Functional Characterisation of Pre-S1/Pre-S2 Deletion Mutants of Hepatitis B virus
Project ended 31 March 2018
Prof Anna Kramvis
- Hepatitis Virus Diversity Research Programme, Department of Internal Medicine, University of the Witwatersrand
Title of the project
Functional Characterization of Pre-S1/Pre-S2 Deletion Mutants of Hepatitis B Virus isolated from Southern African HCC and HIV patients.
Hepatitis B virus (HBV) is the smallest DNA virus infecting man. This virus targets the liver and has been shown to be associated with the development of liver cancer. By comparing the strains found in patients with liver cancer to those found in individuals without cancer, it has been possible to show that the strains from the cancer patients differ from those without cancer. Thus as molecular virologists, the purpose of our research is to determine how these differences in the strains can contribute to the development of liver cancer.
HBV strains with mutations were isolated from South African liver cancer patients. These strains all contained mutations in the preS1/preS2 region of the virus, deletions that were not found in isolates obtained from asymptomatic carriers of the virus. The presence of these strains with deletions has also been described in Japanese and Chinese liver cancer patients and several studies have shown a very strong correlation between these mutants and the development of liver cancer. However, the strains that circulate in Japan and China differ from those found in South Africa, where subgenotype A1, which has unique characteristics, prevails. Moreover, we have also shown that subgenotype A1 has a higher potential to cause liver cancer than other strains and individuals infected with this subgenotype develop liver cancer 6.5 years earlier than individuals infected with other genotypes. In addition, during the course of another study in HBV/HIV infected South Africans, we found five HBV isolates with preS1/preS2 deletions ranging from 11 to 33 codons. These deletions mutants are very similar to those we previously described in HCC patients. This finding has very important implications in the South African context, where HBV/HIV co-infection occurs in 24% of HIV infected individuals. These deletion mutants could be a risk factor for the development of HCC in HIV patients, whose lifespan is being increased following the introduction of highly active antiretroviral treatment (HAART).
Thus our hypothesis is that these mutations in subgenotype A1 could affect viral replication and expression of the viral envelope proteins, leading to endoplasmic reticulum (ER) stress and apoptosis (cell death), which can ultimately contribute to the potential of the virus to cause cancer. The aim of the proposed study is to determine the significance of these mutant isolates of HBV by functionally characterizing them in tissue culture. Replication competent clones of wild-type strains have been constructed and we are busy introducing the preS1/preS2 deletion mutant into these clones. These will be used to transfect liver cells in tissue culture. We will monitor viral replication, surface protein expression and retention at various times post-transfection. Depending on the outcome of these experiments we will determine whether the mutant virus can cause apoptosis and disturb the unfolded protein response of the cells.
The value of the project in the struggle against cancer:
Africa is the only continent, globally, where hepatitis B virus (HBV) infection is still hyperendemic in certain regions, with exposure, as measured by anti-HBc-positivity, of greater than 85% in western Africa, 65% to 85% in eastern Africa and 35% to 75% in southern Africa (1). A quarter of the world’s more than 250 million chronic carriers of HBV reside in Africa and this is probably an underestimate because of under-reporting and lack of extensive surveillance. There is a correspondingly high annual incidence of hepatocellular carcinoma (HCC; liver cancer) per 100 000 of 14.8 in males and 6.2 in females (2), ranking second and fifth of the leading cancer-associated deaths for men and women, respectively (3). In western Africa, HCC is the most commonly diagnosed cancer in males, with the highest annual fatality ratio for any human tumour, of 0.93 (4). HBV infection is the primary aetiological risk factor for the development of HCC in Africans, with two thirds of children and 60% to 80% of adults with the disease being seropositive for hepatitis B surface antigen (HBsAg) (5, 6). Known co-carcinogens of HBV include dietary iron overload (7) and aflatoxin B1 (8). The strains of HBV prevailing in Africa differ both molecularly and functionally from those prevailing out of Africa (9). Thus studies carried out outside Africa cannot necessarily be extrapolated to the African continent.
Subgenotype A1 the prevalent HBV strain in southern-eastern Africa has been to be more hepatocarcinogenic than other strains prevailing in the region (10) and has also been shown to be associated with liver cancer in Indians (11). HBV/HIV co-infection is frequent, with 9% of HIVpositive patients having HBsAg-positive infection (overt) and 15% HBsAg-negative (occult infection) (12) and the majority infected with subgenotype A1 (13). In the same study, pre-S deletion mutants were isolated, four from overt infection (SHH011A, SHH167A, SHH274A and SHH300A) and one from an occult HBV infection (SHH045A) and the majority were pre-S2 mutants. Similar deletion mutants were also found in HCC patients infected with subgenotype A1, both in South Africa (Skelton, PhD thesis) and India (14) and as shown for other genotypes of HBV, are a risk factor for the development HCC (15, 16).
This study is the first study to molecularly and functionally characterize preS deletion mutants isolated from African HCC and HBV/HIV co-infected patients and to show that the they are the majority population in infected individuals. These mutants can replicate in tissue culture and express proteins, with the expression being slower than those by the wild-type strains. The retention of proteins have been shown to affect the unfolded protein response and this can lead to increased cell damage. This increased cell damage can contribute to the hepatocarcinogenic potential of subgenotype A1. These deletion mutants are thus risks factor for the development of HCC and may be useful as biomarkers in determining the risk for development of HCC.
Future plans for the project:
- The study is being continued. We plan to:
Measure the amounts and ratios of extracellular and intracellular envelope proteins by collecting the culture medium and extracting intracellular proteins at various times post-transfection.
- Determine whether the pre-S mutant strains can induce ER stress/apoptosis in transfected cells. Ms Vos (Masters student #4) is studying the expression of HBsAg and HBeAg by the deletion mutants and by the occult strain of subgenotype A1 using cellular fractionation. Ms Pillay (Masters student #3) is carrying out the parallel study on the wild-type strains, that is strains without the deletion mutants.The aim of their studies will be to determine whether HBsAg deletion mutants and the occult strain of subgenotype A1 HBV affect the subcellular localization, expression and release of the different HBsAgs and HBeAg and their relative ratios in vitro.
Following transfection of Huh7 cells with subgenotype A1 replication competent plasmids for the wild-type, occult strain, PreS1 and PreS2 deletion mutants, the objectives of the study will be to:
i. Determine the intracellular expression of LHBs, MHBs, SHBs and HBeAg in the nucleus, cytoplasm and membrane of the cell
ii. Determine the extracellular expression of HBV proteins (HBsAg, LHBs, MHBs, SHBs and HBeAg) and HBV DNA