Discussion
HHV-6 infection and reactivation alter host cell transcriptome, including the miRNAome (
15,
37,
38). miRNAs regulate intraorganellar mechanisms, in which they perform the job of fine-tuning of the functions required to fulfill the metabolic demands of an organ or cell type. Our previous deep sequencing approach (
15) revealed major changes in the expression of a large panel of human miRNAs upon HHV-6A reactivation that hints to a broad range of effects on mitochondria and associated metabolic functions upon viral activation.
In this study, we studied quantitative changes in cellular as well as mitochondrial proteomics upon HHV-6A reactivation and found several significant alterations with close similarity to ME/CFS pathophysiology. PDP1 was downregulated in response to HHV-6A reactivation. PDP1 helps in reverting the negative effects of pyruvate dehydrogenase kinases on pyruvate dehydrogenase. A decrease in PDP1 suggests a potential decrease in functions of pyruvate dehydrogenase, which is also reported in ME/CFS patients (28). We also found a decrease in the SOD2 level that can lead to high amount of ROS within the cell. We have previously shown that HHV-6 infection induces ROS in host cells and alters expression of glutathione reductase (29). These observations strongly support a pathological link between HHV-6 reactivation and ME/CFS.
Hypometabolic state of blood cells is a characteristic feature of ME/CFS (39). Although we did not perform metabolic studies, our study on mitochondrial architecture in presence of CFS patient serum but not controls has revealed a transferrable activity in ME/CFS serum that fragments mitochondria and stimulates a coordinated antiviral state in naive responder cells assayed in vitro. This assay was highly sensitive (0.9–1.00; 95% CI = 0.6–1.0) and specific (0.8–1.0; 95% CI = 0.38–1.0) in identifying serum from patients with ME/CFS. In our studies, both viral reactivation as well as CFS patient serum treatment induced a typical M1 state of mitochondria (35, 36) in host cells, which was also accompanied by a strong proinflammatory state of the cell that protected the host cells against both incoming DNA and RNA viruses, consistent with oxidative shielding (
27). There are many possible ways that a nearby healthy cell can sense potential infectious risk in the environment. Increased IFN response from the virus-transactivated cells can result in secreted IFN that can be sensed by the nearby cells, leading to a hypometabolic state (
40). Several type I and type II IFNs, as well as IFN response genes, are reported to be high in the serum of ME/CFS patients (
41,
42). However, we did not observe any increase in IFN-γ in PBMCs of a small group of ME/CFS patients (
Supplemental Fig. 2B). In addition, we observed significant decrease in ISG expression upon treatment with ME/CFS serum. Hence, the strong antiviral phenotype in our assay system seems to be IFN-independent. Fragmented mitochondria can release broken mitochondrial DNA into the cytoplasm (
43) and to the extracellular space that can induce TLR-mediated cytokine production in nearby cells (
44). Newly synthesized, 8-hydroxyguanosine–containing mitochondrial DNA (ox-mtDNA) can directly activate assembly of the NLRP3 inflammasome (
45). Interestingly excess of certain cytokines can also change mitochondrial metabolism (
46) to prevent further pathogen growth. Certain complex disorders, such as amyotrophic lateral sclerosis, are associated with a hypometabolic state that is characterized by lower mitochondrial size and number (
47–
49).
Autoimmunity is common in ME/CFS (50). Several autoantibodies are capable of inducing a proinflammatory state in target cells (51). Interestingly, HHV-6 is linked to several autoimmune diseases, including multiple sclerosis and Hashimoto thyroiditis (52). Autoantibodies against β2 adrenergic receptors (β2R) were found to be upregulated in a subset of patients with ME/CFS (53). Such Abs belong to a network of natural Abs against adrenergic, acetylcholine (cholinergic), and other GPCR receptors that were shown to be dysfunctional in various autoimmune diseases (54). Autonomic dysregulation is a hallmark of ME/CFS. There is evidence that the β2R-mediated regulation of cytokines by terbutaline is impaired in whole blood immune cells of CFS patients (55). A recent paper showed that influenza replication is inhibited by α2 adrenergic stimulation via cAMP inhibition (56). In contrast β2R stimulation is known to stimulate cAMP. Thus, a disbalance of adrenergic stimulation favoring cAMP downregulation might also be an explanation for our findings. However, similarities between adoptive transfer of HHV-6A reactivation culture supernatant and ME/CFS serum suggests a potential metabolic role in addition to other possibilities.
Lack of a strong HHV-6 and HHV-7 infection in ME/CFS patients in our study and several others has historically cast doubt on the involvement of these viruses in ME/CFS. However, in this study, we show that incomplete HHV-6 reactivation, even in a small fraction of latently infected cells, causes reactivated cells to secrete an activity that can be transferred in serum and produces mitochondrial fragmentation and coordinates a powerful antiviral program in responding cells.
Our studies showed that only IE events, such as the transcription of several small noncoding viral RNAs, were needed to trigger the production and secretion of the mitochondrial fragmentation factor and transferrable antiviral state. No HHV-6 proteins are made during the incomplete reactivation events described in this paper. Specifically, no major change in HHV-6 replication was observed. This explains the failure of anti-herpesvirus drugs in a subset of patients because the HHV-6 polymerase is not expressed during an incomplete virus reactivation, and drugs that target the viral DNA polymerase would have no viral target.
The virus reactivation experiments described in this study show that an antiviral state is produced both in cells with unreactivated and reactivated HHV-6. This seems to be against the viral growth and hence fails to explain the short-term benefits of viral reactivation from the pathogen point of view, unless passive transmission of viral genome to daughter cells after mitosis plays a major role in HHV-6 genome propagation. Decreased IFN response in virus-reactivated cells might provide an advantage for survival of IE RNAs in the host cell cytoplasm. However, in this study, we have tested only the nonproductive transactivation state of the virus. Productive viral infection, with virion production and release, might bring in additional viral factor(s) that damage cellular ability to undergo a hypometabolic state to provide successful virus growth. Additionally, mitochondrial fragmentation often allows virus to acquire persistent or latent state under a hypometabolic state (
57).
In this study, we found that none of 25 patients with ME/CFS had peripheral blood evidence of a fully reactivated HHV-6 or HHV-7 infection, and only 8 of 20 (40%; 95% CI = 0.19–0.64) had evidence of partial reactivation measured by FISH analysis of HHV-6 small noncoding RNA U14 in whole blood.
However, using an in vitro reporter cell assay, we showed that serum from ME/CFS patients contained an activity that produced mitochondrial fragmentation, decreased mitochondrial ATP production, and induced a powerful antiviral state. In 2016, metabolomic analysis of patients with ME/CFS revealed a chemical signature that was similar to the evolutionarily conserved, hypometabolic state known as dauer (39). This dauer-like state was preserved by blocks to healing produced by abnormal persistence of the CDR (36). The CDR has been shown to be directly involved in both healing and the biology of aging (35). In this earlier work, it was hypothesized that the metabolic features of the CDR in ME/CFS patients could protect against certain kinds of infection, but no direct testing for antiviral activity was performed (39). Our current data show that only a small fraction of cells need to be latently infected with HHV-6 to trigger a secretory phenotype that is strongly protective against some of the RNA and DNA virus infection in neighboring and distant cells lacking HHV-6 DNA. The main conclusions of this study are illustrated in the graphical summary (Fig. 6). Larger multicohort studies involving ME/CFS patients from different age groups should be carried out in the future and should include methods for detecting and quantifying both productive and nonproductive (incomplete) viral reactivation events. Furthermore, potential factors affecting mitochondrial dynamics in ME/CFS patients should be systematically evaluated for their ability to induce a powerful antiviral state.
Our mitochondrial reporter-based cell system will provide an opportunity to develop a diagnostic test for ME/CFS as well as provide a platform for further identification of potential factors that define ME/CFS pathophysiology.
And when asked to explain, told that it would be impossible as we unscientific folk would never understand (paraphrased).... sigh and double sigh!!
