Role of heat shock proteins Hsp90 in human adenovirus 5 replication

Role of heat shock proteins Hsp90 in human adenovirus 5 replication

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Title: Role of heat shock proteins Hsp90 in human adenovirus 5 replication

Level of degree:

Degree discipline :

medical sciences

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Instytut Medycyny Doświadczalnej i Klinicznej im. M. Mossakowskiego PAN

Abstract:

Human adenoviruses (HAdV) belong to the Adenoviridae family and are classified to the genus Mastadenovirus. Adenovirus infection is mainly associated with respiratory diseases. In addition to respiratory symptoms, adenoviruses also cause keratoconjunctivitis, hemorrhagic cystitis, gastroenteritis, meningitis, and haemophagocytic syndrome. In immunocompetent hosts, adenoviral infections are usually mild and self-limiting, but in the immunocompromised patient population adenovirus may cause significant morbidity and mortality.During viral replication, a large number of proteins are synthesized. In this process viral proteins require the assistance of cellular factors in proper folding and protection against aggregation. Depending on the group of viruses, during replication heat shock protein Hsp90 performs various functions facilitating the entry of viral particles into the cell, intracellular transport, protein expression and stabilization, and genome replication. In this study, HAdV-5 was identified as a virus which replication process is dependent on the chaperone activity of Hsp90. A specific inhibitor 17-allylamino-17 demethoxygeldanamycin (17-AAG) was used to inhibit the activity of Hsp90. The studies were conducted on human embryonic kidney cell line (HEK293) as well as adenocarcinomic human alveolar basal epithelial cells (A549). 17-AAG has been shown to have a strong dose-dependent inhibitory effect on HAdV-5 replication at concentrations that that are non-toxic to A549 cells. This effect was particularly pronounced when inhibitor was added at the time of infection. Analysis of the time course of HAdV-5 gene transcription showed a significant inhibition of viral gene transcription when 17-AAG was added at the time of infection of the cell culture. The inhibitor is less effective when applied at later time points. An analogous pattern was observed during the synthesis of viral structural proteins. After HAdV-5 genome enters the nucleus, E1A is the first gene to be transcribed and is responsible for inducing the transcription of the remaining viral genes. Studies using the phenomenon of transcomplementation have shown that the reduced amount of DNA, mRNA and structure proteins of the HAdV-5 results from the inhibition of the activity of the early E1A protein. In infected HEK293 cell lines where E1A is constitutively produced, the addition of 17-AAG inhibited HAdV-5 replication to a lesser extent than in A549 cells which do not produce E1A. In addition, immunoprecipitation of protein complexes showed that Hsp90 binds to the E1A protein. In a study using cycloheximide (CHX) and 17-AAG, it was shown that the combination of those inhibitors did not reduce the stability of E1A nor did it increase the rate of degradation of this protein in HEK293 cells. The effect of Hsp90 on the maturation of the newly synthesized E1A is confirmed by the observation that inhibition of Hsp90 causes a disruption of E1A protein synthesis at 2 and 4 hours after infection, despite the lack of inhibition of E1A gene transcription. Interference of HAdV-5 replication, due to inhibition of Hsp90 activity, is a temporary phenomenon. The disappearance of inhibition effect in long-term infection may result fromincomplete disruption of E1A synthesis, resulting in its gradual accumulation in the cell. After 48 hours of culture, the amount of E1A may be sufficient to activate the transcription of the remaining HAdV-5 genes.The results presented in this work show for the first time that Hsp90 is involved in the synthesis of E1A. Since E1A serves as a co-activator of transcription of the adenovirus early genes, the antiviral activity of the Hsp90 inhibitor can be explained by the decreased level of the early protein. The obtained results can be used in the future to design antiviral therapies, which usage will not be associated with the virus resistance.