Oxidative Stress-enhanced Human Cytomegalovirus Replication Via Cyclophilin A

Human cytomegalovirus（HCMV） is a giant cell inclusion body disease pathogen. HCMV, belongs to the beta herpes virus, is common in nature and is characterized by a narrow host range, longer viral replication cycle, especially the progress in the replication of the virus in cell culture medium is very slow. HCMV has 208 open reading frames, encoded more than 200 kinds of polypeptides. It is a large particle complex viruses in nature, which makes the study of viral pathogenesis and host relationship to be complicated and difficult. Cytomegalovirus infection with strict species specificity, man is the only host of the virus. The virus can spread through the vertical or horizontal, and is often shown as no obvious symptoms. When cytomegalovirus in organism who were suffered from repeated blood transfusion, organ transplantation or merger of HIV infection, HCMV will transform from latent state to the state of reactivation and always be characterized by uncontrolled rapid replication, which cause HCMV pneumonia, atypical lymphocytes increased, retinitis, hepatitis, colitis, meningoencephalitis, severe graft rejection and multiple organ failure, and even death.At present, the hypothesis about cytomegalovirus reactivation and replication include cytokine hypothesis, ischemia/hypoxia hypothesis, organ transplant hypothesis, hypothesis of radiation damage, inflammation factors hypothesis, etc. These hypotheses study the effect of some factors on HCMV infection under certain conditions, and cannot solve the basic issue of HCMV replication. And now there are only three kinds of available drugs in clinically control of cytomegalovirus infection, including ganciclovir（GCV） and its precursor valganciclovir（vGCV）, foscarnet（FOS）, cidofovir（CDV）, etc., but the long time use of these drugs will be a severe renal toxicity and antiviral drug resistance strains produce risk. Therefore, the prevention and treatment of cytomegalovirus infection should focus on the exploration of the mechanism of HCMV replication, to find effective measures to prevent and combat viral replication.We find out that all of the crowd of HCMV infection exhibit a common feature which is the redox imbalance and shows high levels of oxidative stress state. Oxidative stress can promote a variety of virus replication in cells, and promote the development of the disease. Further study found that oxidative stress can enhance cell protein expression of cyclophilin A（CyPA） as well as activate p38-MAPK signaling pathway. However, the impact of oxidative stress-activated p38-MAPK pathway mediated by CyPA on HCMV replication is not fully clear.Therefore, we expect to utilize the intracellular and extracellular hydrogen peroxide to stimulate cells formation of oxidative stress environment in vitro. On this basis, we observe the effects of oxidative stress and antioxidant on HCMV infection, to make the relationship between oxidative stress and viral replication clear. Next, we find out the host proteins, CyPA, related to the virus infection under the stimulation of oxidative stress. We can deplete or inhibit of CyPA to observe the effects of CyPA for viral replication, thus explore the relationship between CyPA and virus replication. Mechanically, after find out the law of p38-MAPK signal pathways under the stimulation of oxidative stress, and then we explore the effect of the pathway for HCMV replication, so we can make the relationship between host and virus infection explicit. Thus, this study will serve as a new train of thought and direction for HCMV infection prevention and control of reproduction. The specific research contents and results are as follows:1. Establishment of cellular oxidative stressExtracellular hydrogen peroxide and inactivation of intracellular catalase were utilized to improve cellular hydrogen peroxide to establish cell oxidative stress environment in vitro. After dealing with the ATA, catalase activity in cells is restrained, along with the increase in concentration of hydrogen peroxide. And then, extracellular given hydrogen peroxide, the oxidative stress tracer tests shown the oxidative stress levels rose accompanied with the increasing of concentration of hydrogen peroxide. Next, western-blot detected the increasing of nuclear antioxidant related factor 2, Nrf2, in the nucleus of the cell with the increase of concentration of hydrogen peroxide. It shows cell oxidative stress will be driven by the changes of intracellular and extracellular hydrogen peroxide.2. Oxidative stress enhances HCMV replication through paracrine and autocrine mechanisms in vitro and in vivoBased on the oxidative stress cell model, on the one hand, supplement with extracellular hydrogen peroxide to observe the replication of HCMV, on the other hand, inhibition of cellular hydrogen peroxide enzyme to detect the effects of intracellular hydrogen peroxide on HCMV replication in cells, which will illustrate the relationship between oxidative stress and HCMV replication. Dual luciferase activity tests resulted that hydrogen peroxide can prompt HCMV MIE promoter activity in a dosedependence pattern. Then, the consequences of qRT-PCR, Western-blot, absolute quantitative PCR and viral titer detection shown that oxidative stress can also promote HCMV replication in the cells. Based on this, using NAC to inhibit cell oxidative stress, then observed the effects of HCMV replication under oxidative stress, the results showed cell oxidative stress levels were restrained, after using antioxidant in vitro, and then inhibition the H₂O₂-enhanced MIE promoter activity, which in turn affect the IE gene expression, virus pp72 and pp65 protein expression and viral titer. Similar to in vitro results, NAC could inhibit MCMV in the blood viral load and viral titer in the salivary glands and in the lung tissue after acute infection in vivo. Therefore, these results confirmed the antioxidants could inhibit hydrogen peroxide-promoted HCMV replication. Consequently, these results illustrated the relationship between oxidative stress and viral replication, but it is not clear how the host cell contact between viral replication.3. The effect of CyPA on the H₂O₂-enhanced HCMV replicationOn the basis of cell oxidative stress, an increasing of the host cell CyPA expression was also observed at the genetic level and protein level, as well as the expression of HCMV IE gene, but the expression of CyPA was also decreased when oxidative stress was inhibited, which indicated the CyPA expression under the influence of oxidative stress and suggested that CyPA was involved in HCMV replication. In order to study this relationship, interference by the lentivirus infection was utilized to screen stable CyPA depletion cell line. Based on the CyPA depletion cell, oxidative stress cannot enhance HCMV MIE promoter activity even stimulated with hydrogen peroxide, and at the same time, the virus IE gene expression, protein expression and virus particles were also not affected by oxidative stress. Accordingly, utilizing CsA to inhibit CyPA enzymes activity, we found treated with CsA could not change the oxidative stress circumstance and the oxidative stress-induced CyPA expression. However, CsA could restrain the activity of the H₂O₂-enhanced MIE and subsequent virus IE gene expression and protein expression. In vivo experiment, CsA could inhibit MCMV in the blood viral load and viral titer in the salivary glands and in the lung tissue after acute infection in vivo. The results indicated that CyPA, should be a pivotal cofactor, which acted as an important role in H₂O₂-enhanced HCMV replication.4. p38-MAPK signal pathway regulates HCMV replicationTo find out the mechanism of oxidative stress-enhanced viral replication, we used hydrogen peroxide to boost cell to form oxidative stress circumstance. After indicated times and different concentration of hydrogen peroxide stimulation, western-blot detected the impact of H₂O₂ on p38 activity. The results shown p38 was phosphorylated after 1 hour of hydrogen peroxide stimulation, and with the increase of time the degree of activation of p38 also increased, which lasted to 6 hours at the peak. And at the same time, given 25μM hydrogen peroxide could induce the activation of p38, and the degree of p38 phosphorylated was further enhanced with 50μM H₂O₂, then p38 phosphorylated level has no obvious change even increase the H₂O₂ concentration to 100μM or 200μM. Subsequently, after treatment with NAC and p38 inhibitor SB203580, western-blot results shown oxidative stress-activated p38 phosphorylated was restrained, which determined that oxidative stress can activate intracellular p38 activity. To validation of p38 participated in HCMV replication, the results showed that pretreatment with SB203580 was of no effect on cell oxidative stress level, but could also inhibit H₂O₂-enhanced HCMV replication. To verify whether CyPA was also engaged in the phosphorylation of p38, an inhibition of H₂O₂-activated p38 and H₂O₂-enhanced virus replication were also detected by depletion or inhibition of CyPA.In conclusion, this study comprehensively studied the influence of oxidative stress on HCMV replication, which demonstrated oxidative stress was an essential factor to promote CMV replication in vivo and in vitro. The results revealed the antioxidant was an effective way to control viral replication. The study also preliminary elucidated CyPA was an important cofactor during oxidative stress-enhanced viral replication. Mechanically, p38-MAPK acted as an important signaling pathways between oxidative stress and HCMV replication. Therefore, we put forward the ROSâ†'CyPAâ†'p38-MAPK-HCMV replication pathway, which would provide theoretical basis for further study on HCMV, and the targets based on this pathway could be used to study of the new drugs for controlling HCMV replication and also could provide theoretical support for prevention and treatment of HCMV infection.