Essay: Kaposi’s Sarcoma

1.1 Characteristics of Kaposi’s Sarcoma
Kaposi’s Sarcoma (KS) was firstly described by a Hungarian dermatologist Moritz Kaposi in 1892, and the original five patients of KS all had a fatal outcome with 2-3 years. However, it was later found to be mostly an indolent disease which is common in the Mediterranean region and in parts of Africa. In 1994 KSHV genomic DNA was identified by Chang et al in KS lesions and the whole viral genome had been sequenced by 1996 (1).
However, now KS has become one of the most frequently reported skin tumor diseases and is often an indolent neoplasm. Since American AIDS was identified in 1981 in three KS reported cases, KS has been regarded as a presenting sign of AIDS and it is reported that the incidence of KS was over 20000 times higher in AIDS patients than in general population (2, 3).
HIV infection is likely to promote tumor formation by compromising immune surveillance mechanisms, as well as other immunodeficiency conditions such as iatrogenic immunodeficiency following solid organ transplantation (4, 5). This explains why KS is often indolent but can be lethal when firstly described.
As mentioned, KSHV was first identified in an endothelial tumor but was later proved to cause lymphoproliferative disorders including Primary effusion lymphoma diseases (PEL) and Multicentric castleman’s disease (MCD) (6).
There’re four classes of KS: Classic KS, endemic or African KS, iatrogenic KS associated with immunosuppressive therapies in transplant patients, epidemic or AIDS-related KS. Detailed characteristics of each class of KS will be introduced in the following sections.
1.2 Pathogenesis of Kaposi’s Sarcoma
In KS, spindle cells are the major proliferating cells, named because of its spindle-like shape. Spindle cells are derived from endothelial cells, expressing many markers of endothelial lineage including CD3, CD34, and CD36 (1).
Although KS lesions can be presented at any time, generally, cutaneous KS is thought be begin with a patch stage. The patch stage is demonstrated by a collection of small, irregular endothelial-lined spaces surrounded by normal dermal blood vessels and accompanied with abundant infiltrate of lympocytes. The patch stage is followed by a plaque stage or nodular stage, with a larger portion of spindle cells which form a slit-like vascular channels containing erythrocytes (1) (7). As mentioned above, evidence indicates that an immunodeficiency is required for the progression from the lesion to a real sarcoma.
The histologic appearance of KS is developed from four processes: proliferation, inflammation, reprogramming, and angiogenesis. After inducing the proliferation of spindle cells, KSHV genes have been shown to up-regulate several signal transduction pathways. For example, up-regulated Akt and STAT3 pathways are needed for the lymphatic reprogramming of endothelial cells. In addition to spindle cells, infiltrating T cells, B cells, monocytes are also found in KS lesions with slit-like neovascular spaces. The inflammatory cells and cytokines are important to maintain latency and promote cell survival in order to support viral growth. An inflammatory microenvironment and angiogenesis also help to recruit KSHV-permissive cells to sites of viral infection and therefore viral spreading (1).
Studies showed that only a small percentage of cells is infected in early patch-lesions, while in more progressed lesions, a higher proportion of spindle cells are latently positive. It is said that latency program drives tumorigenesis (1) (8).
Several KSHV-encoded latent proteins are believed to promote lesion pathogenesis (e.g., LANA, vFLIP, vIL-6). vFLIP, a potent activator of NF-κB pathway, which is essential for the survival of PEL cells. vIL-6 induces vascular endotheli¬al growth factor, exerting influences in an¬giogenesis, vascular permeability and formation of PEL effusions. It has been found that LANA binds p53 and antagonize p53 function, and LANA-expressing cells display resistance to p53-dependent apoptosis, contributing to the cell survival (1) (6).
Also, there’s another evidence showing KSHV lytic proteins also contributing to KS tumor development. Clinical studies showed that In the patients infected with HIV and KSHV for many years, the administration of ganciclovir (GCV), which is a drug that only blocks KSHV lytic infection but not latent infection, is able to prevent the onset of new KS tumors (1). Besides KSHV genes, in the case of HIV infection, the HIV-1 Tat protein promotes both incidence and aggressiveness of KS lesions (8).
1.3 Clinical Features of Kaposi’s Sarcoma
As mentioned above, KS has been classified into four different forms: Classic KS, endemic or African KS, iatrogenic KS, and epidemic or AIDS-related KS. KS occurs at various sites of the body, which frequently localized to the skin, while endemic KS was often found in the lower extremities (1).
Classic KS was firstly identified by Moritz Kaposi and described as an aggressive tumor, which was later recognized to be a rare but frequently indolent skin disease that occurred in Mediterranean and eastern European region. Endemic KS was described by the early 1960s, occurring in sub-Saharan Africa. Endemic KS is histologically similar to classic KS but more aggressive. Iatrogenic KS was found in patients receiving immunosuppressive agents, such as solid organ transplant recipients. And endemic KS was associated with HIV infection, in which KS is found to have a much more aggressive outcome, with common dissemination to different organs (1).
Besides visceral KS, patients also have the risk to develop KSHV related lymphoproliferative disorders such as primary effusion lymphoma (PEL) or multicentric Castleman’s disease (9).
After identified in KS, KSHV viral sequences were also identified in PEL. PELs are of B-cell origin, since they commonly present as lymphomatous effusions in body cavities, PEL is also referred to as body cavity based lymphoma (BCBL). PELs usually are immunoblastic in appearance, having a high mitotic rate and variable amounts of apoptotic debris. And the diagnosis is usually made by immunohistochemistry for KSHV viral latency-associated nuclear antigen (LANA) as consistent infection has made KSHV a defining property of PEL (1) (4). PEL is a very aggressive lymphoma, with an average survival period about 6 months after being diagnosed(10).
Multicentric Castleman’s disease is an atypical lymphoproliferative disorder associated with KSHV. In individuals with HIV, almost all patients with MCD have KSHV in involved lymph nodes. Symptoms of MCD include production of excess cytokines, while progression of MCD were reported to correlate with increased viral amount and IL-6 as well as IL-10 levels(1) (4). Systemic symptoms and inflammation responses are often observed in multiple organs within MCD diagnosis(11).
1.4 Epidemiology of Kaposi’s Sarcoma
KSHV infection can occur at any age, studies in Africa showed that a child is also likely to be KSHV positive if the mother or a sibling is positive for the virus (1).
In endemic KS occurring areas, KSHV have been widely found in saliva and in breast milk, indicating that oral/salivary are one of the major routes of endemic KS transmission. Studies in HIV-positive patients also showed that herpesvirus including KSHV viral loads were higher in saliva, which further prove that the importance of oral/salivary route in KSHV transmission (1) (12).
KSHV infection is found to occur at different rates over the world, with a lowest rates in western Europe and America, and a highest one in Africa. ORF K1 is a transmembrane protein that has a high variation in its sequence. Based on ORF K1 polymorphism, KSHV can be divided into four major subtypes: A, B, C, D. Several studies showed that genotype A is usually found in rapidly progressing KS lesions compared to other genotypes (1).
KSHV is necessary for KS to develop, while patients with a progressing KS have a much higher KSHV viral load compared to patients with a regressing KS (13). Other factors are also identified contributing to the KS progression. Immunosuppression including HIV infection and transplant are believed to have the greatest impact on developing KS. Evidences have shown that herpesvirus including EBV and KSHV were more prevalent in HIV-infected patients than in the age- and sex-matched controls (12). Environmental exposures also have an effect on KSHV seroprevalence, for example, volcanic soils, arthropod bites, and living in rural areas all affect are all associated with the incidence of KS (1).
1.5 Treatment and Prevention of Kaposi’s Sarcoma
The treatment methods include cytotoxic chemotherapy, radiotherapy, surgery and Highly Active Anti-Retroviral Therapy (HAART). In patients with limited cutaneous lesions, HAART were found to have an effective outcome with KS lesions typically decreasing and disappearing in a few weeks or months. Chemotherapies are used for patients who do not respond to HAART or have widespread and aggressive KS symptons(9). Cytotoxic agents with antitumor effects including bleomycin, vincristine, vinblastine, etoposide and paclitaxel are often used for patients with AIDS-KS when systemic chemotherapy is indicated(14).
Clinical studies in Tanzania showed that patients with AIDS-KS are treated with a combination of chemotherapy and HAART with a good outcome (2). Also, there’re other researches suggesting that compared with HAART alone, a combination of therapies can inhibit KS progression effectively, including combinations with pegylated liposomal doxorubicin (PLD), liposomal anthracycline, paclitaxel, etc (15). There’re other studies showed that Rapamycin is able to cause the regression of transplant-related KS, as well as antitumorigenic effects in KSHV-infected xenograft models (16, 17).
To date, no ideal vaccine has been developed to Immunize people in risks. Several reports with regression of AIDS-KS suggest that protease inhibitor-based therapy may have better for patients with AIDS than nucleoside or mononucleoside reverse transcriptase inhibitors (1).
2 Characteristics of KSHV
2.1 Introduction of Herpesvirus
Herpesviridae is a large family of DNA viruses that infect animals, among more than 100 herpesviruses that have been discovered, only 9 of them are able to infect humans, including herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), human cytomegalovirus (HCMV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), and Human Herpesviruses 6A, 6B, and 7 (HHV-6A, HHV-6B, HHV-7), and Kaposi’s Sarcoma’s Associated Herpesvirus (also known as HHV-8) (1). Each sub-family of different viruses has different pathogenic effects and growth rates in culture (18). It is reported that almost all patients infected with HIV are also infected with at least one type of human herpesvirus, suggesting the association between HHV infection and immunodeficiencies(19).
A typical structure of herpesviruses includes a linear double-stranded DNA, an icosahedral capsid with a capsomeric structure, an amorphous tegument surrounds the nucleocapsid, and an envelope containing viral glycoprotein on the surface (1).
The herpesviruses apply cellular latency as a mechanism for lifelong persistence in their hosts. During latency, only a limited number of genes are expressed that help viruses to sustain in host. And Production of infectious progeny virus happens during the lytic infection, which is usually accompanied with the destruction of the infected cell (1) (18).
2.2 How Herpesviruses disrupt immune responses
Herpesviruses have been reported to disrupt host immune responses as viruses usually do. Also, different Human Herpesviruses exert different influences on the immune systems.
Genital herpes is a commonly seen sexually transmitted disease which is caused by herpes simplex virus(HSV). HSV-1 is the major cause of orofacial herpes and genital her¬pes is more likely to be caused by HSV-2(20). HSV-1, as a widely spread herpesvirus that infect 50–90 % of the humans over the world, is able to establish a lifelong latent infection and trigger lytic reactivation in epithelial cells and mucosal cells(21). HSV-1 is reported to damp the several stages of the antiviral immunity induced by Toll-like Receptor’s (TLR) pathway, RIG-I like Receptor’s (RLR) pathway. Sen, J, et al described that HSV-1’s tegument protein US3 could inhibit TLR2 signaling by interfering TNF receptor associated factor 6 (TRAF6) activity(22). Also, HSV-1’s tegument protein US11 is shown to target multiple proteins in the RLR’s pathways including RIG-I and MDA5(23). HSV-2 is also a common sub-family of herpesvirus that co-infects over half of HIV-infected adults(24). It is reported by Gray RH, et al that HSV-2 infection is able to deteriorate the health condition of HIV patients by increasing the plasma HIV viral load(25).
Varicella zoster virus (VZV) is the cause of varicella (chickenpox) and zoster (also known as shingles), which is a highly human-specific virus that barely infect other species(26). Experiments done by Ku CC, et al revealed that VZV shows T cell tropism, infecting tonsil T cells with high efficiency(27). Infected T cells were also showed to deliver VZV to skin and leads to the skin lesions after a few weeks (28). Several VZV proteins were reported to suppress while producing a virus-filled lesion at the skin surface. VZV viral protein IE62 and ORF 47 are both shown to reduce the phosphorylation of IFN regulatory factor 3 (IRF3) and therefore inhibit the IFNβ
production (29) (30). In addition, Schaap A, et al reported that VSV ORF66 protein inhibits the activation of STAT1, which also induces the interferon pathways and activate the cellular antiviral states (31).
Similar to VZV’s T cell tropism, Epstein-Barr virus (EBV) has a tropism being transferred to tonsil B cells (1). EBV is a widespread human gammaherpesvirus that is reported to establish a life-long infection in 95% of the people over the world (32). EBV viral gene expression during its latency is highly restricted as compared with being lytic reactivated (33). When persist in B cells, the EBV protein expressions are down-regulated as a strategy of immune escape. For example, RNA structure limits the amount of translated Epstein-Barr Nuclear Antigen (EBNA1), which helps escape from the T cell recognition (34). Also, during lytic reactivation, differentiation and activation of inflammatory dendritic cells are inhibited by EBV proteins BARF1 and BPLF1 (35).
Human herpesvirus6 (HHV-6) was originally identified from AIDS patients with lymphoproliferative disorders, initially characterized as a human B-lymphotropic virus but later found to be a T-lymphotropic virus (36). HHV-6 has several virus encoded genes that have impacts regulating or modifying immune responses. For example, the HHV-6 U21 protein has been reported to down-regulate HLA class I expression on dendritic cells(37). Also, the HHV-6 U83 gene encodes a chemotactic protein with an agonistic effect on human CC chemokine receptors (CCRs) (38).
2.3 Characteristics of KSHV
KSHV is the most recently identified one among all human herpesviruses, in 1994 as mentioned above.
Similar to other herpesvirus, KSHV consists of a nucleocapsid, a proteinaceous tegument layer, a lipid bilayer envelope, and several viral glycoproteins on the surface which assist the KSHV’s entry into the cells(1).
The major mechanism by which KSHV enter the cells is endocytosis(1). Chandran, et al reported that cellular integrin protein is needed for the viral entry as an integrin binding motif is found in KSHV glycoproteins (1). Also, cellular FAK and Pyk2 are found to activate signals that promote the viral entry and create a pro-survival environment in the infected cells (1).
KSHV establish a latent or a lytic state in the infected cells, and the latency is the default program. During latency the viral gene expression is restricted, which is the same as the other human herpesvirus EBV.
Though no virions are produced in the latently infected cells, several KSHV latent genes are expressed to maintain the viral genome and also promote cell survival, thus retain the potential for lytic infection. The latency-associated nuclear antigen (LANA) is the first latent transcripts discovered, encoded by ORF73. LANA is characterized with function in establishment and maintenance of latent viral genes. Also, LANA is involved in promoting cell growth and cell survival as it is found to interact with p53 and the LANA-expressing cell display higher resistance to p53-dependent apoptosis (1). Also, KSHV vFLIP, which is the homologous gene of cellular FLIP, is found to up-regulates the antiapoptotic transcription factor NF-κB, and therefore promote cell survival and protect the viral genomes. vFLIP may also contribute to maintain stable latency as NF-κB also up-regulate inflammatory responses (1). KSHV encodes several viral homologs to cellular interferon regulatory factor (IRF) proteins, vIRF3 is described to have an antiapoptotic function by inhibiting cellular IRF5 functions. While as mentioned above, angiogenesis is induced in order to help virus spread, it is reported that vIRF3 plays a role in angiogenesis in endothelial cells (1).
KSHV lytic reactivation requires the up-regulated expression of genes including ORF9 (DNA polymerase), ORF6 (single-stranded DNA binding protein), ORF40/41 (primase-associated factor), ORF44 (helicase), ORF56 (primase), ORF59 (processivity factor), ORF50 (replication and transcription activator or RTA), and ORF K8 (K-bZIP) (39). RTA, encoded by KSHV ORF50, is the key regulator controlling the switch from latency to lytic reactivation. During lytic infection, the whole KSHV viral genomes is expressed with viruses being synthesized. T cells are critical for the control of KSHV infection, two lytic proteins ORF K3 and ORF K5 are reported to prevent MHC I display, thus inhibit the antigen presentation process (1). Also, KSHV lytic proteins are able to inhibit the activation and functions of type I interferon, prevent apoptosis, modulate inflammatory pathways and shutoff several host genes expression; and therefore promote the accumulation of virions.
2.4 KSHV’s viral homologous genes disrupt immune responses
Many KSHV open reading frames (ORFs) encode proteins that are homologous to human cellular proteins. As we mentioned above, vFLIP and vIRF3 are two homologous genes that have antiapoptotic functions that ensure cell survival. Also, vIRF3 is able to inhibit the interferon induced by IRF3 and IRF7 (1) . And Wies E, et al demonstrated that vIRF3 is required for the proliferation and survival of PEL cells infected by KSHV (40). KSHV encodes other viral IRFs that suppress immune responses. vIRF1 is reported to block IRF-1 and IRF-3 by reduce both the DNA-binding affinity and transcription activity of IRF-1 and also interact with IRF-3 (41). found that KSHV vIRF-1 interact with the cAMP response element binding protein (CREB) binding protein (CBP), which is a cellular coactivator, and thus, inhibiting IRF-mediated gene transcription (42). vIRF2 also exhibits opposite activities in immune responses, inhibiting the IRF1 and IRF3 driven interferon responses and reducing the apoptosis activities (41, 43).
Cellular human IL-6 is demonstrated to activate STAT1/STAT3 through a high-affinity binding subunit (gp80/IL-6Rα) and thus induce antiviral responses, and KSHV viral IL-6. However, KSHV viral IL-6 differs from human IL-6 as it does not need gp80 unit and act by ligating gp130 on a wider range of cells than human IL-6 (44). vIL-6’s reported functions include antiapoptotic activities, inhibiting interferon signalings, promoting the proliferation and survival of PELs (1). KSHV encodes 3 viral MIPs that are shown to exert pro-angiogenic activities on infected cells (45). Also, Yamin R, et al reported that vMIP-II impairs NK cell chemotaxis by binding to CX3CR1 and CCR5 (46). Carrageenan is applied to induce neutrophil migration and vCD200 is found to inhibit neutrophil recruitment in mice with treatment of carrageenan (47). KSHV ORF74 encodes the viral G protein coupled receptor (vGPCR), which is involved in cell growth and survival(48). Montaner S reported that vGPCR activates Akt/TSC/mTOR signaling pathways and promote the growth of endothelial cells (49). Also, Montaner S demonstrated that vGPCR is able to induce the formation of KS-like angioproliferative lesions in mice and therefore contribute to the KS pathogenesis (50). KSHV ORF K6, K4, K4.1 encode three KSHV chemokines vCCL-1, vCCL-2, and vCCL-3, which are shown to effectively inhibit cellular chemokine activities (51). Besides, KSHV viral-chemokines has been demonstrated to promote angiogenesis and the survival of PEL cells (52).
3. Innate Immunity and RLR Pathways
3.1 Human Immune System and How It Works
When being attacked by invaders like a microbe, primitive animals are able to get rid of it by releasing chemicals, while the strong fecundity also ensure the continuity of their species if they fail to expel the microbes(53). However, in humans reproduction is way less frequent and thus, a more efficient defense system against nearly all pathogens is required, which is called immunity.
Innate immunity are the defense mechanisms that are encoded in germline, which is passed down in generations with few refinements. Its function includes detecting and eliminating a wide range of nonselfs and also maintaining the homeostasis human bodies(54). While innate immune responses discern self from nonselfs, adaptive immunity recognize nonself proteins and other molecules with extreme specifities. Different from the innate immunity, the adaptive immune system are the product of gene recombination from lymphocyte development(55).
The two immune systems are associated through the antigen presentation. The phagocytic cells of the innate immune system capture the antigens by recognizing specific molecules, cutting them into small peptides and presenting them to the antigen presenting cells. The antigen presenting cells, for example, dendritic cells package the antigen peptides into major histocompatibility complex (MHC) proteins, which leads to the activation of T lymphocytes of the adaptive immune systems(56, 57). By cooperating together, innate immune system and adaptive immune system eliminate the pathogens with high efficiency and a minimal cost to the host.
3.2 Innate immune system and its viral sensing pathways
After detecting the nonselfs, the innate immune system activates the induction of antiviral responses, which are mainly regulated by type-I interferons (IFNs) and will be introduced later, including inhibiting the entry, the proliferation, and the exit of the viruses. The elimination of the infected cells by natural killer cells (NK cells) can also be induced by IFNs.
The innate immune system detect pathogens through the Pathogen-associated molecular patterns (PAMPs), including extrinsic proteins and viral nucleotides. PAMPs are mainly recognized by pattern recognition receptors (PRRs). RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), Toll-like receptors (TLRs) are the three major pathways through which the immune system sensing PAMPs.
RNA viruses are found to be recognized by two cytoplasmic RNA helicases, retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) (58). Besides RNA viruses, RIG-I like receptors also recognize small RNA encoded by DNA viruses including EBV and KSHV. Kato H, et al suggested that while triphosphate at the 5’end of single-stranded RNA (ssRNA) is recognized by RIG-I, RIG-I can also detect short double-stranded (dsRNA) without 5’triphosphate (59). Indeed, RIG-I tend to selectively detects blunt, short, double-stranded 5’triphosphate RNA, which includes both the two features (60).
RIG-I and MDA-5 have a central RNA helicase domain to recognize viral RNAs, a C-terminal repressor domain (RD) to inhibit the activation of RIG-I at its steady state, and a N-terminal caspase activation and recruitment domain (CARD) that activates downstream signaling once viral RNAs are detected. After being catalyzed, RIG-I and MDA5 interacts with the downstream adaptor molecule interferon-β promoter stimulator 1 (IPS-1), or called mitochondria antiviral signaling protein (MAVS) since it is localized to the mitochondrial membrane (61). The IPS-1 bind to the downstream protein kinase complexes including TRAF3, TANK, and NAP1 (62, 63), which activates the TBK1-IKKi complex and finally leads to the activation of Interferon Regulatory Facotrs including IRF3 and IRF7 (62). Moreover, through FADD, RIP-1 and Caspases 8/10 and IKKα / IKKβ / IKKγ complex, RIG-I is also able to activate NF-κB, which is a key positive regulator of inflammatory responses (64).
All TLRs are transmembrane proteins, with a leucine rich repeat (LRR) recognizing PAMPs, a cytosolic toll/interleukin-1 receptor (TIR) domain, and a transmembrane domain. There are 10 TLRs in humans recognizing bacterial, fungal, protozoan and viral pathogens (65). TLR3, TLR7, TLR8, and TLR9 are expressed in endolysosomes, where they recognize viral necleic acids. TLRs also induce the activation of two major pathways: antiviral cytokines through type I interferon signaling and proinflammatory cytokines through NF-κB signaling. For example, upon PAMP recognition, TLR3 interact with TRIF, while TRIF is capable for activating binding to TRAF6, activating NF-κB and binding to TRAF3, leading to the activation of IRF3.
Besides RLRs and TLRs, NLRs are another family of PRRs that not only recognize PAMPs but also respond to cellular stresses. Several NLR proteins participate in the activation of the caspase-1 which forms the inflammasome complex. The inflammasome complex is capable to induce the activation several proinlammatory cytokines, for example, IL-1β and IL-18 (66).
3.3 Anti-viral functions of type-I Interferon
Interferons are a family of cytokines that induce antiviral activities in not only viral infected cells but also uninfected neighboring cells, triggered mainly by RLRs and TLRs. Type I IFNs in humans can be divided into three subfamilies: IFN-α subtypes, IFN-β subtype, and IFN-ω subtype. In cytosol of most cells, IRF3 are constantly expressed which induces mainly IFN-β, while IRF7 which efficiently induces both IFN-α and IFN-β is expressed at a low level (67).
Type I IFNs receptor complex includes IFN-αR1 and IFN-αR2, and type I IFNs receptors utilize JAK-STAT signaling pathway to induce the interferon-stimulated genes (ISGs). Binding of IFNs to the receptors leads to the phosphorylation of Tyk2 associated to IFN-αR1, which subsequently phosphorylates JAK1 coupled to IFN-αR2. Activated JAK1 then binds to STAT2 and phosphorylates it as well as STAT1. STAT2 and STAT1 are transported to the nucleus, binding to the interferon simulated response element (ISRE) with IRF9 and form a complex called ISGF3, which facilitate transcription of ISGs.
Interferon-stimulated genes are reported to affect almost every stage of the viral life
cycle, including cell entry, genome replication and translation, and also exiting cells.
The murine myxovirus resistance 1 (Mx1) protein was early described to inhibit virus entry. For example, Mx1 are shown to target influenza A viruses, as Mx1 traps incoming viral components like nucleocapsids, and therefore prevents them from reaching their cell nucleus(68). The other Mx protein Mx2 is also reported to inhibit the nuclear entry of reverse-transcribed viral genomes, and Mx2 especially targets HIV-1 and HIV-2(69). Expression of CH25H gene can protect cells against enveloped viruses, as Liu SY, et al proposed that 25HC protein blocks membrane fusion of enveloped viruses by changing the physical property of cellular membranes(70). The proteins of the IFN-inducible transmembrane (IFITM) family are shown to block the entry of a large range of viruses. For instance, IFITM1 inhibits SARS-coronavirus (CoV) and filoviruses including Ebola virus, and IFITM3 has inhibit the entry of influenza A virus with high efficiency (71). The tripartite motif (TRIM) is a large family of proteins with a wide range of functions. TRIM5α is reported to inhibit early stages in HIV-1 infection by binding to viral caspid proteins and causing a early disassembly of the capsid shell, which is described as a premature exposure of the nucleoprotein complex (72).
Several ISGs also inhibit viral replication and translation in infected cells. IFN-induced protein with tetratricopeptide repeats (IFIT) family are reported to block HCV infection by binding to eukaryotic initiation factor3 (eIF3) and inhibit viral RNA translation process(73). Stark GR, et al revealed that 2′-5′ oligoadenylate synthases (OAS) activate the latent nuclease RNase L, leading to the degradation of viral RNA transcripts(74). PKR is another well-studied IFN-induced protein, PKR’s activation leads to the phosphorylation of eukaryotic initiation factor 2α (eIF2α) and consequently blocks the translation of viral mRNAs (75). Recently it has been discovered that human encodes seven APOBEC3 genes that are promising in HIV therapeutics. APOBEC3G is best characterized for encoding a cytosine deaminase that targets reverse transcribed DNA of HIV-1 by deleterious modification (76). Helbig KJ, et al described that viperin, as a highly induced antiviral effectors, is able to inhibits viral RNA replication, for example, the replication of HCV subgenomic replicons(77).
In the late stage of the life cycle, packaged viruses Elimination exit cells through cell lysis, exocytosis, or direct budding from the plasma membrane(78). Some ISGs are also know to inhibit viral assembly and viral egress. Besides its function in inhibiting viral replication, viperin is also reported to inhibiting virus budding of HIV-1 and influenza A by decreasing farnesyl diphosphate synthase (FPPS) and altering membrane fluidity (79, 80). Tetherin is another ISG encoded protein that was shown to inhibit many enveloped viruses, Perez-Caballero D, et al demonstrated that tetherin can tether HIV-1 virion on plasma membrane by membrane anchors (81). Elimination of infected cells is one of the strategies to prevent viral spread. PKR has been proved to be required for apoptosis of cells in response stimulated by Poly I:C in 2000 (82). The tumor suppressor p53 is also an interferon induced protein playing a role in apoptosis, while p53-defecient cells are shown to be defective in VSV-induced apoptosis (83).
4. Mechanisms of How Viruses Block Interferon
4.1 How Viruses Block Interferon
In order to generate a productive and persistent infection, viruses have developed several strategies against human immune defenses. While type I interferon is a crucial cytokine leading to the associated with antiviral states, viruses are reported to modulate IFN activities including virus recognition, activation of IFN, signaling of IFN, and functions of ISGs.
As mentioned above, viruses are detected by receptors including TLRs and RLRs. The adaptor protein TRIF of TLR signaling pathways is reported to be cleaved by 3CD protease-polymer of hepatitis A virus and also 3C protein of enterovirus 71 (84, 85). Also, RLR signaling pathways are inhibited by viruses through different mechanisms. The RIG-I like receptor MDA5 is inhibited by human respiratory syncytial virus (RSV) and HIV protease is found to decrease cytoplasmic RIG-I levels(86, 87). The adaptor proteins of RLR pathway are also antagonized, The influenza A protein PB1-F2 is found to interact with IPS-1 through its transmembrane domain and thus, inhibit IPS-1’s signaling function(88). Another adaptor protein STING (also known as MITA), which also interacts with RIG-I, is found to be cleaved by Dengue virus protease(89).
Type I interferon is mainly induced by IRF3 and IRF7. Several proteins have been reported to directly disrupt IRF3 activities. Okumura A, et al described that HIV-1 protein VPR and vif are able to degrade IRF3 and disrupt interferon signaling (90). Another HIV-1 protein Vpu also interacts with IRF3 and leads to the lysosome-dependent degradation (91). Other viral proteins have also been shown to bind to IRF3 and inhibit its activities, including the V protein of Sendai virus and varicella zoster virus ORF47 protein (92) (30). Disrupting the upstream protein TBK1 or IKK also leads to the inhibition of IRF3. For example, the NSp3 protein of mouse hepatitis virus A59 (MHV-A59) is found to de-ubiquitinate TBK1 and restrict it in inactive conformation(93), while the NP proteins of arenaviruses interact with the kinase domain of IKKε and blocking its binding to IPS-1(94). As mentioned, IRF7 can efficiently induces both IFN-α and IFN-β but is normally expressed at a very low level. It has been demonstrated that some strains of rotavirus protein NSP1 are able to degrade IRF7 through proteasome-dependent pathways, while other strains also target IRF3(95).
IFNs activate ISGs through its receptors (IFNAR) and JAK-STAT pathway. Ren J, et al showed that human metapneumovirus (hMPV) can degrade cellular JAK1 and Tyk2 levels through proteasome-dependent machanisms, while also inhibit the IFNAR expression (96). The C protein of human parainfluenza virus type 1 is reported to inhibit STAT1 activities by blocking its nuclear translocation, and the nuclear translocation of STAT2 can be blocked by RSV NS2 protein (97, 98). ISGs are activated after STAT1/STAT2/IRF9 complex binds to ISRE. The IFITM proteins which block the entry of viruses, is inhibited by miR-130A, a microRNA expressed by Hepatitis C virus(99). OAS and PKR are described above to inhibit viral replication and translation. Several viruses including HCMV, Adenoviruses, HMV are reported to inhibit the expression and the activity of OAS through various mechanisms, for example, MHV protein NS2 is found to cleave 2-5A molecules produced by OAS (100). While PKR block viral RNA translation, Poliovirus cleaves another eukaryotic initiation factor eIF5B to overcome the eIF2α phosphorylation induced by PKR (101).
4.2 How KSHV Blocks Interferon
KSHV is also reported to disrupt viral recognition pathways including TLRs and RLRs. Ahmad H, et al found that RTA shortens the half life of TRIF and therefore degrade it through proteasome-mediated pathways and block TLR induced IFN signaling(102). As mentioned, KSHV encodes several vIRFs including vIRF1, vIRF2 and vIRF3, which is demonstrated to block TLR3-mediated activation of IFN transcription reporters (103). Also, a KSHV tegument protein ORF64 with deubiqutinase (DUB) function, is able to suppress the RIG-I ubiquitination and inhibit further RLR signaling (104).
vIRF1, vIRF2 and vIRF3 target IRF3 and IRF7 mediated IFN induction as previously mentioned. Other KSHV proteins are also found to disrupt type I interferon induction, another KSHV tegument protein ORF45 is able to interact with IRF7 and blocks IRF-7 phosphorylation. Sathish N, et al revealed that ORF45 interacts with the inhibitory domain of IRF-7 and keep it in its inactive form (105). What’s more, a KSHV encoded viral miRNA miR-K12-11 is reported to decrease IKKε expression and therefore inhibit the IKKε mediated IRF3/IRF7 phosphorylation (106).
Besides disrupting IFN induction, vIRF2 is found to inhibit IFN signaling by targeting STAT1 and IRF9, and thus impede the formation of ISGF3 and the transcription of ISGs (107). Also, Burysek L , et al demonstrated that vIRF2 interacts with PKR and thus inhibit the phosphorylation of PKR substrates eIF2α and histone 2A (108). Another viral protein RIF, which is encoded by KSHV ORF10, is found to form inhibitory complexes containing IFNAR, JAK1, Tyk2 and STAT2 transcription factor, consequently inhibiting the phosphorylation of STAT1 and STAT2 and blocking the formation of ISGF3 complex (109).
Blocking the IFN responses is essential for KSHV to release its components into the cell and to transport the viral capsid into the cell nucleus, which are the basis of latent infection. While during latency, the evasion mechanism of innate immune responses ensure the viral genome replication without triggering IFNs. And during lytic reactivation and pathogenic progress, blocking IFN responses promote the survival of the proliferating spindle cells with the virus produced and under this circumstance KSHV lytic genes can reprogram the cellular genome expression. Also with the inhibited interferon responses, KSHV-permissive cells are able to spread to other site of the body and successfully release the viruses to generate a higher level of infection.
4.3 Knockout Viruses that Missing Interferon Blocking Genes
Vpu and Nef are two proteins expressed by HIV-1, with functions of degrading host molecules such as IPS-1 and consequently inhibiting IFN signaling pathways. However, a mutant HIV that does not produce Vpu or Nef is found to lose the ability of degrading IPS-1 and also lead to the induction of IFNs, which suggests that IFN blocking genes are essential for inhibiting the IFN responses(110).
Also, it is previously found that NS1 protein, expressed by Influenza A viruses, is able to disrupt IFN-α/β responses(111). Mutant NS1-truncated virus with are found to induce the expression of IFN-β mRNAs as well as the IFN production, with an inhibition on GFP expression by VSV-GFP in PK15 cells (112). While human respiratory syncytial virus (HRSV) poorly induces interferon, recombinant HRSV lacking the NS1 and NS2 genes induces high level of IFN-α/β in human epithelial cells and macrophages (113).