Báo cáo khoa học: Weapons of STAT destruction Interferon evasion by paramyxovirus V proteins

doi:10.1111/j.1432-1033.2004.04425.x

Eur. J. Biochem. 271, 4621–4628 (2004) (cid:1) FEBS 2004

M I N I R E V I E W

Weapons of STAT destruction Interferon evasion by paramyxovirus V proteins

Curt M. Horvath

Department of Medicine, Evanston Northwestern Healthcare Research Institute, and Departments of Medicine and Biochemistry, Molecular Biology & Cell Biology, Northwestern University, Evanston, IL, USA

tein called V plays a central role in this process. The theme of V-dependent interferon evasion and its variations provide significant insights into virus–host interactions and viral immune evasion that can help define targets for antiviral drug design. Exposure of the viral weapons of STAT destruction may also be instructive for application to STAT-directed therapeutics for diseases characterized by STAT hyperactivity.

Keywords: antiviral; interferon; paramyxovirus; STAT; viral evasion.

The signal transducer and activator of transcription (STAT) family of proteins function to activate gene transcription downstream of myriad cytokine and growth factor signals. The prototype STAT proteins, STAT1 and STAT2, are required for innate and adaptive antimicrobial immune responses that result from interferon signal transduction. While many viruses have evolved the ability to avoid these antiviral cytokines, the Paramyxoviruses are distinct in their abilities to interfere directly with STAT proteins. Individual paramyxovirus species differ greatly in their precise mech- anism of STAT signaling evasion, but a virus-encoded pro-

Interferons: the antiviral cytokines

The interferon (IFN) family, including type I (IFNa, IFNb) and type II (IFNc), refers to a group of cytokines that are capable of modulating diverse biological responses such as immune regulation, tumor inhibition, cell growth arrest, innate antimicrobial responses, and promotion of adaptive immunity. Type I IFNs have long been associated with the ability to diminish virus replication [1], and this antiviral activity is the result of IFN-induced changes in cellular gene expression (reviewed in [2–4]). Cellular response to IFN leads to the establishment of an antiviral state, a process that requires new mRNA and protein synthesis of many IFN- stimulated gene (ISG) products that contribute to the antiviral responses required to limit diverse virus families.

Immediate responses to virus infection result in rapid transcriptional activation of type I IFN, typified by the

single human IFNb gene (Fig. 1). This IFN induction is initiated by diverse virus replication intermediates, including dsRNA as well as other Toll-like receptor (TLR) ligands [5,6]. In response to these signals, serine/threonine kinases activate immediate-responding transcription factors inclu- ding interferon regulatory factor (IRF) 3, AP1(ATF2/ c-Jun), and NFjB, which rapidly mobilize to the IFNb enhancer where they collaborate to recruit a series of transcriptional coactivators that remodel the enhancer chromatin and enable RNA pol II transcription [7]. The newly synthesized IFNb is secreted from the primary infected cell and signals to adjacent cells through direct binding to a transmembrane type I IFN receptor on the cell surface. The receptors are phosphorylated by associated Janus family tyrosine kinases, leading to receptor tyrosine phosphorylation. Latent STAT2 in association with IRF9 [8,9] binds to these docking sites, and becomes phosphor- ylated, followed by the recruitment and tyrosine phosphory- lation of latent STAT1 [10]. The STATs heterodimerize via SRC homology 2 (SH2) domain–phosphotyrosine inter- actions, and together with the STAT-associated IRF9, assemble into a heterotrimeric complex known as the IFN- stimulated gene factor 3, ISGF3 [11–15]. ISGF3 rapidly accumulates in the nucleus, binds to conserved IFN- stimulated response element (ISRE) sequences on IFNa/ b-stimulated gene promoters, and increases their transcrip- tion rates. One ISG target is IRF7, which combines with IRF3 to amplify the IFN response by inducing the expression of the numerous IFNa genes [16–18].

Despite the negative selective pressure exerted by IFN signaling on viruses, the very existence of successful infectious and pathogenic viruses in IFN-competent hosts demonstrates their ability to resist host defenses. In fact, many well-characterized virus adaptations allow them to

Correspondence to C. M. Horvath, Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Pan- coe Pavillion, Room 4401, 2200 Campus Drive, Evanston, IL 60208, USA. Tel.: +1 847 491 5530, E-mail: horvath@northwestern.edu Abbreviations: CTD, C-terminal domain; CRM1, chromosomal region maintenance 1; DDB1, UV-damaged DNA binding protein 1; E1, Ub-activating enzyme; E2, Ub-conjugating enzyme; E3, Ub- ligating enzyme; IFN, interferon; ISG, IFN-stimulated gene; ISRE, IFN-stimulated response element; NES, nuclear export signal; NDV, Newcastle disease virus; SH2, SRC homology 2; STAT, signal trans- ducer and activator of transcription; SV5, simian virus 5; TLR, Toll- like receptor; Ub, ubiquitin; VDC, V protein-dependent degradation complex; VIP, V interaction protein. (Received 7 January 2004, revised 6 February 2004, accepted 7 October 2004)

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Fig. 1. IFN biosynthesis and antiviral signal transduction induced by virus infection. Left cell illustrates IFNb biosynthesis. In response to virus infection, Toll-like receptor (TLR) signal transduction, or intracellular dsRNA, pathogens activate IRF3 and NFjB transcription factors which combine with ATF2/c-jun to initiate IFNb transcription. IjB, inhibitor of NFjB. Right cell illustrates the cellular response to the released IFNb. Interaction with specific cell surface receptor initiates a tyrosine phosphorylation signaling program. Upon tyrosine phosphorylation, latent STAT1 and STAT2 (which can be preassociated with IRF9 [8,9]) heterodimerize to induce the trimeric IRF9–STAT1–STAT2 complex, ISGF3. ISGF3 binds to response elements in the promoters of target antiviral genes, and increases the rate of transcription. JAK1, Janus kinase 1; Tyk2, tyrosine kinase 2.

virus IFN evasion strategies have revealed unique abilities to directly target STAT components of IFN signal trans- duction. While phenotypically similar, the molecular mech- anisms of STAT-directed IFN evasion are as diverse as the viruses themselves. These similarities and differences are highlighted in the descriptions below.

Fig. 2. Targets for virus evasion of IFN antiviral responses. Vulnerable access points for virus evasion include: (1) Induction of IFN biosynthesis, (2) Interaction between IFN and receptor, (3) IFN signal transduction and (4) Activities of antiviral effectors.

Paramyxoviruses and their V protein

evade IFN-induced innate antiviral responses through a number of access points vulnerable to viral invaders [3,19] (Fig. 2). For some viruses, the common early steps of IFN induction are targeted by general inhibitory mechanisms that can occur via dsRNA sequestration or signaling interference to antagonize IRF3 and NFjB pathways, protein kinase inhibition by protein–protein interaction, or TLR signaling interference by viral products. In other cases, virus-encoded soluble IFN receptors or receptor antagonists block cytokine signalling. Viruses can also block specific antiviral effectors to preserve key cellular machinery needed for their replication. Recent investigations of paramyxo-

Paramyxoviruses encompass a large family of enveloped, negative strand RNA viruses that cause zoonotic diseases including significant human pathogens like measles virus

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findings demonstrate that a fundamentally important activity associated with a variety of paramyxovirus V proteins is direct interference with STAT protein function, but individual genera within the family exhibit remarkably diverse mechanisms of STAT inhibition.

Fig. 3. Paramyxovirus coding strategies for accessing the conserved V protein CTD. Diagrams illustrate the open reading frames generated in alternate mRNAs that encode C-terminally unique P and V proteins. Coloring indicates common translational reading frames. CTD, cysteine-rich, V-specific C-terminal domain. (A) Coding strategy used by the Rubulavirus genus. The colinear mRNA encodes the V protein from a single translational reading frame, but site-specific addition of two nontemplated guanine nucleotides (+2G) generates a second (cid:1)edited(cid:2) mRNA encoding the P protein from two overlapping reading frames. (B) Coding strategy used by the Henipavirus, Morbillivirus, and Respirovirus genera. The colinear mRNA encodes the P protein, but site-specific addition of a single nontemplated guanine nucleotide (+1G) generates a second (cid:1)edited(cid:2) mRNA encoding the V protein from two overlapping reading frames. In Sendai virus, both transcripts encode a third overlapping open reading frame that encodes a nested set of C proteins (C¢, C, Y1, and Y2). (C) Comparison of the amino acid sequences in the V-specific C-terminal domain of several paramyxoviruses. Boxes highlight conserved amino acids.

Rubulavirus V proteins: STAT ubiquitin ligases

and mumps virus, or the more treacherous Nipah virus and Hendra virus. The large family Paramyxoviridae is subdi- vided into several genera, including the Rubulavirus, Hen- ipavirus, Morbillivirus, and Respirovirus groups. All of these viruses share common structural, biochemical, and genetic elements including the single-stranded RNA genome that encodes a small number of proteins, including surface glycoproteins and several subunits of an RNA-dependent RNA polymerase (reviewed in [20]). One locus contains a polycistronic gene that encodes two or more viral proteins from overlapping ORFs that code for the phosphoprotein, P, a second protein named V (Fig. 3), and in some species additional overprinted proteins called C, W, X or Y. Due to a unique coding strategy involving generation of alternate mRNAs via cotranscriptional insertion of nontemplated guanine nucleotides [21], the paramyxovirus P and V proteins are amino coterminal but have unique C-termini. Paramyxovirus V proteins are readily identifiable by a highly conserved cysteine-rich domain at their C-termini derived from the overlapping ORF [20–22]. This conserved C-terminal domain (CTD) is approximately 50% identical among all paramyxovirus V proteins and contains seven invariant cysteine residues. This domain enables the V protein to bind two atoms of zinc, a stoichiometry similar to that found in some cellular zinc-binding proteins [22,23]. Aside from this outward resemblance, it is important to note that V proteins have no cellular homologues and that the spacing of CTD cysteine residues is not consistent with known cellular zinc-binding domains including the RING, PHD, or LIM motifs [24]. Paramyxovirus host evasion has been ascribed to this locus, and a diverse range of host evasion activities, including IFN signaling inhibition [25], prevention of apoptosis [26,27], cell cycle alterations [28], inhibition of double-stranded RNA signaling [27,29], and prevention of IFN biosynthesis [26,27,29] have been ascribed to paramyxovirus V proteins. A number of recent

The STAT proteins are well known to cycle between active and inactive states as the result of reversible post-transla- tional modification, namely tyrosine phosphorylation and dephosphorylation [30–32]. The estimated half-lives of STAT1 and STAT2 are in the order of days rather than hours [32–34], but this long half-life can be greatly reduced upon infection with Rubulavirus species or following expression of the Rubulavirus V protein. In the prototype example, STAT1 protein accumulation was found to be dramatically reduced by infection of cells with simian virus 5 (SV5) [25]. This STAT1 targeting was conferred by the sole expression of the SV5 V protein, and similar STAT degradation properties were soon found to be shared by V proteins from a variety of Rubulaviruses [2,25,34–40]. Chemical proteasome inhibitors can prevent STAT degra- dation by Rubulavirus V proteins [25,34]. Moreover, expression of the Rubulavirus V proteins induces polyubi- quitylation of specific target STATs [34,39–41]. Character- ization of bacterially expressed SV5 and type II human parainfluenza virus (HPIV2) V proteins in vitro revealed an intrinsic ability to catalyze the transfer of ubiquitin (Ub) in a reaction that required ATP, Ub-activating enzyme (E1), and Ub-conjugating enzyme (E2). This intrinsic enzymatic activity meets the definition of a Ub-ligating enzyme (E3), but the in vitro reaction failed to fully recapitulate the native reaction because it was substrate-independent and gener- ated only mono-Ub transfer rather than a poly-Ub chain [41]. In intact cells that express V protein, the complete

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STAT targeting by polyubiquitylation results in protea- some-dependent degradation.

range determinant for this virus [35]. SV5 does not replicate efficiently or cause STAT1 degradation in the mouse [45,46], where the murine STAT2 protein is unusually divergent in amino acid sequence [47–49]. Expression of human STAT2 in mouse cells (cid:1)rescues(cid:2) the defective STAT1 targeting and provides the virus with a replication advantage [35]. The Newcastle disease virus (NDV, a member of the Avulovirus genus that is restricted to avian species) also encodes a V protein that can antagonize the avian IFN system [50]. Like SV5, the NDV IFN inhibition is species-restricted. The molecular basis underlying this NDV species-specificity has not yet been revealed, but it is interesting to speculate that avian STAT2 might be involved.

It is striking that, in spite of the high amino acid sequence identity between V proteins and similar abilities to target STAT proteins for proteasomal degradation, Rubulavirus species differ in their specificity. While the SV5 V protein can target STAT1 for polyubiquitylation and proteasomal degradation, HPIV2 V protein targets STAT2 [34], and mumps virus V protein can eliminate both STAT1 [42] and STAT3 [40]. Affinity purification of Rubulavirus V proteins from host cells identified strikingly similar patterns of V interaction protein (VIP) partners (Fig. 4) but in detail each species also exhibited unique superimposed VIP patterns. These differences in the VIP composition have been suggested to account for differential V protein activities and target specificity [40,41].

Fig. 4. Schematic diagram of the Rubulavirus VDC ubiquitin ligase complex. In this model, the box representation of the SV5 V protein serves as a nucleation site for protein inter- actions that coordinate the transfer of ubiqu- itin (Ub) via Ub-conjugating enzymes (E2) to the specific STAT protein target (in this case, STAT1). Colored ovals represent the cellular V interaction protein components required for complete E3 Ub ligase activity to generate polyubiquitylation of STATs, some of which are identified as DDB1, Cullin 4A, STAT1, STAT2. Also depicted are the Ub-activating enzyme, E1, and polyubiquitylation leading to degradation via the proteasome. This model is illustrative only, is not drawn to scale, and does not accurately portray protein inter- action sites or stoichiometry.

Henipavirus V proteins: STAT sequestration in high molecular mass cytoplasmic complexes

Nipah virus and Hendra virus are the two known species of a recently emerged and deadly paramyxovirus genus, Henipavirus, that was responsible for outbreaks of res- piratory disease and fatal encephalitis in humans and livestock in Malaysia and Australia [51,52]. Both Henipa- virus species were demonstrated to share V-dependent IFN signaling evasion properties with other paramyxoviruses [50,53–55].

Nucleotide sequencing of

The STAT-targeting machinery consists of V protein- dependent degradation complexes (VDCs) that contain the V protein and VIPs including STAT1 and STAT2 (and STAT3 in the case of mumps virus). A number of additional cellular proteins including DDB1, a UV-damaged DNA binding protein [40,41,43,44], and members of the Cullin family of ubiquitin ligase subunits including Cullin 4A are also required [40,41] (Fig. 4). RNA interference experiments demonstrate that DDB1 and Cullin 4A are required for STAT1 degradation by SV5, lending support to the model that the VDC is a coalition of virus-encoded and host factors that together function as a STAT-directed E3 ubiquitin ligase enzyme [41].

Somatic cell genetics and biochemical analysis has revealed that all of the Rubulavirus V proteins require the participation of a nontarget STAT for their in vivo E3 Ub ligase activity [36]. SV5 can only target STAT1 in cells that express STAT2, while HPIV2-mediated STAT2 degrada- tion fails in the absence of STAT1. For mumps virus, STAT1 targeting requires cellular STAT2, but STAT3 targeting is STAT2-independent [40]. A powerful confirma- tion for the role of STAT2 in STAT1 destruction by SV5 was provided by the discovery that STAT2 acts as a host

the Henipavirus genomes revealed many similarities with other paramyxoviruses, including a polycistronic gene encoding a V protein CTD [52,56]. In comparison to the STAT-degrading Rubulavirus V proteins, the V proteins of Nipah virus and Hendra virus share (cid:1) 50% amino acid identity within the CTD (Fig. 5). The Henipavirus V protein N-terminus is larger and entirely unique compared to other paramyxovirus proteomes and has no obvious homology to any cellular protein. This sequence divergence between Henipavirus and Rubulavirus V proteins indicates an alternate mechanism of IFN signaling inhibition. The Henipavirus V proteins are overall (cid:1) 58% identical in amino acid sequence, with (cid:1) 83% identity between amino acids 1–140, (cid:1) 44% identity

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between amino acids 141–405, and (cid:1) 80% identity within the CTD (amino acids 406–457). This sequence conserva- tion accounts for the functional similarity in the IFN evasion activities of the Nipah virus and Hendra virus V proteins. Both Henipavirus V proteins have been demon- strated to subvert IFN responses by sequestering STAT1 and STAT2 in high molecular mass cytoplasmic complexes without inducing their degradation [53,54]. This complex formation prevents IFN-induced STAT tyrosine phos- phorylation [53].

for STAT2 binding, and association with STAT2 conse- quently requires a large overlapping binding site between residues 100–300. Evidence from site-directed mutagenesis suggests that contact between STAT2 and Nipah V requires a conserved peptide including amino acids 230–237. Hence, in intact cells, a coordinately assembled trimeric V–STAT1– STAT2 complex forms that inhibits IFN signal transduc- tion. As these protein interaction domains are absolutely required for V protein IFN evasion activity, they are prime candidates for therapeutic intervention with Henipavirus outbreaks.

two regions important

The region of STAT1 bound by Nipah V was also determined. Nipah V binds to STAT1 but not to STAT3, and only binds to chimeric STAT1–STAT3 fusion proteins when the C-terminal region was derived from STAT1. The results indicate that a STAT1 fragment containing the linker domain and SH2 domain is the target site for Henipavirus V for STAT protein interaction, activation, dimerization, and DNA binding.

Fig. 5. Henipavirus V protein sequence conservation and domain structure. (A) Comparison of Nipah virus and Hendra virus V proteins to SV5 V and HPIV2 V. Percent sequence identities were determined using NCBI BLAST algorithms. (B) Illustration of functional domains mapped in the Nipah virus V protein. (Adapted from [55], see text for details.)

Morbillivirus V proteins: inhibition of STAT nuclear translocation

In addition to the ability to bind to both STAT1 and STAT2, the Henipavirus V proteins exhibit nuclear–cyto- plasmic shuttling behavior that depends on chromosomal region maintenance 1 (CRM1)-dependent nuclear export signals. Not only does this shuttling affect the steady-state subcellular distribution of the V protein, but it also alters the distribution of the latent STAT1. STAT1 is typically observed in both the cytoplasm and nucleus of unstimulated cells, and expression of the Henipavirus V protein efficiently relocalizes the latent STAT1 protein to the cytoplasm [53,54]. Unexpectedly, despite the high degree of sequence conservation within the cysteine-rich CTD, it is dispensable for IFN signaling inhibition [50].

Dissection of Nipah V protein functional domains revealed insights into the molecular mechanisms underlying Henipavirus IFN evasion and explained the dispensable role of the CTD [55]. Three V protein activities, nuclear export, STAT protein interaction, and IFN signaling inhibition, all map to the N-terminal portion (Fig. 5B). A novel nuclear export signal (NES) was identified within Nipah V amino acids 174–192. Deletion or substitution within the NES prevents V protein cytoplasmic accumulation and also prevents redistribution of latent STAT1 to the cytoplasm. However, the ability to thwart IFN-dependent STAT1 and STAT2 nuclear translocation remains intact regardless of NES mutation, suggesting that the shuttling behavior of the V protein has a distinct role in Henipavirus biology.

Measles virus, a prototype species of the Morbillivirus genus, encodes a V protein distinct from both the Rubulavirus and Henipavirus genera, sharing only (cid:1) 20% overall amino acid sequence identity. Despite the divergence, measles virus V protein is an efficient inhibitor of IFN signal transduction but acts via a mechanism distinct from either Rubulavirus or Henipavirus V proteins [58]. Measles virus V protein expression effectively prevents both IFNa/b and IFNc- induced transcriptional responses. The measles virus V protein does not degrade STATs or prevent IFN-induced STAT protein activating tyrosine phosphorylation, but effectively prevents IFN-induced STAT1 and STAT2 nuclear import. Unlike the Henipaviruses, measles V does not shuttle between nucleus and cytoplasm, and conse- quently does not alter the distribution pattern of latent STAT1.

Dissection of the V protein domains involved in IFN evasion activity and STAT protein interactions revealed that these functions also map within Nipah V amino acid residues 100–300. STAT1 binds independently to residues 100–160, and this interaction site is the primary evasion motif, sufficient to block IFN signaling responses. The amino coterminal P and W proteins [57], or artificial fusion proteins that share this sequence motif [55] can also prevent IFN signaling. Moreover, STAT1 binding is a prerequisite

Affinity chromatography demonstrated that the measles V protein copurifies STAT1, STAT2, STAT3, and IRF9, but not the cellular components required for Rubulavirus VDC ubiquitin ligase function, in agreement with its distinct mechanism of action. In addition, measles V binds to an IFN receptor subunit (IFNAR2.2 or bLong; H. Palosaari and C. M. Horvath, unpublished observations) and a signaling

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adaptor, RACK1 [59], possibly indicating multivalent receptor interactions. The measles V-dependent binding of STAT3 partially inhibits signaling by IL6 and v-Src, which, in conjunction with the mumps virus STAT3 degradation, further suggests a role for STAT3 in antiviral responses.

inhibitors will not only yield new therapeutic targets and vaccination strategies for the control of the infectious diseases themselves, but will also undoubtedly provide insights into new ways to regulate hyperactive cytokine– JAK-STAT signaling that is characteristic of neoplastic and inflammatory diseases.

Acknowledgements

The ability to prevent STAT nuclear import is also observed in measles virus-infected cells, where a dramatic redistribution of cellular STAT proteins is also observed. In measles-infected cells, a portion of the STAT1 and STAT2 proteins are redistributed to cytoplasmic aggregates that also stain for the viral nucleocapsid protein and nucleic acids [58]. Similar intracellular aggregates are observed for many viruses, including mumps virus, where STAT2 is condensed to cytoplasmic bodies. It is enticing to speculate that these bodies represent intracellular sites of virus replication or assembly, possibly indicating that the STATs may also play some role in measles virus replication.

The author is grateful to all the members of the Horvath Laboratory, and wishes to acknowledge the contributions in the study of V proteins made by Jean-Patrick Parisien, Cristian Cruz, Christina Ulane, Jason Rodriguez, Heidi Palosaari, and Tom Kraus. Paramyxovirus research in the Horvath Laboratory is supported by NIH grants AI-50707 and AI-55733.

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