Role of HIV-1 replicative fitness on HIV pathogenesis and disease progression in untreated patients

Disease progression varies significantly in HIV-infected patients and is dependent on a number of host genetic, immune and virological factors. One of them is HIV replicative fitness that is associated with virulence, pathogenicity, drug-resistant mutations and disease progression. Based on disease progression we divide patients into rapid progressors, chronic progressors and long term nonprogressors also called controllers. The CD8+cytotoxic T lymfocytes (CTL) response is the major contributor to viral control after HIV-1 infection. It was shown that CTL escape mutations are frequently selected in gag p24 (capsid) and gp120 but evidence of these mutations are found in the whole HIV proteome. Several recent studies have reported that CTL escape mutations can result in a loss of viral fitness. Here, we intend (i) to establish method for measuring HIV replicative fitness in therapeutically naïve patients taking into account all three major genes (gag, pol, and env), (ii) to evaluate the contribution of various virus genes to HIV replicative fitness in chronic progressors and controllers, and (iii) finally to apply these results in a pilot study to correlate HIV replicative fitness with disease progression in the absence of antiretroviral treatment. Furthermore, we will analyze host genetic and immune factors associated with slow disease progression. Our goal is to elucidate if monitoring of HIV replicative fitness and correlation with genetic and immunological parameter brings benefit to the physician in HIV/AIDS disease management.

HIV capsid as a new therapeutic target

HIV-1 life cycle can be divided to early phase during which the infection is established, and late phase, characterized by the production of new virions. In both of these phases plays HIV capsid protein a critical role. In the early phase, after fusion of the viral membrane and cellular cytoplasmic membrane the capsid core is released into the cytoplasm of the target cell. HIV-1 uncoating is a necessary step of HIV-1 infection and includes the transition between capsid abundant reverse transcription complex in which reverse transcription occurs, and preintegration complex which is competent to integrate into the host genome and that lacks capsid. The molecular mechanism underlying the destabilization and uncoating of HIV-1 core in the cytoplasm of infected cell and timing of steps leading to productive infection are still unclear. We plan to analyze temporal course of uncoating and its possible trigger. In addition, we will search for small molecules that could interfere with core disassembly and thus represent a new class of antivirotics.

Next to regulated disassembly of the capsid core the capsid protein plays also critical role in the late phase of life cycle during the assembly of the immature capsid and after proteolytic cleavage of Gag polyprotein by protease, the capsid protein is the building block of the mature core of the infectious virus. Here, in collaboration with Dr. Jan Konvalinka's group, we will search for potent HIV assembly inhibitor.

Epigenetic control of HBV replication

Hepatitis B is, despite the availability of an efficacious vaccine, a major health problem worldwide with the prevalence depending on the geographic area. An estimated 2 billion people have been exposed to hepatitis B virus (HBV) infection, of which about 350 million remain chronically infected. This makes HBV the most important cause of liver disease. More than 90% of immune-competent individuals clear acute HBV infection, but chronicity develops frequently in immune-compromised patients. The molecular basis of HBV persistence lies in a key replication step in HBV life cycle. Upon entering of the virion in the hepatocyte the viral genome is uncoated and as a relaxed circular DNA transported to the nucleus, where it is converted into a covalently closed circular DNA (cccDNA). The cccDNA is the template for transcription leading to production of progeny. The cccDNA forms with cellular histones viral minichromosome that is resistant to enzymatic digestion or to current antiviral compounds. Many molecular mechanisms were proposed as a reason leading to HBV latency and HBV reactivation. Next to host immune responses, coinfections, sequence variations, also epigenetic changes were proposed as a key factor. It was shown that methylation of HBV DNA impairs HBV replication, HBV protein synthesis and HBV virion production. Methylation of cccDNA is associated with low serum HBV DNA in patients with liver cirrhosis. Furthermore, hypermethylated HBV DNA sequences are frequently detected in patients with occult HBV infection. We will analyze the effect of methylation and acetylation on HBV replication initially in cell culture and in the future in patients chronically infected with HBV. As HBV core protein was shown to interact with cccDNA we will also analyze the impact of HBV core protein on HBV replication, methylation, and acetylation of cccDNA. In addition, we will search for proteins interacting with HBV core protein especially for transcriptional factors known to influence methylation and acetylation.