represents amino acids 257–337 of Beclin-1 -representing the region within the beclin 1 evolutionarily conserved domain (ECD) that binds Nef, counteracts Nef mediated inhibition of autophagy and decreases viral replication. The presence of both Nef and Gag at the site of the formation of the phagophore can be explained by the interaction of Nef with Gag.

Domains of Beclin-1 (top) and potential interaction with Nef (bottom)

Consequently, the formation of the phagophore may or may not dependent on Gag. It is interesting though that the expression of Nef increases the levels of Gag, which makes sense if Nef prevents the degradation of Gag by autophagy and allows the targeting of Gag-Pol to the site of viral assembly (the plasma membrane) instead. Alternatively, the Nef-Gag complex located at the ER might be directed to the site of viral assembly and the inhibition of the degradation of this complex by autophagy be a mechanism to increase traffic of the complex to the plasma membrane. Indeed, the transport of ATP from the amphisome to the plasma membrane in vesicles in a VAMP7 dependent manner has been reported.
Furthermore, in cells infected with a pseudotyped VSV- Δ Nef-Gag virus, Gag accumulates at the ER instead the plasma membrane, but the effect of chemicals inhibiting autophagy has not been determined (yet).

Based on results obtained by the author of this post in the past, in the presence of HIV Vpr, Nef positive punctae are largely absent if Vpr is expressed. HIV Vpr has been show to induce mitochondrial damage and thus potentially not only induce apoptosis but also mitophagy. If this is the case, then Nef might facilitate and not block the engulfment of mitochondria in LC3 positive vesicles whilst preventing degradation of damaged mitochondria in this specific case. Since Nef itself induce the oxidative response and depolarisation of mitochondria under hypoxia indicated by results published in 2011, the accumulation of depolarised mitochondria in Nef expressing cells might lead to apoptosis and incomplete mitophagy itself.
In SupT1 cells stably expressing an inducible Nef plasmid, the expression of both Nef and Vpr damages mitochondria as indicated by loss of mitochondrial potential and induce mitochondria dependent apoptosis. Indeed both the expression of Vpr and Nef (as well as the combined expression) induce apoptosis, although it is not clear if this is due to inhibition of the clearance of damaged mitochondria by mitophagy via inhibiting mitophagy or not.

Nef and Vpr in SupT1 cells stably expressing inducible Nef plasmid: Mitochondrial depolarisation
(CCCP: positive control)

Expression of Nef in the context of a proviral construct does not prevent apoptosis

HIV-1 Tat

HIV-1 not only infects cells of the immune system such as macrophages or T lymphocytes but also enters the brain early during the infection, infecting- an albeit limited number of- microglia and astrocytes. Long-term survival of these cells has been implicated in serving as a viral reservoir and be one of the obstacles in treating HIV-1 positive patients. As outlined in previous posts, one of the mechanisms preventing apoptosis of cells infected with viruses in general is the expression of antiapoptotic proteins while autophagy represent a second mechanism to counteract apoptosis. As outlined above, the Nef protein of HIV inhibits autophagy and the inhibition of autophagy by Influenza Virus M2 protein has been implicated in inducing apoptosis. Regarding HIV-1 Nef, preliminary data obtained by the author of this blog indicate that the expression of the proviral pNL43-Δ Nef nor pNL43- Nef* does prevent apoptosis in transfected SupT1 cells (whether these constructs inhibit autophagy or not was not tested) suggesting that under these conditions Nef does not contribute to the induction of apoptosis. It should be noted however, that the expression of Nef by itself induces caspase dependent apoptosis in various cell lines.

Among the proteins that regulate the interplay between autophagy and apoptosis, it has been reported that the expression of BAG3 enhances autophagy whilst inhibiting apoptosis as well as degradation of proteins via the proteasomal pathway. Indeed, BAG3 expression is upregulated in both glial and T lymphocytes infected with HIV-1. Following infection of the host cell with HIV the viral genome is imported into the nucleus where it is integrated into the host genome. In the absence of any stimulation, the integrated genome is not expressed (latent phase). In order to stimulate gene expression, the viral enhancer and promoter elements contained in the viral long term element (LTR) located at the end of integrated provirus need to be activated by HIV Tat (trans-activator of transcription), one of the earliest genes being expressed. Tat interacts with cellular histone acetyltransferases (HATs) in particular p300/CBP, that are recruited to the viral LTR, thereby acetylating nucleosomes within the promoter region.

In addition to activating the expression of viral genes, Tat also activates the expression of cellular genes. Following the transfection of a plasmid allowing the expression of HIV-1 Tat into normal human astrocytes as well as U87MG cells expressing GFP-LC3, the formation of mature autophagosomes can be observed as evidenced by an increase in GFP-LC3 positive punctae as well as LC3-II. Additionally, these punctae co-localise with lysosomal markers (although no control experiment using Chloroquine nor E-64d has been conducted). Since the levels of BAG3 are increased following the transfection with Tat and cells transfected with siRNA targeting BAG3 do not show an increase in mature autophagosomes it has been proposed that Tat either increases BAG3 levels independent of inducting BAG3 expression (as evidenced by qRT-PCR). Furthermore, the increase in BAG3 protects cells from apoptosis thus linking the induction of autophagy by Tat to the inhibition of apoptosis. Although the mechanism of how Tat stabilises BAG3 is not known, it might be possible that Tat expression facilities the accumulation of misfolded proteins in the ER and that BAG3 is stabilised as part of the ER stress response. Indeed, Tat has been reported to induce the ER stress response under hypoxia.

Nef, Gag, and Tat induce autophagy whilst Nef also inhibits
the fusion of the autophagosome with the lysosome

In summary, the inhibition of Nef of the autophagy pathway might explain the accumulation of endosome like structures and vacuoles in infected cells. Short peptides composed of the Beclin-1 interaction of Nef but lacking the ability of Nef to localise to the Golgi might stimulate autophagy and decrease viral titers in patients infected with HIV-1 Δ Nef. Vice versa, peptides derived from the domain of Beclin-1 interacting with Nef have been shown to inhibit the replication of West Nile Virus (WNV) and Chikungunya Virus (CHIKV) in mice as well as HIV-1 in vitro. In these cases the peptide tested, Tat-Beclin1, binds viral proteins that otherwise bind cellular Beclin-1 and thus inhibit viral proteins that inhibit autophagy. In other words, it increases autophagy. Personally I would like to test this peptide in cells expressing the coronaviral nsp-6 and/or PLP2.

In the case of Tat, Tat might induce the accumulation of (misfolded) proteins in the ER under hypoxia conditions and maybe under normal atmospheric conditions and thus induce BAG3 mediated autophagy. Unfortunately, a paper describing the inhibition of ER stress apoptosis by HIV-1 Nef had to be retracted.

Further reading

Kyei GB, Dinkins C, Davis AS, Roberts E, Singh SB, Dong C, Wu L, Kominami E, Ueno T, Yamamoto A, Federico M, Panganiban A, Vergne I, & Deretic V (2009). Autophagy pathway intersects with HIV-1 biosynthesis and regulates viral yields in macrophages. The Journal of cell biology, 186 (2), 255-68 PMID: 19635843

Haller C, Müller B, Fritz JV, Lamas-Murua M, Stolp B, Pujol F, Keppler OT, & Fackler OT (2014). HIV-1 Nef and Vpu are Functionally Redundant Broad-Spectrum Modulators of Cell Surface Receptors Including Tetraspanins. Journal of virology PMID: 25275127

Shoji-Kawata S, Sumpter R, Leveno M, Campbell GR, Zou Z, Kinch L, Wilkins AD, Sun Q, Pallauf K, MacDuff D, Huerta C, Virgin HW, Helms JB, Eerland R, Tooze SA, Xavier R, Lenschow DJ, Yamamoto A, King D, Lichtarge O, Grishin NV, Spector SA, Kaloyanova DV, & Levine B (2013). Identification of a candidate therapeutic autophagy-inducing peptide. Nature, 494 (7436), 201-6 PMID: 23364696

Geist MM, Pan X, Bender S, Bartenschlager R, Nickel W, & Fackler OT (2014). Heterologous Src homology 4 domains support membrane anchoring and biological activity of HIV-1 Nef. The Journal of biological chemistry, 289 (20), 14030-44 PMID: 24706755

Dinkins, C., Pilli, M., & Kehrl, J. (2014). Roles of autophagy in HIV infection Immunology and Cell Biology DOI: 10.1038/icb.2014.88

Dinkins, C., Arko-Mensah, J., & Deretic, V. (2010). Autophagy and HIV Seminars in Cell & Developmental Biology, 21 (7), 712-718 DOI: 10.1016/j.semcdb.2010.04.004 Yang YP, Hu LF, Zheng HF, Mao CJ, Hu WD, Xiong KP, Wang F, & Liu CF (2013). Application and interpretation of current autophagy inhibitors and activators. Acta pharmacologica Sinica, 34 (5), 625-35 PMID: 23524572

Kang R, Zeh HJ, Lotze MT, & Tang D (2011). The Beclin 1 network regulates autophagy and apoptosis. Cell death and differentiation, 18 (4), 571-80 PMID: 21311563

Zhong Y, Wang QJ, Li X, Yan Y, Backer JM, Chait BT, Heintz N, & Yue Z (2009). Distinct regulation of autophagic activity by Atg14L and Rubicon associated with Beclin 1-phosphatidylinositol-3-kinase complex. Nature cell biology, 11 (4), 468-76 PMID: 19270693

Minoia M, Boncoraglio A, Vinet J, Morelli FF, Brunsting JF, Poletti A, Krom S, Reits E, Kampinga HH, & Carra S (2014). BAG3 induces the sequestration of proteasomal clients into cytoplasmic puncta: implications for a proteasome-to-autophagy switch. Autophagy, 10 (9), 1603-21 PMID: 25046115

Gamerdinger M, Kaya AM, Wolfrum U, Clement AM, & Behl C (2011). BAG3 mediates chaperone-based aggresome-targeting and selective autophagy of misfolded proteins. EMBO reports, 12 (2), 149-56 PMID: 21252941

Carra S, Seguin SJ, & Landry J (2008). HspB8 and Bag3: a new chaperone complex targeting misfolded proteins to macroautophagy. Autophagy, 4 (2), 237-9 PMID: 18094623

Bruno, A., De Simone, F., Iorio, V., De Marco, M., Khalili, K., Sariyer, I., Capunzo, M., Nori, S., & Rosati, A. (2014). HIV-1 Tat protein induces glial cell autophagy through enhancement of BAG3 protein levels Cell Cycle DOI: 10.4161/15384101.2014.952959

Romani B, Engelbrecht S, & Glashoff RH (2010). Functions of Tat: the versatile protein of human immunodeficiency virus type 1. The Journal of general virology, 91 (Pt 1), 1-12 PMID: 19812265

Tiede LM, Cook EA, Morsey B, & Fox HS (2011). Oxygen matters: tissue culture oxygen levels affect mitochondrial function and structure as well as responses to HIV viroproteins. Cell death & disease, 2 PMID: 22190005