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Hepatitis B Research Review

 

 

 

 

Welcome to the Hepatitis B Research Review! This monthly blog shares recent scientific findings with members of Baruch S. Blumberg Institute (BSBI) labs and the hepatitis B (HBV) community. Technical articles concerning HBV, Hepatocellular Carcinoma, and STING protein will be highlighted as well as scientific breakthroughs in cancer, immunology, and virology. For each article, a brief synopsis reporting key points is provided as the BSBI does not enjoy the luxury of a library subscription. The hope is to disseminate relevant articles across our labs and the hep B community. 

Interferon-inducible MX2 is a host restriction factor of hepatitis B virus replication Journal of Hepatology

  • This paper from Fudan University in Shanghai, China reports the interferon-induced GTPase MX2 as a host protein which inhibits HBV replication. Interferon alpha (IFN-α) is a type 1 interferon used in a subset of HBV-infected patients to help eradicate the virus. IFN-α treatment results in the activation of hundreds of genes known as interferon-stimulated genes (ISGs). Which ISGs are most important in eliminating HBV infection remain largely unknown. GTPases are a large family of hydrolase enzymes which convert guanosine triphosphate (GTP) to guanosine diphosphate (GDP). GTPases act as molecular switches in an array of cellular process including signal transduction, cell division and differentiation, and protein translocation. The myxovirus resistance (Mx) proteins are highly conserved, dynamin-like, large GTPases. Humans have two MX proteins: MX1 and MX2, both of which are known ISGs. While MX1 is known to have broad-spectrum antiviral activity against RNA viruses, MX2 has only recently been shown to inhibit human immunodeficiency virus 1 (HIV-1), hepatitis C virus (HCV), and hepesviruses. MX2 antiviral activity against HIV-1 and herpesviruses is mediated through MX2 binding to the capsid of invading viruses whereby it likely inhibits the uncoating of viral DNA. In HCV, MX2 was found to interact with non-structural protein 5A (NS5A) thereby inhibiting its localization to the endoplasmic reticulum (ER). MX1 has been reported to inhibit HBV replication by inhibiting nuclear export of viral RNas and/or trapping the HBV core protein indirectly. This study investigates the anti-HBV activity of MX2. First, the group compared the anti-HBV activity of MX2 to four other innate immune restriction factors: HNRNPU, SAMHD1, MOV10 and A3G. They co-transfected these genes along with the HBV genome into HUH-7 cells and then assessed HBV replication via Southern blot. MX2 was found to inhibit HBV replication the most, with 44% of viral DNA compared to the empty vector control. The group then used siRNA, Southern blot, Western blot, fractionation, and mutagenesis studies to elucidate the anti-HBV role of MX2. Overall, they found that MX2 significantly reduces HBV RNA levels and indirectly impairs cccDNA formation. MX2 was found to contribute substantially to the anti-HBV affect of  IFN-α. Both the GTPase activity and oligomerization status of MX2 were found to be important in conferring its anti-HBV affect. In the future, MX2 and its related pathways may be exploited to help prevent the formation of and even eliminate cccDNA in those infected with HBV.

An HBV-encoded miRNA activates innate immunity to restrict HBV replication – Journal of Molecular Cell Biology

    • This paper from the Tianjin Medical University in China explains how an HBV-encoded microRNA (miRNA) activates the innate immune system in humans infected with the virus. miRNAs are short (21-25 nucleotides) sequences of mRNA which are mainly involved in post-transcriptional silencing of genes. miRNAs are produced in plants, animals, bacteria, and viruses. Typically, miRNA acts to silence protein translation from a messenger RNA (mRNA) by binding to the 3′ untranslated region (UTR) of the mRNA. This binding may result in the destabilization or cleavage of the mRNA or inhibit the function of the ribosome during translation. This group has identified an miRNA from the HBV genome called HBV-miR-3 which they have previously reported inhibits HBV replication by targeting the HBV mRNA transcript. In this paper, the group first shows that HBV-miR-3 is produced in an amount proportional to virus infection in vitro. They also show that HBV-miR-3 is secreted from cells in exosomes. Next, using both patient serum samples and in vitro assays, the group found a positive correlation between HBV-miR-3 production and IFN-α signaling pathways. In patient serum, levels of HBV-miR-3 positively correlated with levels of the hepatitis-related parameters alanine aminotransferase (ALT), aspartate transaminase (AST) and type I IFNs (IFN-α and IFN-β). In cell culture, they observed an increased expression of  the IFN-α-induced antiviral effectors OAS-1, MX1, IFIT2 and IFIT3 in the context of HBV-miR-3 production. Further experiments indicated that HBV-miR-3 promotes IFN-α production by suppressing the expression of suppressor of cytokine signaling 5 (SOCS5), allowing for signal transducer and activator of transcription 1 (STAT1) to be activated by phosphorylation. Finally, the group shows that HBV-miR-3 released from infected cells in exosomes  promotes polarization of the M1 macrophage phenotype. M1 or “classically activated” macrophages secrete high levels of pro-inflammatory cytokines and thereby fight pathogenic infections. Taken together, these results show that aside from directly limiting HBV replication, HBV-miR-3 also indirectly limits HBV infection by activating the host innate immune system. The virus may do this in order to adopt host miRNA-mediated antiviral machinery and thereby alleviate pathogenesis so that persistent and latent infection can continue. In the future, levels of HBV-miR-3 may be used as a diagnostic marker for HBV infection and may shed light on novel antiviral approaches.

Innate and adaptive immunity associated with resolution of acute woodchuck hepatitis virus infection in adult woodchucks – PLOS Pathogens

    • This paper from Georgetown University in Washington, DC is a “woodchuck paper”. That is, it is an in vivo study of woodchucks infected with Woodchuck Hepatitis Virus (WHV). WHV infection is used as a model system for HBV infection in humans because WHV is similar to HBV. This type of study is beneficial, especially when studying the immune response to hepadnaviruses, because humans infected with HBV are typically asymptomatic in the early stage of infection and because it is not advisable to obtain liver biopsies from these patients. The woodchuck infection model offers a controlled infection with WHV at a known time-point, which can be monitored by regular blood tests and liver biopsies. When studying the immune response to hepadnaviruses, liver biopsies are necessary because the liver is the site of the infection. About 95% of adults infected with HBV “clear” the virus; that is, their immune system is able to fight off the virus completely, giving them life-long immunity. The other 5% become chronic carriers of HBV and are at a high risk for liver cirrhosis and hepatocellular carcinoma (HCC). However, 95% of infants infected with HBV become chronic carriers. Differences in the immune systems of adults vs infants have been attributed to this drastic difference in chronicity, but what specific components of the immune system are important in staving off chronic infection remain unknown. Overall, the data presented here indicate that there is an early, non-cytolytic control of WHV replication mediated by interferon gamma (IFN-γ) produced mainly by natural killer (NK) cells. This was followed by an adaptive immune response characterized by antibody production, a T-cell response, and cytolytic action of cytotoxic T lymphocytes (CTLs). This adaptive immune response led to both the decline of WHV as well as symptoms of acute hepatitis B (AHB) including sinusoidal and portal inflammation in the liver.

Differential alternative splicing regulation among hepatocellular carcinoma with different risk factors BMC Medical Genomics

    • This paper from the University of Utah School of Medicine in Salt Lake City, Utah uses bioinformatics to examine how different risk factors for hepatocellular carcinoma (HCC) correlate with differential alternative splicing (AS) of tumor mRNAs. After a primary (precursor) mRNA transcript is produced in the nucleus by RNA polymerase, the transcript must “mature” by having regions called “exons” removed in a process called splicing. Splicing results in an mRNA transcript consisting entirely of “introns”. The mRNA is then capped at its 5′ end with a 7-methylguanosine residue and polyadenylated at its 3′ end with about 200 adenylate residues (poly-A tail). This mature mRNA is able to exit the nucleus and be translated into protein by a ribosome. Alternative splicing (AS) describes how one genomic region may code for many different protein variants (isoforms) by differential spicing of the primary mRNA transcript. A common mechanism of AS is “exon skipping”, where exons are included in some mature transcripts but not others. HCC has various risk factors including alchohol consumption and infection with hepatitis B or C viruses (HBV and HCV). This study used data from The Cancer Genome Atlas (TCGA) and  the Genomic Data Commons (GDC) Data portal to analyze 218 patients with primary HCC associated with HBV (n = 95), HCV (n =47), or alcohol (n = 76). They used RNA sequencing (RNA-Seq) data to examine differences in AS between three groups: HBV vs. HCV, HBV vs. alcohol, and HCV vs. alcohol. 143 genes were identified with differential AS across these groups and these genes were found to be mainly involved in immune system, mRNA splicing-major pathway, and nonsense-mediated decay pathways.Of the 143 AS genes identified, eight and one gene were alternatively spliced specific to HBV and HCV respectively. The human leukocyte antigen genes HLA-A and HLA-C had differential AS in HBV-related HCC compared to both HCV- and alchohol-related HCC. HLA ptoteins are part of the major histocompatibility complex (MHC) class 1 surface proteins which present foreign antigens to the immune system. Also, exon 3 of  the gene encoding inositol hexakisphosphate kinase 2 (IP6K2) was skipped more often in HBV-related HCC than in other groups. IP6K2 is known to be involved in cancer metastasis. This study represents the first investigation into how different risk factors of HCC may affect the AS status of specific genes.

The Cytosolic DNA-Sensing cGAS–STING Pathway in Cancer (Review) Cancer Discovery

    • This review from the Memorial Sloan Kettering Cancer Center in New York City covers current understanding of the cGAS-STING pathway in the context of cancer. While it is well known that the cGAS-STING pathway is an evolutionarily-conserved  antiviral signaling platform, how this pathway is involved in tumorigenesis remains unclear. In preneoplastic (early tumor) cells, cGAMP produced in response to DNA damage is exported out of the cell to activate STING in neighboring antigen-presenting cells (APC). This activation results in the release of type 1 interferon (IFN) from the APC, which cross-primes natural-killer and CD8 T-cells to kill the preneoplastic cells. In this context, the cGAS-STING pathway plays a role in tumor surveillance by activating innate immunity to create “hot spots” of inflammation. However, there is also evidence that activation of the cGAS-STING pathway can contribute to tumorigenesis.  In advanced, metastatic tumor cells, chronic activation of STING by chromosomal abnormalities leads to suppressed production of IFN and the upregulation of Nf-kB-driven pro-survival genes. This can drive chronic inflammation of the tumor as well as its metastasis to other locations in the body. Activation of the STING pathway in tumor cells may also allow for their immune evasion by inducing autophagy and upregulating expression of programmed death-ligand 1 (PD-L1). Another interesting finding mentioned in this review is a STING-independent form of cGAS activation which may drive tumorigenesis during cell division. During mitosis, cytoplasmic cGAS may bind to repeat sequences in the centromere regions of chromosomal DNA. Once bound, cGAS may interrupt the repair of sister chromatids by homologous recombination, causing aneuploidy in daughter cells, a hallmark of tumor cells. Of additional interest, mentioned in this review are several recent findings regarding the cGAS-STING pathway, including: cGAS can be activated by extracellular DNA entering the cell in exosomes; cGAS can be activated by “micronuclei” which are small nuclear compartments in the cytoplasm formed by chromosomal instability; cGAS-DNA complexes turn into a liquid phase to produce cGAMP; STING dimers oligomerize to form tetramers when activated; palmitoylation of STING has been proposed to recruit TANK binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3).

Lay Summary: 
This month, the innate immune system was the focus of HBV research. Scientists hope to find how the innate immune system interacts with HBV during viral infection and proliferation. Doing so will shed light on host factors which lead to chronic infection and inform antiviral strategies. Notably, this month a human protein, MX2 was found to have potent anti-HBV activity by preventing cccDNA formation. Also, a microRNA encoded by HBV called HBV-miR-3 was found to activate the human innate immune system to limit HBV replication. This month, a paper studying woodchuck hepatitis virus (WHV) traked activation of the innate immune system as well as he adaptive immune system in an acute infection model. Also this month, concerning hepatocellular carcenoma (HCC), the alternative splicing of mRNA in tumors was found to vary in HCC patients based upon their risk factor (HBV, HCV, or alcohol). Finally, a review was published this month concerning STING, an innate immune protein which is not activated by HBV infection but which may prove a valuable tool for cancer treatment.  

Meet our guest blogger, David Schad, B.Sc., Junior Research Fellow at the Baruch S. Blumberg Institute studying programmed cell death such as apoptosis and necroptosis in the context of hepatitis B infection under the direction of PI Dr. Roshan Thapa. David also mentors high school students from local area schools as part of an after-school program in the new teaching lab at the PA Biotech Center. His passion is learning, teaching and collaborating with others to conduct research to better understand nature.

Is a Cure for Hepatitis B Coming? Experts Say Yes

How far are we from finding a cure for hepatitis B? We are close, said Timothy Block, PhD, president and co-founder of the Hepatitis B Foundation and its research arm, the Baruch S. Blumberg Institute. He points out that hepatitis C, once thought to be incurable, is today cured with new combination treatments.

Image courtesy of suphakit73 at FreeDigitalPhotos.net.
Image courtesy of suphakit73 at FreeDigitalPhotos.net.

Experts believe a cure for hepatitis B will also soon be developed. And the need for a cure has never been greater, with more than 240 million people worldwide living with chronic hepatitis B, causing 1 million deaths per year from related liver failure and liver cancer.

“Treatments are available,” explained Block, “but we have become a little too comfortable with the medications that are currently approved for use.” While these drugs are effective, interferon has many side effects and daily antivirals require lifelong use. These drugs work in only half of the infected population and reduce death rates by only about 40 to 70 percent.

What will a cure look like?

The available antivirals are similar and combining them offers no advantage. They have limited effectiveness against cccDNA, the seemingly indestructible “mini-chromosome” of the hepatitis B virus that continues to produce virus particles in infected liver cells, even in people being treated. A cure, therefore, would have to destroy or silence cccDNA and provide long-term immunity. Because one-drug treatments can lead to drug resistance, a cure would almost certainly involve combination therapy, similar to hepatitis C. Continue reading "Is a Cure for Hepatitis B Coming? Experts Say Yes"

HBV Journal Review – September 2014

ChrisKHBF is pleased to connect our blog readers to Christine Kukka’s monthly HBV Journal Review that she writes for the HBV Advocate. The journal presents the
 latest in hepatitis B research, treatment, and prevention from recent academic and medical journals. This month, the following topics are explored:

  • New Study Finds HBV Genotype E Responds Poorly to Entecavir
  • HBV Genotypes Help Tell the Human Story of Slavery in the Americas
  • Researchers Find Tenofovir Increases Hip Bone Loss in Older Patients
  • Decline in HBV RNA Indicates Who Loses HBeAg During Antiviral Treatment
  •  Shortened Vaccination Schedule May Get More Drug Users Immunized
  • Primary Care Doctors Rarely Screen Patients for Cirrhosis
  • Tenofovir or Telbivudine Recommended for Pregnant Women with High Viral Loads
  • Access to Healthy Food Vital for HBV Patients, but Many Live in Food “Deserts”
  • Scientists Create Viable Liver Cells in a Lab for HBV Research
  • Nerve Damage Prompts Warning Against Telbivudine-Interferon Combo Treatment

HBV Journal Review

September 1, 2014
Volume 11, Issue 9
by Christine M. Kukka

New Study Finds HBV Genotype E Responds Poorly to Entecavir
Experts know some hepatitis B virus (HBV) strains called genotypes respond better to interferon treatment than others, but now scientists are discovering that genotypes respond differently to antiviral treatment too.

HBV genotypes are found in different regions of the world and each evolved over centuries to have slightly different molecular make-ups with unique traits. Some carry a higher risk of liver damage and cancer, while other genotypes are less virulent.

In a recent study, Italian researchers compared how well patients with genotypes A, D and E fared after three years of treatment with the antiviral entecavir (Baraclude). All of the patients tested negative for the hepatitis B “e” antigen (HBeAg-negative). The scientists measured hepatitis B surface antigen (HBsAg) levels and HBV DNA (viral load) every three months during the first year of treatment and then every six months over the study period.

They found the rates of HBsAg declines resulting from antiviral treatment varied markedly between genotypes. They extrapolated how many years of entecavir treatment each genotype required before a patient would clear HBsAg and achieve undetectable viral load.

HBV genotype A: It would take on average 15.6 years of entecavir treatment for an HBeAg-negative patient with HBV genotype A to lose HBsAg. This genotype is found in northern Europe, North America, India and southern Africa.

HBV genotype D: It would take 17 years for genotype D patients to lose HBsAg. This strain is found primarily in Russia, the Middle East, the Mediterranean region, and India.

HBV genotype E: This genotype, found in Central Africa, responded the most poorly to entecavir. Scientists estimated it would take 24.6 years for these patients to lose HBsAg, according to the report published in the August issue of the Journal of Medical Virology.

Source: www.ncbi.nlm.nih.gov/pubmed/25131947

HBV Genotypes Help Tell the Human Story of Slavery in the Americas
Because HBV genotypes develop in specific regions around the world, their distribution around the world today can help tell the story of mass human migrations, including the enslavement and forced migration of millions of Africans to Brazil since the 1500s.

Read the HBV Journal Review in its entirety here.