3.1. HTLV-1 Viral mRNA Exhibits NMD-Initiating Features
HTLV-1 is a complex retrovirus with multiple proteins coded in a single genomic RNA. The overall sensitivity of HTLV-1 retroviral RNA to NMD has been known for a few years [
42,
43]. Although the molecular determinants of RNA features triggering NMD sensitivity have not yet been clearly defined [
78], we can at least suggest that this sensitivity may have multiple origins; the viral RNA can be directly as well as indirectly targeted by the NMD machinery.
Direct regulation: As described above, the half-life of unspliced HTLV-1 RNA is increased under NMD inhibition. How can this sensitivity be explained? Since the mRNA is un-spliced, it should not have bound an EJC, especially in the 3′UTR, which is expected to hamper its sensitivity to NMD. However, according to the above findings and our knowledge of HTLV-1 RNA organization, the 3′UTR size stands out as a possible factor. While the median human 3′UTR size is ~750 nt [
79], the gag mRNA 3′UTR is~4000 nt, making it a suitable target for NMD. For instance, Garcia et al. showed that two RNA(+) viruses had genomic and sub-genomic RNA NMD sensitivity because their 3′UTRs ranged from 1 kb to 2.5 kb [
37]. In contrast, the secondary structure of RNA and its mRNP composition can also compete for the long 3′UTR to have an effect on sensitivity [
40,
67,
80]. Another noteworthy parameter of HTLV unspliced mRNA, and that of other retroviruses, is the –1 frameshift (–1FS). HTLV-1 has two successive –1FS in the un-spliced mRNA region, allowing for the synthesis of three different polyproteins: GAG, GAG-PRO and GAG-PRO-Pol fusion proteins. The low frequency of these (–1FS) maintains the correct ratio of these three viral proteins. By slowing ribosome reading and preventing translation termination, a frameshift can promote NMD. On the other hand, Hogg et al. suggested that retroviral readthroughs and frameshiftings destabilize UPF1 accumulation in the 3′UTR and impair NMD by redirecting the ribosome and avoiding the in-frame stop codon, even at a rate of approximately 1% [
81]. In the case of HTLV-1, the treatment of infected cells by okadaic acid led to increased levels of unspliced viral RNA associated with UPF1 [
43]. Okadaic acid prevented the dephosphorylation of active UPF1 molecules and suspended NMD in the latest steps of mRNA decay. These observations strongly suggest that UPF1 is not stripped from the unspliced viral RNA and that this RNA is subjected to active decay.
Indirect regulation: In cells transfected with an HTLV-1 molecular clone, the knockdown of UPF1 led to a 3-to-4-fold homogeneous upregulation of all viral RNAs in the steady state [
42]. This homogeneity might suggest that the transcriptional activator(s) of HTLV-1 can be controlled by NMD, leading to the indirect regulation of viral mRNA. Supporting this hypothesis, CREB-2/ATF4 has been shown to be involved in LTR transactivation, regulated by NMD and stabilized by Tax at the post-transcriptional level [
42].
While the sensitivity of HTLV-1 to NMD is well documented, additional work is needed to clearly demonstrate which stop codons are the most likely to induce NMD. Moreover, to maintain its capacity to replicate, HTLV-1 had to evolve solutions to evade the NMD threat. These solutions are discussed in the next section.
3.2. HTLV-1 Protection against NMD
The arms race involving HTLV-1 and NMD led to the evolution of two viral countermeasures. The viral proteins Tax and Rex were shown to target the NMD process in what is called “trans-inhibition”, incapacitating the decay of viral as well as cellular NMD targets. This evidence emerged from observations that HTLV-1-infected lymphocytes were able to specifically stabilize globin mRNA with a PTC [
42]. Nakano et al. also showed that, in HeLa cells co-cultivated with HTLV-1-infected cells, this inhibition was maintained as long as
Tax/Rex mRNA was expressed [
43].
Tax was first described as the main viral transactivator [
82]. It has the capacity to bind multiple host factors in the nucleus and in the cytoplasm, leading to dysfunctions promoting cell transformation [
83,
84] Tax-dependent NMD inhibition was initially investigated due to its interaction with the translation initiation factor eiF3E/INT6 [
85], known to interact with UPF2 and to be involved in NMD [
86]. In addition to this interaction, lNT6 was observed to be delocalized from the nucleus to the cytoplasm by Tax. This study also revealed contacts between Tax and several NMD factors and a direct interaction between Tax and the helicase UPF1. A complementary study introduced interesting details on Tax: first, Tax can bind to the helicase domain of UPF1 at the exit of the RNA binding channel, preventing UPF1 loading onto its target. Second, when UPF1 is already bound to RNA due to its action in NMD, Tax binding blocks ATP hydrolysis and helicase activity, freezing UPF1 on RNA. These observations suggest a broad effect on UPF1 with the capacity to impact NMD at different steps [
87]. When analysing viral mRNA, it is difficult to dissociate the transactivation role of Tax on the viral promoter from its post-transcriptional effect via NMD. Therefore, a mutant form of Tax specific for NMD interference must be engineered. Nevertheless, when Tax is expressed alone or from a provirus, the half-lives of host mRNAs, such as creb-2/atf4, growth and arrest DNA damage-inducible 45 (Gadd45A) and smg5, are stabilized as a consequence of NMD trans-inhibition.
The Rex protein was also shown to inhibit NMD. Similarly to Tax, several host mRNAs known to be NMD sensitive had increased half-lives upon Rex expression. Rex is known to bind viral RNA at the RxRE motif. Upon binding to RxRE, Rex controls viral mRNA splicing. It also contacts the CRM1 export system to ensure the nucleo-cytoplasmic shuttling of the unspliced viral mRNA [
88,
89,
90]. To date, the mechanism of NMD inhibition by Rex has not been described. It has also not yet been investigated whether the HTLV-1 RNA secondary structure provides a first line of defence against NMD (
Figure 3).
3.3. When Does NMD Inhibition Occur during HTLV-1 Infection?
During infection, HTLV-1 is spread in two different ways: viral propagation is initially dependent on cell-to-cell transmission, then it evolves towards polyclonal and monoclonal expansion (reviewed elsewhere [
12]). Cell-to-cell infection depends on virion production. These virions are composed of structural proteins translated from singly spliced mRNA (ENV) and unspliced viral mRNA (GAG). Tax, as the viral transactivator, is indispensable for the production of this unspliced mRNA. Additionally, the modulation of splicing, leading to the stabilization of viral unspliced mRNA as well as their nuclear export, depends on Rex. By targeting the gag unspliced mRNA, NMD prevents virion formation. Supporting this hypothesis, knockdown of UPF2 was associated with increased levels of the p24 and p19 GAG protein [
43]. Interestingly, HIV gag mRNA and GAG protein expression were also shown to be affected by UPF2 and SMG6 expression in the context of virus reactivation [
91]. Hence, it is understandable that NMD, which does not require induction by type I interferon and thus acts as a cell-intrinsic antiviral barrier, plays a role in the early steps of HTLV-1 infection. The results of an analysis of viral mRNA kinetics showed a clear separation between the early phase when Tax and Rex (p21 and p27) are produced and the later phases characterized by the increase in other mRNAs, including gag mRNA [
92]. This finding suggests that the stabilization of
gag mRNA and the production of the gag protein necessitate the formation of a favourable environment: NMD inhibition might contribute to this initial condition. Notably, the results from kinetics experiments performed with RNA(+) virus infections suggest that the impact of NMD inhibition on viral RNA is greater in the early steps of the infection [
37,
39]. Finally, it is striking how the same factors, Tax and Rex, are involved in both the production and the protection of viral particles.
The second mode of provirus amplification is clonal expansion, which depends on the proliferation of a few selective clones. This oncogenic behaviour emerges from the modulation of multiple cellular processes by non-structural viral proteins such as Tax and HBZ; notably, Tax plays an essential role in this cellular transformation by generating instability and bypassing checkpoints: it inhibits DNA repair, disrupts cell cycle progression, and affects autophagy. It also modulates transcription through the modification of the epigenetic landscape and transcription complex composition and deregulates signalling pathways, including NF-κB (inducing its constitutive activation) and innate immune pathways (with an immunosuppressive effect) (reviewed in [
4,
12]). Knowing that Tax is the immunodominant HTLV-1 antigen in the T-cell response, it is considered that the progression of an infected clone is then favoured by the progressive inhibition or occasional bursts of expression of Tax [
93], which confers a survival advantage through escape from the strong cytotoxic T-lymphocyte (CTL) immune response [
94]. The other viral oncoprotein HBZ that triggers a less efficient immunity is continuously expressed in infected cells and plays important roles in viral latency and the proliferation of infected cells. Interestingly,
hbz RNA appears to play specific roles in T cell proliferation (reviewed in [
95]).
Although it is widely accepted that HTLV-1 infection leads to cell proliferation, it has been frequently observed to cause apoptosis and senescence of lymphoid and non-lymphoid cells in a Tax-dependent manner. It has been proposed that cells responding by senescence or apoptosis have very high levels of expressed Tax and Rex, are associated with robust replication of the virus and are subjected to great oncogenic pressure. This viral hyperactivity might be dependent on the site of integration, stimulating LTR transactivation and thus may be specific to each clone. Senescence induction has been shown to be linked with the stabilization of the p27 protein and cyclin-dependent kinase inhibitor p21CIP/WAF1 mRNA because of NF-κB hyperactivation ([
96,
97,
98] reviewed in [
99]). NF-B activation is a hallmark of Tax expression and HTLV-1-infected cells, and multiple steps in the NF-B pathway were shown to be regulated directly or indirectly by Tax. For example, NIK (MAP3K14), the NF-κB inducing kinase that activates IKKα, is highly expressed in HTLV-1-infected cells [
100]. Moreover, p21, p27 and NIK transcripts are known to be NMD targets. Their mRNA levels are tightly controlled, and NMD inhibition experiments induced their stabilization [
71,
101,
102]. In lung inflammatory myofibroblast tumors, decreased NMD leads to the increased expression of the transcript for the NIK protein kinase, which activates the NF-B pathway and promotes cytokine expression and inflammation. Supporting this observation, the stabilization of the NIK mRNA half-life was observed in a separate Tax and Rex-dependent manner [
43,
87]. Additionally, it is worth noting that NMD is involved in the apoptotic response induced by high stress levels; more precisely, it has been demonstrated that maintained NMD inhibition induces cell death [
103,
104]. Gadd45 isoforms promote apoptosis by upregulating the mitogen-activated protein kinase (MAPK) signaling pathway upon the activation of diverse forms of stress, including that from TNFα and DNA damage. Gadd45α and β transcripts are dependent on canonical as well as non-canonical NF-κB pathway activation and are upregulated in Tax and HTLV-1 expressing lymphocytes [
105]. Gadd45α and β mRNA are also sensitive to NMD due to their respective 3′UTRs. This regulation of Gadd45 by NMD is evolutionarily conserved from flies to mammals. It was recently shown that the upregulation of Gadd45 isoforms due to NMD inhibition is a major contributor to NMD-associated programmed cell death [
106]. Altogether, these observations suggest that, in the context of HTLV-1 infection, high levels of Tax and Rex, viral-induced senescence/apoptosis and NMD inhibition might be correlated (
Figure 4). However, NMD inhibition in vivo does not always lead to the activation of apoptosis; for example, the absence of the key NMD factors UPF1 and SMG1 induces embryonic cell death, but UPF3-null mice are viable. Similarly, during hematopoiesis, NMD inhibition by UPF2 knockdown prevents hematopoietic stem cell and progenitor survival, whereas mature cells are only mildly affected [
107,
108,
109,
110]. Thus, whether NMD inhibition can activate apoptosis might be dependent on the degree of the NMD inhibition, the cell type and differentiation state.
While in the early steps of infection when HTLV-1 directly benefits from NMD inhibition to produce infectious particles, it is conceivable that, in a second step, this NMD trans-inhibition also participates in the selection of a clone with attenuated expression of Tax and Rex that is more suited to avoid the immune response and thus to maintain infection.
3.4. HTLV-1-Associated Pathologies. How Can NMD Inhibition Impact the Host in the Long Term?
As introduced above, HTLV-1 is the etiological agent of ATL and HAM/TSP. ATL is a malignant lymphoproliferative syndrome established after decades of latency and characterized by genetic instability combined with checkpoint adaptation. A favorable environment enabling malignant proliferation is dependent on the establishment of an immunosuppressive state. The viral protein Tax plays a major role in these steps. HAM/TSP is an immune-mediated inflammatory disease associated with the accumulation of HTLV-1-specific CD8+ T cells and infected CD4+ T cells in cerebrospinal fluid and neural tissues. It is characterized by a chronic inflammatory state due to elevated cytokine expression and production (reviewed in [
111]). We wondered whether NMD downregulation, induced for the early infective stage of HTLV-1, may play a role in the later steps of the infection and could converge with ATL or HAM/TSP onset.
In 2015, genomic sequencing of 400 ATL samples showed that the mutation rate for ATL was relatively high compared to other hematologic malignancies, with an average of 2.3 mutations per mega base in coding regions [
26,
112]. Notably, transacting T cell-specific transcription factor GATA3, which is required for multiple steps of T-cell differentiation in both developing thymocytes and mature T cells, is commonly affected by nonsense and frameshift mutations. The authors suggest that these mutants confer altered protein function (possibly dominant negative functions due to truncated proteins), rather than GATA3 haploinsufficiency. Most CCR4 and CCR7 mutations in ATL-related proteins cause truncation of the cytoplasmic domain with gain of function. Moreover, more than half of ATL cases have either nonsense or frameshift mutations in the components of the class I MHC. Although the NMD sensitivity of mRNA resulting from these hotspot mutations has not yet been analysed, these mutations seem to be promising targets, and NMD inhibition by Tax and Rex might be an important parameter to consider. Of course, due to its quality control function, NMD defects contribute to genetic instability; in combination with alternative splicing, NMD tunes the level of many DNA repair factors [
75,
113,
114]. Pancreatic squamous carcinoma cells have mutations in the
upf1 gene allowing the synthesis of mutated dominant negative p53, functionally correlating NMD inhibition with cancer [
115]. Loss-of-function or overexpression of NMD proteins is also associated with several other cancer types, including colorectal cancer, hepatocellular carcinoma and neuroblastoma [
116,
117,
118]. NMD is also involved in the adaptation to stress response [
119,
120]. Hypoxia, amino acid depravation and reactive oxygen species production downregulate NMD, which leads to the stabilization of transcripts such as activating transcription factor ATF4, ATF3, ATF6, CCAAT-enhancer binding protein homologous protein (CHOP) and TNF receptor-associated factor 2 (TRAF2), which re-establish homeostasis by the integrated stress response (ISR). It is now acknowledged that tumour cells must adapt to microenvironment stresses such as these to proliferate. In this context, it has been shown that NMD downregulation plays a role in this adaptation, promoting tumorigenesis [
121]. Moreover, it was also recently proposed that NMD tunes the immune response. Impaired NMD in mice with forebrain-specific UPF2KO triggers immune response activation and results in exacerbated neuroinflammation. The latter symptom was partially reversed upon UPF2 restoration [
122]. In Arabidopsis, NMD downregulation due to bacterial infection has been shown to control the turnover frequency of numerous TIR domain-containing, nucleotide-binding, leucine-rich repeat (TNL) receptor mRNAs, inducing innate immunity. However, maintained NMD inhibition by silencing NMD components deregulates homeostasis, leading to an autoimmunity phenotype characterized by stunting, spontaneous formation of necrotic lesions, and elevated salicylic acid levels [
123]. The role of NMD in cancer and immunity likely depends on the tissues implicated and the associated genomic stress, but the effects of its inhibition in HTLV-1 unrelated examples likely converge with HLTV-1 phenotypes.
However, to clarify whether there is an effective link between NMD and HTLV-1-associated pathologies, the status of NMD in the later stages of the infection and in patient cells, ATL and HAM/TSP, has yet to be analysed. The absence of (+) strand transcription in most ATL cells due to epigenetic repression and genomic alterations raises the following questions: Is the burst of (+) strand expression sufficient to inhibit NMD? HBZ, the protein expressed from the HTLV-1 minus strand, is a critical component of cell proliferation and tumorigenesis and maintains constant expression during infection, in contrast to Tax and Rex; could it also be involved in NMD inhibition? NMD is also sensitive to bivalent cation concentrations [
124], and interestingly, ATL patients show hypercalcemia; does this suggest that NMD can be constitutively inhibited? Without additional experimental data, these questions remain unanswered.
