Studies On The Effects On Cell Apoptosis And The Antagonistic Function To Host Restriction Factors Of Significant Viral Disease Pathogens

Viral disease is a disease caused by a viral infection. In recent years, severaloutbreaks of avian influenza virus, enteroviruses have brought serious harm to China.In addition, the number of AIDS patients increased year by year, which also broughtnew challenges for the country’s public health career. Virus infection is oftenaccompanied by an organism immune system disorders, causing abnormal apoptosis,inhibition of the anti-viral agents, so that the body makes pathological changes.Furthermore it has a wide spread epidemic. Therefore, the viral disease has become aresearch hotspot in the world today.Apoptosis is a process controlled by a series of genes in ontogeny, subject tovarious death stimuli to active death process. Apoptosis requires co-stimulation ofendogenous apoptosis signal and exogenous apoptotic signal, thereby activating cellsuicide program encoded therein. Apoptosis occurs in eukaryotic cells. The apoptosisis also a dynamic process which eliminate abnormal cells and to maintain theirenvironmental stability. It plays a very significant role in many pathological andphysiological adjustment processes. Therefore, it is important for the qualitative andquantitative detection of apoptosis clinical medicine or basic research.A typical model that virus on the host restriction factor inhibiting is that HIVaccessory protein Vif degrades APOBEC3family protein. Vif can inhibit the anti-viralactivity of APOBEC3protein family, which is cytosine deaminase. The mechanism isthat accessory protein Vif has the ability to interact with host cell protein ElonginB,ElonginC and Cul5to form an E3complex, and APOBEC3family proteins weredegradated by the ubiquitin pathway. In this study, we take the current popular enterovirus CA16and HIV as examples,analyzing the mechanism that impact of the virus on the human organism.CA16-infected patients will exhibit many complications, and the abnormal cellapoptosis will lead to many diseases. Therefore, we applied the detection of cellmorphology, nuclear staining, DNA Ladder method and Annexin V-PI staining inorder to assess CA16-induced apoptosis. And we tried to look for the key protein toCA16-induced apoptosis and CA16-induced apoptosis.In recent years, HIV-related protein, the VPX, to SAMHD1and Vpr protein aresubject to more extensive research. The paper suggests that, Vpr is a HIVapoptosis-related protein and the Vpr having CRL4(DCAF1) E3connection.Therefore, we make a guess to the same connection mode protein, Vpx. Whether theVpx is another apoptosis-related factors in HIV? Since we knew that the Vpx andSAMHD1the closely related, whether Vpr also associated with SAMHD1?Therefore, this paper did some work on finding the new area that Vpx-mediated thedegradation of SAMHD1. We hope that we could provide some theoretical data tothe research of HIV apoptosis or the HIV mechanism.This paper includes the following aspects:1. The study of viral disease pathogens that cause apoptosis.We used the CA16to infect HEK293T, Magi, Vero, HepG2and A172,5kinds ofcells and by classic apoptosis detection methods at different times after infection tojudge apoptosis. The results showed that CA16can induce Magi, Vero, HepG2andA172to apoptosis, however, impacted less on HEK293T.48hours after infection,HEK293T still had intact nuclei and cell morphologyMeanwhile, we applied the Real-Time PCR method to assess the replication of thevirus in different cells. The experimental results showed that all the virus inintracellular above can copy. Thus, we can also conclude that the CA16can notinduce the HEK293T to apoptosis, which has nothing to do with viral replication.The occurrence of apoptosis may be associated with a particular protein. Afterreferring to the papers of EV713C and2A proteins promote apoptosis, we constructed the CA162A and3C protein plasmids, and we transiently transfectedthem into Vero cells to assess weather they will induce apoptosis. Experimental resultsshowed that these two proteins are not able to cause apoptosis in Vero. Thus, weconjectured that CA16-induced apoptosis may exist alone functional protein, or someother proteins that interact with2A and3C, inducing apoptosis.There are several ways to apoptosis; we explored the CA16-induced apoptosis ofA172. Since the CA16had no significant difference in induction of apoptosis ofneuronal and non-neuronal cells, therefore, we also hoped that the A172, for example,will play a guiding role in looking for CA16induced apoptosis pathway in othernon-neuronal cells. We harvested the cells at different times after infection, and toidentify the relationship between the A172apoptosis and caspase by immunoblotting.Experimental results showed that CA16induced A172apoptosis were associate withCaspse-8, and the apoptotic pathway is the death receptor pathway.2. The research of the viral disease pathogens antiviral mechanism.The SAMHD1protein, associated with HIV, including SAM domain and HDdomain. SAMHD1can suppress the infection of CD4+T cells and human myeloidcells as well as HIV, SIV. Vpx connects with SAMHD1C-terminal. By the CRL4(DCAF1) E3ligase, Vpx promotes SAMHD1degradation by the proteasome.However, the SAMHD1degradation area which mediated by Vpx is not very clear.Therefore, the area of its degradation is crucial. In this paper, we designed someSAMHD1N-terminal deletion mutants. We co-transfected these mutants and Vpx intoHEK293T cells, and tried to find the Vpx degradation SAMHD1area throughmethods such as Western blot. The experimental results demonstrated that theconnection area of SAM and HD and HD domain are very important for Vpxmediated degradation. The SAM region and N terminal region in SAMHD1are thenecessary areas that Vpx mediated degradation.SAMHD1contains a classical nuclear localization signal (NLS), however, ourexperimental results showed that NLS mutation was in the cytoplasm, and couldinhibit the degradation of Vpx. Therefore, this article also focused on the NLS area. We made SAMHD1NLS mutations to study the relationship between Vpx andSAMHD1K11A, as well as the position of SAMHD1K11A. The results showed thatSAMHD1K11A was found in the cytoplasm and suppress being degraded by Vpx.When the presence of wild-type SAMHD1, SAMHD1K11A will be re-positioned inthe nucleus, and, at the same time, restore the sensitivity of Vpx. UnlikeSAMHD1K11A, other SAMHD1mutants are within the nucleus. Therefore, wespeculated that SAMHD1contains an addition nuclear targeting mechanism otherthan NLS.In summary, this study laid the foundation for the mechanism of viral diseases,meanwhile, provided new direction to targeted therapy for viral diseases.