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Foreword
The publication of “Hepatitis B virus in Human Diseases” coincides with the fiftieth anniversary of the discovery of the virus [1]. This is a good time, therefore, to reflect on where we have come from, and are going.
We now know that Hepatitis B virus (HBV) is a small DNA virus that has chronically infected hundreds of millions of people worldwide and is responsible for nearly a million liver disease-related deaths a year [2, 3]. However, it is worth noting that the discovery of hepatitis B was not an obvious one, but was made in a very exciting time. Although science progressed with great speed in the 1940s, 1950s, and early 1960s, most of this progress was in molecular biology, as compared with medical science. For example, and to put the period in context, DNA was identified as the information molecule in all cells [4] and its structure of DNA was solved by Watson and Crick [5]. The genetic code was translated by Nirenberg and Matthaei [6] and the genetic regulation of protein synthesis was discovered by Jacob and Monod [7]. These fundamental discoveries were made possible by rigorous application of the scientific method. If medical science progress was slower, it may have been because it focused on detailed descriptions of disease rather than applications of the scientific method to discover etiology. Discovery of HBV came after a series of clever insights standing on what could have been taken as unrelated discoveries and observations.
Baruch S Blumberg, who received the 1976 Nobel Prize in Medicine or Physiology for his role in the discovery of hepatitis B and chronic viral diseases, had studied biochemistry with Alexander Ogston at Oxford University in the late 1950s. There he learned that British research used a “scientific” method to investigate medical and biological problems. His work began with the study of protein polymorphisms in peripheral blood. Initially, Blumberg and Allison determined if people who received transfusions made antibodies to antigens on polymorphic blood proteins [8]. Later Blumberg and Alter continued to test this hypothesis using serum from multiply transfused patients, to identify more polymorphic proteins. One such protein, they called “Australia” antigen, named for the location of individual in whose blood it was found [1]. Careful testing of sera from patients with a variety of diseases eventually led to finding the association of Australia antigen with
one type of viral hepatitis, once called “serum hepatitis,” and is now called hepatitis B. Surprisingly the Australian antigen was located on the surface of the virus itself and is now called HBsAg. The complicated natural history of hepatitis B disease in people made the discovery of its etiology all the more remarkable. Blumberg viewed it as a vindication of both non-goal-oriented research and the application of the scientific method to human disease.
Realization that Australia Antigen, originally recognized only by precipitin lines in Ouchterlony gel plates [1], was associated with hepatitis [9] and the hepatitis B virus envelope protein [10, 11] led to development of an assay to screen donor bloods and to invention of an effective vaccine [12]. The screening assay rapidly led to clearance of the blood supply of virally contaminated blood. Harvey Alter led the call to test all blood to be used for transfusions for the presence of “Australia” antigen based on these early observations and thus affected an important translation of basic scientific findings into an important clinical application in an unprecedentedly brief time.
The first HBV vaccine was produced from viral antigen derived from chemically inactivated virus, isolated from the blood of infected carriers [12], and approved for use by the US FDA in 1981. This was replaced in the 1990s with vaccines made from HBV recombinant envelope protein, isolated from yeast or mammalian cell culture (CHO cells), thus avoiding the concerns surrounding use of human HBV carrier blood [13]. These vaccines have been effective in interrupting perinatal transmission (which is a form of “post-exposure” protection), as well as other “horizontal” transmissions of the virus [14]. The effectiveness of these vaccines, all of which are formulations of purified HBsAg proteins (rather than live, replicating virus), is, and in itself, as surprising as it is important and instructive. Indeed, the discovery of HBV and development and use of an effective vaccine is one of the great accomplishments in medical and public health of the last century.
Realization that HBV is associated with hepatocellular carcinoma (HCC) must also be considered a great scientific accomplishment [15–17]. The demonstration that vaccination against HBV resulted in reducing the incidence of HCC both showed the public health benefit of vaccination [18] and providing final, definitive evidence of a cause and effect relationship between the virus and the cancer.
However, how HBV causes HCC remains elusive. The mechanism of oncogenesis almost certainly involves the necro-inflammatory pathogenesis associated with most chronic hepatitis B, but there is no specific viral oncogene, and replication of the virus in hepatocytes does not usually kill the infected cell [14]. Indeed, how the virus replicates has also generated some surprises. For example, the discovery that, although HBV is a DNA virus, it replicates through an RNA intermediate, and uses a virus-specified reverse transcriptase, is one of the major non-obvious findings in virology of the last part of the twentieth century [19]. It is also the molecular basis for the action for the small molecule direct acting hepatitis B antivirals [20] that are changing the natural history of the disease.
Parenthetically, one of the more dramatic demonstrations of how intervention with only polymerase inhibitors (in this case, lamivudine) can affect the natural history of chronic hepatitis was reported by this book’s co-editor, Dr. Liaw [21].
The discovery of hepatitis D, a “viroid” that requires hepatitis B co-infection for it to complete its replication cycle, and exacerbates chronic hepatitis B, must also be considered to be another enormously significant medical and scientific finding that is a part of the hepatitis B story [22]. Hepatitis D continues to be a major, although often overlooked health threat.
Hepatitis B remains a vital topic for study: with somewhere between 250 and 350 million people chronically infected with the virus, and as many as 25 % may die from liver disease (liver cirrhosis or hepatocellular carcinoma), without beneficial intervention [3].
Thus, chronic hepatitis B (but not hepatitis D) is now treatable. The polymerase inhibitors, and interferons, are enabling achievement of viral suppression and improvement of liver function. Indeed, discussion about these advances is found in this book. However, a medical cure for hepatitis B is not yet available, and continued research is still needed to achieve a cure of the infection and prevent HCC, its most deadly outcome. We went 25 years from the discovery of hepatitis C to a definitive cure. The part of the story that tells of a cure for hepatitis B, even after 50 years, still needs to be written.
Philadelphia, PA, USA
Literature Cited
T.M. Block
W.T. London Philadelphia, PA, USA
1. Blumberg BS, Alter HJ, Visnich S. A “New” antigen in leukemia sera. J Am Med Assoc. 1965;191:101–6.
2. Block TM, Mehta AS, Fimmel CJ, Jordan R. Molecular viral oncology of hepatocellular carcinoma. Oncogene. 2003;22(33):5093–107.
3. El Serag H, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology. 2007;132:2557–76
4. Avery OT, Macleod CM, McMarty M. Studies on the chemical nature of the substance inducing transformation of the pneumococcal types: induction of transformation by a desoxyribonucleic acid fraction isolated from pneumococcus type III. J Exp Med. 1944;79:137–58.
5. Watson JD, Crick FH. Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid. Nature. 1953;171:737–8.
6. Nirenberg MW, Matthaei JH. The dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic polyribonucleotides. Proc Natl Acad Sci U S A. 1961;47: 1588–602.
7. Jacob F, Monod J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961;318–56.
8. Allison AC, Blumberg BS. An isoprecipitation reaction distinguishing human serum protein types. Lancet. 1961;1:634–7.
9. Blumberg BS, Gerstley BJS, Hungerford DA, London WT, Sutnick AI. A serum antigen (Australia antigen) in Down’s Syndrome leukemia and hepatitis. Ann Int Med. 1967;66:924–31.
10. Bayer ME, Blumberg BS, Werner B. Particles associated with Australia antigen in the sera of patients with leukaemia, Down’s syndrome and hepatitis.1968;1057–9. Foreword
11. Dane DS, Cameron CH, Moya Briggs. Virus-like particles in serum of patients with Australiaantigen-associated hepatitis. Lancet 1970;295(7649):695–8.
12. Blumberg BS, Millman I. Vaccine against viral hepatitis and process. US Patent Office, Patent No.3,636,191. 18 Jan 1972.
13. McAleer WJ, Buynak EB, Maigetter RZ, Wampler DE, Miller WJ, Hilleman MR. Human hepatitis B vaccine from recombinant yeast. Nature. 1984;307:178–80.
14. Ganem D, Prince AM. Hepatitis B virus infection—natural history and clinical consequences. N Engl J Med. 2004;350(11):1118-29.
15. Beasley RP, Hwang LY, Lin CC, Chien CS. Hepatocellular carcinoma and hepatitis B virus. A prospective study of 22,707 men in Taiwan. Lancet. 1981;2(8256):1129–33.
16. Smith JB, Blumberg BS. Viral hepatitis post necrotic cirrhosis and hepatocellular carcinoma. Lancet. 1969;294(7627): 953.
17. Vogel CL, Anthony PP, Mody N, Barker LF. Hepatitis-associated antigen in Ugandan patients with hepatocellular carcinoma. Lancet 1970;296(7674):621–4.
18. World Health Organization. Media Centre: Hepatitis B. July 2013. Available at: http://www. who.int/mediacentre/factsheets/fs204/en/.
19. Summers J, Mason WS. Replication of the genome of a hepatitis B-like virus by reverse transcription of an RNA intermediate. Cell. 1982;29(2):403–15.
20. Keefe E, Zeuzem S, Koff RS, Dietrtich DT, Esteban-Mur R, Gane EJ, Jacobson IM, Lim SG, Naoomov, Marcellin P, Piratvisuth T, Zoulim F (2007) Report of an international workshop: roadmap for management of patients receiving oral therapy for chronic hepatitis B. Clin Gastroenterol Hepatol 2007;5(8):890–7.
21. Liaw YF, Sung JY, Chew WC, Farrell G, Lee CZ, Yuen H, Tanwandee T, Tao QM, Shue K, Keene ON, Dixon JS, Gray F, Sabbat J. Lamivudine for patients with chronic hepatitis and advanced liver disease. N Engl J Med 2004;351:1521–31
22. Rizzetto M, Canese, MG, Arico S, Crivelli O, Trepo C, Bonino F, et al. Immunofluorescence detection of a new antigen-antibody system (delta\anti-delta) associated with hepatitis B virus in liver and serum of HBsAg carriers. Gut. 1977;18:997–1003.
Preface
The discovery of Australia antigen 50 years ago, with subsequent link to hepatitis B virus (HBV), has opened up a golden era of hepatitis research. Tremendous advances in both basic and clinical aspects of HBV have been achieved in the past five decades. However, HBV remains a major public health problem worldwide and is still the leading cause of hepatocellular carcinoma, one of the most deadly cancers. The field of HBV infections continues to evolve, allowing maximal benefit for patients. This is manifest in higher response rate to antiviral therapy and prevention of liver disease complications in infected patients, and better prophylaxis for noninfected ones. The HBV research field is currently living a new momentum with major discoveries on its life cycle for instance with the discovery of the receptor for virus entry or the identification of key cellular enzymes involved in the formation of viral cccDNA, and research efforts for the identification of novel treatment targets towards a real cure of the infection.
To mark and celebrate the fiftieth anniversary of HBV discovery, world renowned HBV experts have reviewed the development/advancement in their respective fields, as compiled in this book. Thanks to the contribution of these experts, this textbook has provided a comprehensive, state-of-the art review of this field. The different chapters review new data about basic and translational science including the viral life cycle, the immunopathogenesis of virus-induced chronic hepatitis, the mechanism of virus-induced liver cancer, and their potential applications for the clinical management of patients. The book also provides a comprehensive review of the clinical aspects of this chronic viral infection with important chapters on the global epidemiology, the natural history of the disease, and the management of special patient populations. Important chapters on the management of antiviral therapy and the recent international guidelines for the treatment of hepatitis B should help clinicians in their daily decisions when treating patients. Finally, the book reviews the current state of the art regarding immunoprophylaxis to prevent the spread of the virus and its major clinical consequences. The new advances and perspectives in the development of improved antiviral treatments are discussed as they may pave the way towards novel therapeutic concepts which, together with mass vaccination programs, should significantly impact the disease burden hopefully in a near future.
The content of this book may have shed light on and may help in the development of new viewpoints and approaches in hepatitis B research and clinical hepatology. Hopefully this book should serve as a valuable resource for students, clinicians, and researchers with an interest in hepatitis B. In this regard, we would like to express our deep appreciation to the authors of this book. We would also like to acknowledge our collaborators who helped in the reviewing of the chapters:
Birke Bartosch, David Durantel, Boyan Grigorov, Julie Lucifora, Eve-Isabelle Pecheur (INSERM Unit 1052, Cancer Research Center of Lyon, Lyon, France)
Chia-Ming Chu, Edward J Gane, Jia-Horng Kao, Ming-Whei Yu
Lyon, France Fabien Zoulim Taipei, Taiwan Yun-Fan Liaw
Contributors
Laura Belloni IIT Centre for Life Nanoscience (CLNS), Rome, Italy
Antonio Bertoletti, M.D. Emerging infectious Diseases, Duke-NUS Graduate Medical School, Singapore, Singapore
Singapore Institute for Clinical Sciences, A*STAR, Singapore, Singapore
Mei-Hwei Chang, M.D. Department of Pediatrics, and Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan
Chao-Long Chen, M.D. Department of Surgery, Kaohsiung Chang Gang Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan
Liver Transplant Center, Kaohsiung Chang Gang Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan
Chien-Jen Chen Genomics Research Center, Academia Sinica, Nankang, Taipei, Taiwan
Graduate Institute of Epidemiology and Preventive Medicine, National Taiwan University, Taipei, Taiwan
Chia-Ming Chu, M.D. Liver Research Unit, Chang Gung Memorial Hospital, Taipei, Taiwan
Chang Gung University College of Medicine, Taipei, Taiwan
Markus Cornberg Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany
Maura Dandri, Ph.D. Department of Internal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
German Center for Infection Research, Hamburg-Lübeck-Borstel Partner Site, Hamburg, Germany
Contributors
Stevan A. Gonzalez, M.D., M.S. Division of Hepatology, Annette C. and Harold C. Simmons Transplant Institute, Baylor All Saints Medical Center, Fort Worth, TX, USA
Francesca Guerrieri IIT Centre for Life Nanoscience (CLNS), Rome, Italy
Luca G. Guidotti Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy
Department of Immunology and Microbial Sciences, The Scripps Research Institute, La Jolla, CA, USA
Jianming Hu, M.D., Ph.D. Department of Microbiology and Immunology-H107, The Penn State University College of Medicine, Hershey, PA, USA
Tsung-Hui Hu, M.D., Ph.D. Division of Hepato-Gastroenterology, Department of Internal Medicine, Kaohsiung Chang Gang Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan
Liver Transplant Center, Kaohsiung Chang Gang Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan
Hui-Han Hu Genomics Research Center, Academia Sinica, Taipei, Taiwan
Matteo Iannacone Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy
Vita-Salute San Raffaele University, Milan, Italy
Harry L.A. Janssen Toronto Centre for Liver Diseases, University Health Network, Toronto, ON, Canada
Department of Gastroenterology and Hepatology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands
Mark Kane, M.D. Mercer Island, WA, USA
Hideaki Kato Nagoya City University Graduate School of Medicine, Nagoya, Japan
Pietro Lampertico, M.D., Ph.D. “A. M. and A. Migliavacca” Center for Liver Disease, Gastroenterology and Hepatology Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Università degli Studi di Milano, Milan, Italy
Daniel Lavanchy, M.D., M.H.E.M. Ruelle des Chataigniers 1, Denges, VD, Switzerland
Mei-Hsuan Lee Graduate Institute of Epidemiology and Preventive Medicine, National Taiwan University, Taipei, Taiwan
Massimo Levrero IIT Centre for Life Nanoscience (CLNS), Rome, Italy
Dipartimento di Medicina Interna e Specialità Mediche (DMISM), Sapienza Universita’ di Roma, Policlinico Umberto I, Rome, Italy
Cancer Research Center of Lyon (CRCL), INSERM U1052, Lyon, France
Yun-Fan Liaw, M.D. Liver Research Unit, Chang Gung Memorial Hospital, Taipei, Taiwan
Chang Gung University College of Medicine, Taipei, Taiwan
Samuel Litwin Fox Chase Cancer Center, Philadelphia, PA, USA
Jessica Liu Genomics Research Center, Academia Sinica, Taipei, Taiwan
Victor C.K. Lo, M.A.Sc., C.C.R.P. Francis Family Liver Clinic, Toronto Western Hospital, Toronto, ON, Canada
Stephen Locarnini Research and Molecular Development, Victorian Infectious Diseases Reference Laboratory, Peter Doherty Institute, Melbourne, VIC, Australia
Marc Lütgehetmann Department of Internal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
Institute of Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
Michael P. Manns, M.D. Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany
William S. Mason Fox Chase Cancer Center, Philadelphia, PA, USA
Masashi Mizokami Research center for Hepatitis and Immunology, National Center for Global Health and Medicine, Chiba, Japan
Natalia Pediconi Department of Molecular Medicine, Sapienza University, Rome, Italy
Robert P. Perrillo, M.D. Division of Hepatology, Annette C. and Harold C. Simmons Transplant Institute, Baylor University Medical Center, Dallas, TX, USA
Teresa Pollicino Division of Clinical and Molecular Hepatology, University Hospital of Messina, Messina, Italy
Department of Pediatric, Gynecologic, Microbiologic and Biomedical Sciences, University Hospital of Messina, Messina, Italy
Giovanni Raimondo Division of Clinical and Molecular Hepatology, University Hospital of Messina, Messina, Italy
Department of Clinical and Experimental Medicine, University Hospital of Messina, Messina, Italy
Peter Revill Research & Molecular Development, Victorian Infectious Diseases Reference Laboratory, Peter Doherty Institute, Melbourne, VIC, Australia
Christoph Seeger Fox Chase Cancer Center, Philadelphia, PA, USA
Christoph Höner zu Siederdissen Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany
Masaya Sugiyama Research center for Hepatitis and Immunology, National Center for Global Health and Medicine, Chiba, Japan
Camille Sureau Laboratoire de Virologie Moléculaire, Institut National de la Transfusion Sanguine, Paris, France
Mauro Viganò Liver Unit, Ospedale San Giuseppe, Università degli Studi di Milano, Milan, Italy
Tassilo Volz Department of Internal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
Hwai-I Yang Genomics Research Center, Academia Sinica, Taipei, Taiwan
Fabien Zoulim, M.D., Ph.D. Cancer Research Center of Lyon (CRCL), INSERM U1052, Lyon, France
Hepatology Department, Hospices Civils de Lyon, Lyon, France
Université de Lyon, Lyon, France
Chapter 1 Hepatitis B Virus Virology and Replication
Jianming Hu
The Virus and Classification
Discovered 50 years ago as an antigenic “polymorphism” in an Australian aborigine—the “Australia antigen” [1], the hepatitis B virus (HBV) remains today a major global pathogen that causes acute and chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma [2]. Owing to its unique replication strategy, as will be detailed below, HBV is classified into its own family, Hepadnaviridae [3], along with related animal viruses. The latter include the woodchuck hepatitis virus [4], particularly useful as a model for studying HBV pathogenesis, and the duck hepatitis B virus (DHBV) [5], particularly useful for studies on viral replication. HBV and DHBV represent, respectively, the type member of the two separate genera within the family, the mammalian and avian hepadnaviruses that infect a number of mammalian and avian species. All hepadnaviruses share strict species and tissue tropism— mostly restricted to hepatocytes in their respective hosts, and a unique life cycle— replicating a double-stranded (DS) DNA genome via an RNA intermediate and are thus sometimes called retroid viruses or para-retroviruses [3, 6].
The Virions and Subviral Particles
The complete HBV virion is a sphere with a diameter of ca. 40–45 nm, first visualized using transmission electron microscopy (EM) (the so-called Dane particle) [7] and more recently, and with much greater detail, using cryo-EM [8, 9].
J. Hu, M.D., Ph.D. (*)
Department of Microbiology and Immunology-H107, The Penn State University College of Medicine, 500 University Dr., Hershey, PA 17033, USA
Y.-F. Liaw, F. Zoulim (eds.), Hepatitis B Virus in Human Diseases, Molecular and Translational Medicine, DOI 10.1007/978-3-319-22330-8_1
The virion has an outer envelope, which is studded with viral envelope (surface) proteins and surrounds an icosahedral capsid that, in turn, encloses the DS DNA genome. The complete virion is extremely infectious, with one virion being able to cause productive infection in chimpanzees that, in addition to humans, are susceptible to HBV [10]. On the other hand, a huge excess (100–100,000-fold over the complete virion) of noninfectious viral particles that contain no viral genome are also produced during infection. These so-called subviral particles include the classical spheres and filaments, which are ca. 22 nm in diameter and contain only the outer envelope layer of the virion, and the recently discovered empty virions, which contain both the outer envelope and the inner capsid shell but no viral DNA or RNA [11, 12].
Viral DNA Structure and Genome Organization
All hepadnaviruses share a peculiar virion DNA structure (Fig. 1.1) [13–15]. The DNA is small (3.2 kbp for HBV and 3.0 kbp for DHBV) and is held in a circular configuration via complementarity at the 5′ ends of both DNA strands, the length of complementarity being ca. 200 nucleotides (nt) in HBV and 60 nt in DHBV. Neither of the two strands of this relaxed circular DNA (rcDNA) is covalently closed. Whereas the (−) strand, i.e., the DNA strand complementary to pgRNA, has a short (ca. 9 nt) terminal redundancy (r), the other strand, the (+) strand, is heterogeneous in length with 3′ ends terminating hundreds of nt before completion. The rcDNA is further modified by a covalently linked protein (the terminal protein or TP) [16], later shown to be part of the viral reverse transcriptase (RT) or polymerase (P) protein [17] that is used to prime (−) DNA synthesis, and a capped, 18 nt-long RNA oligomer attached to the 5′ end of (+) DNA resulting from its use as a primer to initiate (+) strand DNA synthesis [14, 15] (see section “Reverse Transcription and NC Maturation” below).
Four distinct classes of viral mRNAs, all 5′ capped and 3′ polyadenylated, are encoded by the viral DNA. The genomic PreC/C mRNA is in fact longer than the DNA template (i.e., overlength), being 3.5 kb in length. The subgenomic PreS1, PreS2/S, and X mRNAs are approximately 2.4 kb, 2.1 kb, and 0.7 kb long, respectively. All viral mRNAs share the same 3′ sequences as represented by the shortest X mRNA, since they all terminate at the single polyadenylation signal. Transcription of these four groups of mRNAs is driven by four different viral promoters, respectively, the core, PreS1, PreS2/S, and X promoters that are further regulated by two viral enhancers, enhancer I upstream and overlapping with the X promoter and enhancer II located upstream of the core promoter (Fig. 1.1). A total of seven viral proteins are produced from these mRNAs using four open reading frames (ORF) (Fig. 1.1). The PreC/C mRNAs encode the viral core or capsid (C) protein and the slightly longer PreC protein using the same ORF, and the P protein using an alternative reading frame. As will be detailed below, the shortest of these genomic RNAs also serves as the template for reverse transcription to reproduce rcDNA
Fig. 1.1 HBV DNA structure and genome organization. The inner circle represents the virion rcDNA, and the dashes represent the region of the (+) strand DNA that is yet to be synthesized. The 5′ ends of the (−) and (+) strands are indicated. The small, filled circle represents the P protein covalently attached to the 5′ end of the (−) strand, and the short wavy line, the capped RNA oligomer attached to the 5′ end of the (+) strand. The vertical bars on rcDNA denote the direct repeats 1 and 2 (DR1 and DR2). The short terminal redundancy (r) on the (−) strand is denoted by the flap attached to the P protein (for clarity, it is not labeled in the figure). The promoter and enhancer positions are indicated. The middle circle of shaded boxes represent the four open reading frames (ORFs) corresponding to the precore/core, X, polymerase, and surface proteins, with their C-terminal ends denoted by the arrows. The outer circle of wavy lines represent the viral RNAs. Filled squares at one end of the mRNAs denotes heterogeneous 5′ ends (PreC/C, PreS2/S, and X mRNAs), the thin vertical line represents the precise 5′ end of the PreS1 mRNA. The arrows at the other end of the lines denote the 3′ ends of the mRNAs with polyA tails (AAA). BCP basal core promoter, Pol polymerase, PolyA polyadenylation
during replication and is thus termed pregenomic RNA or pgRNA. The PreS1, PreS2/S, and X mRNAs encode, respectively, the large (L) envelope protein, and the middle (M) and small (S) envelope proteins, and the X protein. All three envelope (or surface) proteins are encoded within a single ORF, which is entirely embedded within the alternative P ORF (Fig. 1.1). The P gene also overlaps with the 3′ end of the C gene and 5′ end of the X gene at its 5′ and 3′ ends respectively. In addition, all transcriptional regulatory elements including the promoters, enhancers, and the polyadenylation signal overlap with the protein-coding sequences. The genomic organization of hepadnaviruses is thus characterized by extreme economy.
Structure and Functions of Viral Proteins
The Envelope
Proteins
Of the three HBV envelope proteins, the smallest, S, is 226 residues long and is the most abundant. M contains a N-terminal extension, relative to S, called the PreS2 region, which is 55 residues long (Fig. 1.1). L is the longest and contains yet another N-terminal extension called the PreS1 region, which is 108 (or 119 depending on the strains) residues long. In addition to being major constituents of the virions, the envelope proteins are also secreted in large excess to the blood stream of infected people as spheres and filaments in the absence of capsids or genome, as mentioned above. Indeed, it was the abundance of these particles that allowed the discovery of HBV as the Australian antigen, i.e., hepatitis B surface antigen (HBsAg). The spheres contain mostly S and some M, and the filaments have in addition some L, which is enriched in virion particles [18]. Both L and S are required for virion secretion but M is dispensable [19]. In particular, the PreS1 region in L contains determinants required for both capsid envelopment during virion formation as well as receptor binding during entry (see below) [20]. This dual role of PreS1 is facilitated by its dynamic dual topology in the virions [21, 22]. Immediately following translation in the endoplasmic reticulum (ER) membrane, all PreS1 is located on the cytosolic side allowing it to interact with the capsids to fulfill its role in virion formation; as the virions traffic through the cellular secretory pathway, ca. 50 % of PreS1 is translocated from the interior of the virions to the exterior to allow it to bind the cell surface receptor. How this dramatic gymnastic feat is accomplished remains an enigma.
The C Protein and e Antigen
The C protein is 183 (or 185 depending on the strains) residues long. C can be divided into two structural and functional domains. The N-terminal 140 residues form the assembly domain (NTD) that is suffi cient to mediate capsid assembly [ 23 , 24 ]. The C-terminal domain (CTD) is dispensable for capsid assembly but plays essential roles in packaging of pgRNA into replication-competent nucleocapsids (NCs) and in reverse transcription of pgRNA to rcDNA. The C protein rapidly forms dimers, which are the building blocks for capsid assembly. Two morphological capsid isomers, with either 120 ( T = 4, the major isomer) or 90 dimers ( T = 3), are formed [ 25 , 26 ]. The functional signifi cance, if any, of this dichotomy is unknown. The arginine-rich CTD is highly basic and has nonspecifi c nucleic acid binding activity [ 27 ]. It also harbors multiple nuclear localization signals (NLSs) [ 28 – 30 ] that may be important for delivery of NCs to the nucleus (see section “Intracellular Traffi cking and Uncoating” below).
Moreover, CTD is heavily phosphorylated when expressed in mammalian cells, with three major sites of phosphorylation all displaying the Ser-Pro motifs [28, 31] plus three to four additional minor sites of phosphorylation [32]. As will be described below, CTD phosphorylation plays critical roles for C functions in viral replication. As HBV does not encode any viral kinase, it has to usurp host protein kinases for C phosphorylation. A number of cellular kinases, including protein kinase C (PKC) [33], cyclin-dependent protein kinase 2 (CDK2) [34], serine-arginine protein kinase (SRPK) [35] have been reported to phosphorylate C or specifically its CTD. Among these, CDK2 has been shown to associate with and phosphorylate the CTD, in particular, its Ser-Pro sites (consistent with the known substrate specificity of CDK2 as a well-known proline-directed kinase), and is incorporated into the capsids (see below) [34, 36]. As will be detailed below, CTD phosphorylation is highly dynamic and a dramatic dephosphorylation of CTD is shown to accompany viral DNA synthesis in the DHBV NCs. The cellular phosphatase(s) responsible for C dephosphorylation remains to be identified.
The precore (PreC) protein is translated from its own mRNA (PreC mRNA), which differs from the C mRNA (pgRNA) by a 5′ extension some 30 nt long. The sequence of PreC is thus essentially the same as C, except for an additional 29 amino acids at its N-terminus [37, 38]. However, these two proteins are functionally very different; unlike C, PreC is entirely dispensable for viral replication and mutants unable to express this protein are frequently selected late during persistent infection [38]. The first 19 residues of PreC comprise a secretion signal that induces its translocation into the lumen of the ER, where the signal sequence is cleaved off by a host cell signal peptidase. The remainder of PreC undergoes further proteolytic processing (e.g., by furin) in the host cell secretory pathway to remove the highly basic CTD in C, resulting the secretion of a heterogeneous population of soluble, dimeric proteins [39, 40], defined serologically as the hepatitis B e antigen (HBeAg) (Fig. 1.2, 9c) [41]. While dispensable for viral replication, PreC/HBeAg appears to play an important role in vivo for establishing persistent infection by regulating host immune response against the related and highly immunogenic C protein [ 42 ]. Also, serum HBeAg has proven to be a useful marker to monitor viral replication as its presence tends to correlate with high levels of viral replication and its loss usually signifies a decrease in viral replication [38].
The Reverse Transcriptase
The HBV RT or P protein is a multifunctional protein that plays a central role in viral replication. P is 832 or (or 845 depending on the strains) residues long and can be divided into four separate domains, from the N-terminus: TP, the spacer, the RT domain, and the RNase H domain [43–47]. TP harbors the invariant Tyr residue essential for priming reverse transcription [48–50] (see section “Reverse
Fig. 1.2 HBV life cycle. The replication cycle of HBV is depicted schematically. (1) Virus binding and entry into the host cell (large rectangle). (2) Intracellular trafficking and delivery of rcDNA to the nucleus (large circle). (3) Repair of rcDNA to form cccDNA, or integration of dslDNA into host DNA (3a). (4 and 4a) Transcription to synthesize viral RNAs. (5) Translation to synthesize viral proteins. (6) Assembly of the pgRNA-containing NC, or alternatively, empty capsids (6a). (7) Reverse transcription to make the (−) strand DNA and then rcDNA. (8) Nuclear recycling of progeny rcDNA. (9) Envelopment of the rcDNA-containing NC and secretion of complete virions, or alternatively, secretion of empty virions (9b) or HBsAg spheres and filaments (9a). Processing of the PreC protein and secretion of HBeAg are depicted in (9c). The different viral particles outside the cell are depicted schematically with their approximate titers indicated: the complete or empty virions as large circles (outer envelope) with an inner diamond shell (capsid), with or without rcDNA inside the capsid respectively; HBsAg spheres and filaments as small circles and cylinder. Intracellular capsids are depicted as diamonds, with either SS [(−) strand] DNA (straight line), viral pgRNA (wavy line), or empty, and the letters “P” denoting phosphorylated residues on the immature NCs (containing SS DNA or pgRNA) or empty capsid. The dashed lines of the diamond in the rcDNA-containing mature NCs signify the destabilization of the mature NC, which is also dephosphorylated. The soluble, dimeric HBeAg is depicted as grey double bars. The dashed line and arrow denote the fact that HBeAg is not always secreted during viral replication. The wavy lines denote the viral RNAs: C, mRNA for the C and P protein (and pgRNA); S and LS, mRNAs for the S/M and L envelope proteins, respectively; PreC, mRNA for the PreC protein and following
Transcription and NC Maturation”), and together with the RT domain, are required for specific binding of pgRNA for its encapsidation into NCs. pgRNA packaging requires also the RNase H domain but none of the known enzymatic activities of P [51, 52]. The spacer region is the least conserved of the four domains and is dispensable for all known functions of P. However, its coding sequences have to be retained to encode the PreS1 region in the overlapping S ORF, which is essential for the virus as discussed above. The RT domain harbors the polymerase active site essential for DNA polymerization [44, 45], particularly the Tyr-Met-Asp-Asp motif conserved across all RT proteins including those in retroviruses and retrotransposons. The RNase H domain is responsible for degrading the pgRNA template during (−) strand DNA synthesis [44, 45, 53].
The HBV DNA polymerase activity was discovered early on, before it was realized that HBV replicates via reverse transcription, via the so-called endogenous polymerase assay [54] whereby a DNA polymerase activity in the virions was shown to carry out DNA synthesis using the endogenous virion DNA as a template. It was only a decade later that Summers and Mason made the landmark discovery that a DNA virus (i.e., DHBV) replicates through reverse transcription of an RNA intermediate [6]. However, biochemical studies on this important enzyme have proven difficult to date, and no high-resolution structures of P are yet available. As will be detailed below (section “NC Assembly”), the discovery that P requires specific host factors for its folding and functions provides at least a partial explanation to this difficulty.
The X Protein
The 154 residue-long hepatitis B X protein (HBx) is the smallest HBV protein but is arguably the least understood. There is general agreement that X is required for viral replication in vivo [55] and perhaps contributes to viral pathogenesis (for a recent review, see ref. [56]). Numerous reports have suggested a large number of functions for X in the regulation of viral and host gene expression [57], DNA damage repair [58], Ca2+ signaling [59], cell cycle [60], apoptosis [61], and autophagy [62, 63]. As there is no evidence that X has DNA binding activity, it is thought to affect gene expression through host protein interactions, which are probably also
Fig 1.2 (continued) processing, HBeAg. Boxed letters denotes the viral proteins translated from the RNAs. The filled circle on rcDNA denotes the P protein attached to the 5′ end of the (−) strand (outer circle) of rcDNA and the arrow denotes the 3′ end of the (+) strand (inner circle) of rcDNA. ccc cccDNA, dsl double stranded linear DNA, HNF hepatocyte nuclear factor, HSP heat shock protein, PPase phosphatase, rc rcDNA, TF transcription factor. For simplicity, the synthesis of dslDNA (the minor genomic DNA form) in the mature NC, its secretion in virions, and infection of dslDNA-containing virions are not depicted here, as are the functions of X. See text for details
important for the various other viral or cellular effects attributed to X. It remains to be clarified how the various activities attributed to X are related to each other, and how they in turn relate to viral replication and/or pathogenesis (esp., hepatocarcinogenesis). As these activities are uncovered usually using different systems and assays, which are often less than physiologically optimal due to experimental limitations, and the role of X in viral replication or pathogenesis is likely regulatory and indirect, the interpretation of the results obtained, which can be in apparent conflicts, is by no means straight-forward. Recent attempts at standardization of experimental systems and assays and the development of more physiologically relevant systems will hopefully help clarify the functions of HBx in viral replication and pathogenesis [64].
Viral Life Cycle
As a para-retrovirus, the HBV life cycle (Fig. 1.2) shares a number of similarities to conventional retroviruses, including, of course, the central role of reverse transcription. However, HBV and hepadnaviruses in general have indeed a rather unique replication strategy, which is different from the conventional retroviruses in a number of important aspects including the initiation of reverse transcription and NC assembly, genome maintenance, and virion morphogenesis.
Entry
The strong species and tissue tropism of hepadnaviruses are in part underpinned by viral entry. Until recently, the only cells in culture that are reported to support HBV infection reproducibly are primary hepatocytes from humans [65] and the small primate tupaia [66], and one human hepatoma cell line HepaRG, which requires differentiation in vitro for even the rather low infection efficiency achieved [67]. Very recently, hepatocyte-like cells differentiated from induced human pluripotent stem cells [68], and a newly established human hepatoma cell line HLCZ01 [69], are reported to support limited HBV infection.
After many false starts, the primary entry receptor for HBV was finally identified in 2012 as a hepatic bile acid transporter, sodium taurocholate cotransporting polypeptide (NTCP) (Fig. 1.2, step 1) [70]. This breakthrough allows the establishment of convenient hepatoma cell lines such as HepG2 and Huh7, which have been the mainstay for studying other aspects of HBV replication and can now support infectious entry via NTCP reconstitution. On the other hand, NTCP is insufficient to render mouse hepatocytes susceptible to HBV infection [71, 72]. This result, though disappointing, is not unexpected given previous observations that another essential, intracellular, stage in the viral life cycle, the formation of the covalently closed circular DNA (cccDNA), is also defective in mouse hepatocytes (see section “Nuclear Recycling of rcDNA and Amplification of cccDNA”). It is clear that
additional host factors are required for the early stages of the viral life cycle beyond cell attachment and entry.
The viral requirements for entry are much better defined. Specifically, the N-terminal 48 residues of L as well as its N-terminal myristylation are both required for NTCP binding and infection [20, 70, 73, 74]. In addition, a region in S within the conserved “a” determinant (the major antigenic loop) is also required for infection via binding to the cell surface heparin sulfate proteoglycans and mediating the initial (nonspecific) viral attachment to the cells [75, 76]. This observation helps explain the high conservation of this antigenic determinant among HBV genotypes/ strains. A role for glycosylation of the envelope proteins has also been reported recently [77]. Intriguingly, HBV binding to NTCP, in addition to mediating viral infection, may also inhibit the transport function of NTCP and alter cellular gene expression [78], raising the possibility that this initial virus-host interplay may contribute to viral pathogenesis.
Intracellular Trafficking and Uncoating
The next essential step in the HBV life cycle after entry into susceptible host cells is to deliver the virion rcDNA into the nucleus (Fig. 1.2, step 2). Relatively little is understood here due to the lack of convenient and efficient infection systems until very recently. It is thought once the viral envelope and cellular membrane fuse to release the internal NC, the latter traffics towards the nuclear membrane, through interactions with cellular importins mediated by NLSs located on the C CTD [36, 79, 80]. As HBV can efficiently infect nondividing hepatocytes in the liver and NC is too large to pass through the nuclear pore complex (NPC), it has been proposed that NC interacts with components of the NPC leading to the arrest of NC and release of its rcDNA content into the nucleus [81] for cccDNA formation (see next).
cccDNA Formation
cccDNA is the first new viral DNA species detected upon infection [82] and is essential to initiate and sustain viral replication, as it is the only viral transcriptional template that can direct the expression of all viral RNAs and proteins. As with NC trafficking/uncoating, little is currently understood about this critical stage of HBV infection due to the lack of convenient experimental systems, until recently, that can support efficient HBV infection (for a recent review, see ref. [83]). It is clear, however, that the conversion of rcDNA to cccDNA in the nucleus (Fig. 1.2, step 3), as well as the preceding stages of entry and uncoating, must be highly efficient during natural infections since one DNA-containing virion particle is able to establish a productive infection in susceptible hosts [10, 84]. As will be detailed below (section “Nuclear Recycling of rcDNA and Amplification of cccDNA”), cccDNA can also be formed from progeny rcDNA synthesized de novo, via an intracellular amplification
pathway. This process, which bypasses the entry process, has been used, with limited success so far, to study cccDNA formation.
Viral RNA Synthesis
Once formed, the nuclear episomal cccDNA functions as the equivalent of a provirus in retroviruses and is used as the template to transcribe, by the host RNA Pol II, all the viral RNAs (Figs. 1.1 and 1.2). Viral transcription is dependent on ubiquitously expressed as well as liver-enriched transcriptional factors (hepatocyte nuclear factors or HNFs) (Fig. 1.2, step 4), which contributes to the liver (hepatocyte) specificity of viral replication [85–94] (for a recent review, see ref. [95]). cccDNA is organized into mini-chromosomes with host cell histones and potentially other host and viral proteins [96, 97]. Transcription from the cccDNA mini-chromosomes, like that from host chromosomes, is subject to epigenetic regulation, which may be further modulated by the viral regulatory protein, X [98–100]. X has been reported to be critical for transcription from cccDNA during infection [57], and apparently functions only on episomes (like cccDNA) but not integrated DNA, in a DNA sequenceindependent manner [101]. This sequence independence is consistent with the lack of DNA binding activity of X but if and how X specifically affects viral transcription remains an enigma. It has been suggested that HBx is recruited onto the cccDNA mini-chromosomes [98] but how this is accomplished in a DNA sequence specific fashion also remains unclear.
An interesting feature of HBV transcription is its dimorphic response to sex hormones, being stimulated by androgen [102, 103] and suppressed by estrogen [104]. Why HBV has evolved such a sexual dimorphism is an interesting unresolved question but this phenomenon likely contributes to the well-known male predominance of HBV replication and carcinogenesis.
All HBV mRNAs described above are unspliced, which have to be exported from the nucleus to the cytoplasm in order to be translated or packaged into NCs in the case of pgRNA. As eukaryotic mRNA export is usually coupled to splicing, HBV has evolved a mechanism of exporting its mRNAs out of the nucleus in a splicing-independent manner, which relies instead on a cis-acting RNA sequence called the post-transcriptional regulatory element (PRE) [105] encoded by viral DNA sequences overlapping enhancer I (Fig. 1.1).
Viral Protein Synthesis
Another interesting feature of HBV gene expression is that all viral promoters, except PreS1, lack an canonical TATA box and as a result, all viral RNAs, except the PreS1 mRNA, have heterogeneous 5′ ends, which is used to translate distinct proteins from closely related mRNA species. The heterogeneous 5′ ends of the
over-length genomic RNAs, PreC/C mRNA, bracket the translation initiation codon of the PreC protein. The longer PreC mRNAs containing the PreC initiation codon are translated to produce the PreC protein and ultimately the secreted HBeAg (Fig. 1.2, step 5), as described above. The shortest, C mRNA, missing the PreC initiation codon, is translated to produce both the core and RT proteins, the latter from an internal AUG in a different reading frame from C (Fig. 1.1). As mentioned above, the C mRNA is also pgRNA, serving as the template for reverse transcription to produce progeny viral DNA (section “Reverse Transcription and NC Maturation”). The M and S envelope proteins are similarly translated from the PreS2/S mRNAs, which have heterogeneous 5 ′ ends bracketing the PreS2 initiation codon. Thus, the longer RNAs containing this initiation codon are translated to produce M and the shorter ones lacking it are translated to produce S. This gene expression strategy effectively increases further the coding capacity of the highly compact HBV genome.
NC Assembly
The next stage in the viral life cycle ensues once C and P are translated from their shared mRNA, which doubles further as the template for reverse transcription (pgRNA) as alluded to above. These three components, the C and P protein and their shared mRNA (pgRNA) are all the viral factors needed for intracellular HBV replication (i.e., in the absence of virus secretion or infection). Assembly of the replication-competent NC requires the incorporation, into the same capsid, of both pgRNA—the template for reverse transcription, and P—the catalyst for DNA synthesis, by assembling C protein dimers. HBV has evolved to satisfy this dual (the P protein and pgRNA) packaging requirement by initiating NC assembly via the formation of a specific pgRNA-P ribonucleoprotein (RNP) complex, which then serves to trigger NC assembly (Fig. 1.2, step 6). A short structured RNA signal, called ε, located at the 5′ end of pgRNA (Fig. 1.3, step 1) was identified as the RNA packaging signal that mediates the packaging of pgRNA into NCs [106, 107]. ε was later found to be recognized specifically by the P protein (Fig. 1.3), not C, and P and pgRNA packaging are mutually dependent [51, 108, 109].
ε forms a conserved stem-loop structure with an apical loop and two short stems separated by an internal bulge (Fig. 1.3). To form the RNP complex, the internal bulge but not the apical loop is required [50, 110, 111]. However, for ε to serve its RNA packaging function, both the internal bulge and apical loop are required [112–114]. Furthermore, a closely spaced 5′ cap next to ε is also critical for pgRNA packaging in HBV [115] but dispensable for RT-ε interaction [50, 110, 111]. This requirement for a closely spaced 5′ cap in pgRNA packaging provides a satisfying explanation for the failure of the other copy of ε, which is located at the 3′ end of all viral RNAs (Figs. 1.1 and 1.3), to serve as a functional RNA packaging signal. Similarly, the requirements from P for pgRNA packaging go beyond those required for ε binding. Whereas only parts of the TP and RT domains are required for ε
Fig. 1.3 HBV reverse transcription pathway. The pathway is depicted schematically from bottom to top on the left so as to match the Southern blot image of replicative viral DNAs extracted from intracellular NCs shown on the right. (1) The pgRNA (dashed line) harbors a large terminal repeat (R) that bears the RNA packaging signal, ε, and DR1. (2) P protein binding to ε triggers proteinprimed initiation of (−) strand DNA synthesis at ε and packaging of pgRNA into NCs (not depicted). (3) (−) strand template switch to the 3′ DR1 and continuation of (−) strand DNA synthesis. (4) Degradation of pgRNA as (−) strand DNA synthesis proceeds, leaving a capped RNA oligomer containing DR1. (5) Translocation of the RNA oligomer to DR2 to prime (+) strand DNA synthesis. (6) (+) strand template switch from the 5′ to the 3′ end of the (−) strand DNA facilitated, in part, by the short terminal repeat (r) on the (−) strand DNA, circularizing the DNA, and continuation of (+) strand DNA synthesis for a variable length generating rcDNA. The boxed numerals 1 and 2 denote DR1 and DR2. An polyA tail, nasc nascent
binding [110, 111, 116–118], pgRNA packaging additionally requires P sequences extending into most of the RNase H domain [51, 52, 116, 119]. Interestingly, four conserved Cys, three within the C-terminal portion of the initially defined spacer region and one in the RT domain, were found to be required for both ε binding and pgRNA packaging [116, 120].
In addition to the viral P protein and ε RNA, host cell factors play an important role in RNP formation, and thus in pgRNA packaging (and protein-primed initiation of viral reverse transcription or protein priming, see section “Reverse Transcription and NC Maturation” below). In particular, the chaperone proteins heat shock protein 90 (Hsp90) and Hsp70, and other co-chaperones, are required to establish and maintain a P conformation active in ε binding (Fig. 1.2, step 6) [121–125]. One structural effect on P elicited by chaperone action is the exposure of a site in TP that may directly bind ε RNA [126]. Deletion of the RNase H domain and the C-terminal
J. Hu
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Title: Mäenpään isäntä
Kyläromaani kapinaviikilta
Author: Veikko Korhonen
Release date: December 4, 2023 [eBook #72308]
Language: Finnish
Original publication: Helsinki: Otava, 1919
Credits: Juhani Kärkkäinen and Tapio Riikonen
START OF THE PROJECT GUTENBERG EBOOK MÄENPÄÄN ISÄNTÄ ***
MÄENPÄÄN ISÄNTÄ
Kyläromaani kapinaviikoilta
Kirj.
VEIKKO KORHONEN
Helsingissä, Kustannusosakeyhtiö Otava, 1919.
I.
Helmikuun alkupäivät olivat ohi vierimässä. Ilmat olivat harvinaisen leutoja, päivä paistoi kuin maaliskuussa, ja vesi tipahteli räystäiltä.
Illansuussa hanki koveni, ja lapset luulivat jo kevään tulleen. Kelkat haettiin kätköistä esiin, ja mäkirinteillä helisi hilpeänä lasten ilonpito.
Talojen pelloilla ja metsä vainioilla olivat työt täydessä käynnissä. Mutta se tapa, jolla nyt töitä suoritettiin, oli omituista ja entisyydestä poikkeavaa.
Talojen isännät ja pojat liehuivat synkkäkatseisina, aivan kuin koettaen karkoittaa pahoja ajatuksia. Rengit ja päivätyöläiset seisoskelivat ryhmissä ja kiihkeästi neuvottelivat. Mitä? Sitä he eivät vielä tienneet oikein itsekään, talon omat miehet sitä vähemmin.
Jotakin suurta piti tapahtua, ja ehkä jo piankin. Odotettiin kuin ukkosen ilmaa kesällä helteisenä päivänä, vaikka ei vielä pilviäkään näy, mutta muuten painostaa.
Ja nytkin painosti.
Mäenpään isäntä oli ollut murahaudalla kuormia luomassa ja palasi sieltä nyt päivälliselle. Rengit ja verotyöläiset olivat jo menneet aikaisemmin pihaan hevosineen ja rekineen. Olivat laulaneet kuin
uhitellen mennessään internatsionaleaan ja äänekkäästi naureksineet. Puheista ei saanut selvää, mutta niissäkin oli uhitteleva sävy. Äsken työssä ollessaan ja palatessaan tyhjien rekien kanssa murakuopalle vaihtoivat he keskenään merkitseviä silmänluonteja ja suupielissä karehti pirullinen hymy.
Mäenpään isäntä, Juho, käveli raskaasti viljelyksien läpi kiemurtelevaa tietä lapio olallaan. Jalat tuntuivat kovin raskailta. Tällaista oli jatkunut jo monta päivää. Kukaan ei tiennyt mitä tästä oikein seuraisi. Tilanne oli koko maassa uhkaava, ja varsinkin liikeseudut kuohuivat. Mutta täällä sydänmaan loukossa hän oli omineen luullut saavansa olla rauhassa. Oli koettanut olla aina ja nyt varsinkin ystävällisissä väleissä työmiestensä kanssa, mutta viime päivien kokemukset osoittivat, että se ei hyödyttänyt mitään. Uhkamieli näytti kasvavan, ja työnteko oli melkein kokonaan mennyttä. Jos siitä huomautti, niin sai vihasta välähtävät silmäykset ja pistopuheet osakseen. Täytyi viimeiseltä vain tyytyä katsomaan ja odottamaan mitä seuraisi.
Posti-illat varsinkin olivat Juholle olleet kaikkein tukalimpia. Rengit ja päivätyöläiset repäisivät lehtikimpusta »Työmiehen» ja »Raatajan voiman» ja alkoivat äänekkäästi saarnata perheen lukurauhasta välittämättä. Jos joku perheen jäsenistä pyyteli hiljaa lukemaan, sai tavallisesti töykeän vastauksen.
— Hyvää se tekee teillekin, kuunnelkaa vain…
Juho tavallisesti hiipi sanomalehtensä kanssa kamariinsa, jossa emäntäkin näkyi tutkivan omaa lehteään. Mutta väliseinä ja ovi eivät estäneet kuulemasta, mitä pirtissä puhuttiin ja saarnattiin.
Ja nämä ilta-istunnot olivat viimeisinä posti-iltoina jatkuneet myöhään yöhön. Työmiesten joukossa oli muutamia sellaisiakin, jotka eivät ennustaneet hyvää nykyisestä asiain menosta ja sosialistilehtien kirjoituksista, ja heidän kanssaan syntyi aina kiistaa. Välistä se paisui suuriääniseksi meluksi.
Ja kun tuli tieto ensimäisistä kapinaliikkeistä, alkoivat salaiset neuvottelut nurkkajuurissa, työmailla, työväentaloilla ja joka paikassa. Töistä ei tullut enää mitään. Mieliala näytti vain kiihtyvän hetki hetkeltä.
Sellainen päivä oli ollut tänäänkin? Oli jo lausuttu Juholle raakuuksia ja uhkauksiakin. Ne olivat vielä heikosti peitettyjä, mutta tarkoitus oli selvä. Eikä näyttänyt olevan pelastuksen toivoa mistään päin. Sillä Juhokin saattoi hyvin arvata, että kapina tulisi yleiseksi. Kenenkään henki ei voisi olla turvattu, omaisuudesta puhumattakaan. Siltä varalta oli kyllä joku kuukausi sitten perustettu tällekin paikkakunnalle salaa suojeluskunta, mutta mitä sekään voisi, kun ei saatu aseita. Eivät edes omaa henkeään voisi säilyttää. Paikkakunnan punaiset olivat saaneet jo tietää suojeluskunnan perustamisesta ja salaa uhkailleet, että kunhan saadaan aseita, niin näytetään »lahtareille».
Nepä ne saivatkin aseita. Huhuiltiin jo, että oman pitäjän kirkonkyläänkin olisi tuotu aseita ja kätketty ne työväentalolle. Heillä kuului olevan käsipommejakin…
Tien käänteessä Juho tapasi Hakamaan isännän. Tervehdykset vaihdettiin, mutta ei niin reippaasti kuin ennen. Alakuloiselta näytti Hakamaankin isäntä. Rinnakkain siinä kävelivät hetkisen ääneti.
— Outoihin aikoihin tässä mekin vielä elettiin, virkkoi Hakamaan Mikko. — Veljessotaan valmistellaan molemmin puolin. Ei näytä olevan enää mitään muutakaan keinoa jäljellä.
— Eipä näytä… Jos omien punaisten kanssa saisikin vielä jotenkuten sovinnon aikaan, niin ryssät jäävät. Niitä he eivät suostu pois laskemaan, vielä vähemmin täältä ajamaan… Ja ne on saatava maasta pois. Ne saastuttavat muuten kohta kaikki koti nurkat.
— Millä ne ajetaan, kun ei ole aseita, hymähti Mikko. — Suojeluskuntia on perustettu, mutta ei ole hankittu ajoissa aseita. Kyllä ryssät sen sijaan aseistavat punaiset kiireestä kantapäähän.
Tässä sitä nyt ollaan, valmiina ryöstettäviksi ja murhattaviksi. On oltu liian kärkkäitä uskomaan viimeiseen asti, että punaiset eivät kehtaa omia kansalaisiaan vastaan nousta taistelemaan. Nyt se nähdään, että kehtaavat.
Tultiin jo Mäenpään pihaan. Isäntä pyysi naapuriaan istumaan, mutta tämä sanoi vain menevänsä nyt kotiin. Lupasi tulla toiste juttelemaan.
— Tulisit vaikka tänä iltana, pyyteli Juho. — Nykyiset puhteet ovat liian pitkiä, eikä tarvitsisi kuunnella aina niiden saarnaamista.
— Kieltäisit saarnaamasta.
— Ei uskalla.
— … olla isäntä omassa talossaan!
— Ei omassaankaan. Kyllä kai heillä on jo nuorat punottu meidänkin pään varalle. Riistäjiä ollaan ja lahtareita.
— Mh… pr… kyllähän ne nyt räyhäävät, mutta kuinkahan pitkältä…
— Mikäpä sen tietää. Vieläkö elänee isäin henkeä yhtään nykypolvessa.
— Ja jos ne työläisten kimppuun kävisivät, niin kyllä sitten isäntäkin jo kiittäisi.
— Pitäkää vain ryssäin kanssa yhteyttä, kivahti Tuomas. Veljeilkää minkä jaksatte ja antakaa naistennekin veljeillä. Pian se
lysti loppuu!
Kiistaa jatkui. Oli muutamia työmiehiä, vankempaa polvea, jotka asettuivat vastustamaan toisia. Ja nämäpä kinasivatkin vielä suuriäänisesti keskenään.
Ilta oli jo hämärtynyt ja lamput sytytettiin, ensin pirtissä ja hetkistä myöhemmin toisissa huoneissa. Posti tuli ja heitti kirjeet ja sanomalehdet pöydälle.
»Työmies» lennähti veromiesten käsiin. Isäntä tapaili myöskin lehteään, mutta ei sitä löytänyt. Olisiko joku sen ottanut? Ei näkynyt kellään. Juhon kasvot värähtivät. Joko se nyt alkaa?
Joku miehistä huomasi isännän hämmästyksen ja purevasti virkkoi:
— Taisi jäädä porvarilehdet tulematta.
Juho meni kamariinsa. Tuomas ja Lauri olivat heittäytyneet piippujaan polttelemaan tuvan sänkyyn.
Joku miehistä luki »Työmiehestä» Työväen pääneuvoston vallankumousjulistusta ja kehoitusta punakaarteille olemaan joka hetki valmiina. Viipurissa oli jo taistelu alkanut. Senaatin vangitsemismääräys oli annettu, ja kaikki oli reilassa.
Tuomas puraisi huultaan ja meni sisähuoneisiin. Lauri oli mennyt isänsä huoneeseen ja virkkoi:
— Nyt se siis alkaa.
— Siltä se näyttää.
Juhon ääni värähteli hieman. Mikä hetki hyvänsä tässä saattoi olla viimeinen. Tuomas ja Lauri kuuluivat suojeluskuntaan ja olivat tietysti velvollisia menemään sinne, missä ensiksi tarvittiin. Talo jäi hänen varaansa. Kukapa takaa, eikö punaiset täälläkin ryhdy ryöstämään ja polttamaan.
Lauri oli mennyt Tuomaan kamariin. Sinne oli ulko-ovesta pistäytynyt naapuritalon poika, Hakalan Timo.
— Mitä kuuluu? kysäisi Lauri.
Timo osoitti pöydälle, jossa oli paperilappu. Lauri otti sen käteensä.
»Tänä yönä kello 1 valmiina ennemmin määrätyssä paikassa. Kolmen päiväneväs mukaan.»
Timo otti lapun ja kätki poveensa.
— Nyt se siis alkaa.
— Nyt se alkaa, ja meillä on jo aseitakin, kuiskasi Timo.
— Mitä?
— Oman Mikko-sepän tekemiä tikareita. Pojat ovat ommelleet jo tupetkin niihin. Ehkä saadaan muutamia kiväärejäkin.
— Onko haulikot otettu mukaan? kysyi Tuomas.
— On, virkkoi Timo ja riensi ulos.
— Siellä tavataan.
— Siellä.
Pojat menivät Juhon kamariin. Siellä oli naapurin Mikko neuvottelemassa, pitäisikö poikia lähteä kyytimään hevosella.
— Kun saisi hevoset valjaisiin niin, etteivät punaiset huomaisi.
— Huomatkoot. Nyt sitä mennään.
— Mutta aamulla saattavat jo lähteä rankaisuretkille, kun tietävät.
— Tietäväthän ne sen kumminkin. Joka talossa on jo aamiaispöydässä miestä ja kahta vähemmän.
— Voidaan sitä mennä suksillakin.
— Kai se niin on parasta.
— Ja älkää te täällä hätäilkö, varoitteli Tuomas. — Kotinurkkia varten järjestetään seisova joukko.
— Mukaan minäkin lähtisin, jos en olisi näin vanha, virkkoi Mikko.
— Tarvitaan niitä miehiä täälläkin. Meillä täällä voi olla työtä parhaiksi kotinurkkia säilytellessä, arveli Juho.
Naapurin isäntä toivotti hyvää yötä ja painui ulos. Pojat jäivät vielä hetkiseksi isänsä kanssa puhelemaan. Puhuttiin talon yhteisistä asioista siltä varalta, että matkalle-lähtijöistä sattuisi joku jäämään tai vanha isä kotinurkilla kaatumaan. Pelko ei tuntunut erikoisesti ketään vaivaavan. Pojista vain tuntui, niinkuin painostava tunne olisi jo jotakuinkin lauennut. Pääsihän nyt toimimaan. Toimintaa odotellessa oli aika tuntunut pitkältä.
Emäntä laitteli evästä reppuihin hiljaa päivitellen sitä, että oli
tällaisiin aikoihin eletty. Sodasta oli tähän asti vain puhuttu, nyt se syttyi omilla kotinurkilla. Mutta eihän tällaistakaan jaksanut enää kestää. Parasta kai oli, että miehet lähtivät selvitystä tekemään.
Kunpa vain terveinä palaisivat!
Kello löi pirtissä rämisten kaksitoista. Pojat sitoivat reput selkäänsä ja hyvästelivät. Äidin kasvoille vierähtivät kirkkaat pisarat. Muuta ei voinut puhua kuin Jumalan siunausta toivotella ja pyytää pian takaisin tulemaan.
— Seisokaa miehinä viimeiseen asti maanne ja kotinne puolesta, virkkoi
Juho kättä lyöden pojilleen.— Lyökää ryssiä niinkuin isät ennen löivät.
Säästäkää mikäli mahdollista omia kansalaisia, varsinkin niitä, jotka on pakotettu mukaan lähtemään. Syylliset saakoot armotta rangaistuksen.
— Pian kai sitä nähdään, virkkoi Lauri helpottaakseen eron tunnelmaa.
Yö oli leuto ja tähdet tuikkivat tummasta korkeudesta. Sukset luistivat, ja matkaa joudutti paisuttava innostus yhteisestä asiasta.
Sinä yönä valmistauduttiin lähtöön joka talossa. Samana yönä peitettiin kirkonkylän työväentalon ikkunat, ettei valoja näkyisi kylään; harjoitettiin punakaartia ja jaettiin aseita.
II.
Mäenpään veljekset hiihtivät peräkkäin tähtikirkkaassa yössä. Hiihtäessään naapuritalojen peltoja he arvailivat, joko kaikki toiset olivat lähteneet taipaleelle. Siinä oli Hakala ja Tienpää. Isännän kamarin ikkunassa vain näkyi tulta. Uni ei tullut näinä helmikuun öinä. Aikoihinpa tosiaankin oli eletty! Talontyöt ne nyt jäävät, kun miesten täytyy sotateille suoriutua. Kotiin vain jää vanhukset ja lapset. Naisetkin uhkaavat lähteä keittämään ja haavoja sitomaan.
Kaksi hiihtäjää sukelsi esiin metsästä.
— Nuija!
— Tappara!
Ne olivat tunnussanat, jotka vaihdettuaan miehet lähenivät ystävällisesti ja jatkoivat nyt matkaa rinnakkain hiihtäen.
— Ei sitä ole ennen vielä tällaisia retkiä käyty.
— Eipä ei. Nyt on aika jo lähteä. Veri tässä alkoikin jo sakoa.
— Mitenkä luulet tässä käyvän? kysyi naapurinpoika Tuomaalta.
— Mikäs siinä? Lyödään ryssät liiskaksi ja tehdään punaiset vaarattomiksi. Silloinhan se on selvää.
— Niin, puhdasta jälkeä sitä nyt tehdään. Ennen ei palata kotinurkille.
— Saisi nyt vain aseita.
— Kyllä hallitus aseita puuhaa.
— Mikä sen tietää, jos ovat jo vankeina.
— En usko: Kyllä ne varansa pitävät.
Lauri oli toisten huomaamatta jäänyt jäljelle ja hiihtänyt erään metsätien varteen ladon kupeelle. Punamäen Kaisu oli luvannut tulla puoli yhden aikoina sanomaan hyvästit Laurille. Viestintuojan mukana oli Kaisu lappusen lähettänyt Laurille, jonka tiesi mukaan lähtevän.
Siellähän tyttö jo odottikin ladon kupeella. Painoi kainosti päänsä alas, kun Lauri hyppäsi suksiltaan.
— Nytkö sinä sitten menet? kysyi tyttö hiljaa.
— Niin, enkö sitten saisi mennä?
— Kyllä sinun on mentävä. Muutoin et mies olisikaan! Kunpa minäkin pääsisin mukaan!
— Sinä? Mitä siellä sinä tekisit?
— Kaipa siellä olisi työtä tällaisellekin.
Tytön ääni värähti.
— Mitä sinä Kaisu nyt noin…
Lauri laski kätensä tytön olkapäälle. Tyttö näytti tähtien valossa aivan kalpealta.
Tuli hetkisen kestävä hiljaisuus. Lauri tunsi itsensä tällä kertaa kovin neuvottomaksi. Hän olisi tahtonut sanoa jotakin rohkaisevaa tytölle, mutta tunsi itse olevansa rohkaisun tarpeessa. Tuntui hieman oudolta lähteä ensi kertaa taisteluun, joka kenties tulisi kestämään kauankin.
— Tämä taitaa sitten olla meille viimeinen tapaaminen, virkkoi tyttö hiljaa.
— Miten niin? Voihan tämä piankin selvitä, ja kyllä minä terveenä palaan.
— Sen minä uskon, mutta muuten… sinä et välitä minusta enää tämän jälkeen.
— Nyt minä en yhtään sinua ymmärrä.
Tyttö loi kirkkaat silmänsä Lauriin ja virkkoi hiljaa:
— Kyllähän sinä tiedät, miten punaisia kotoväkeni ovat, ja nyt… tytön ääni takertui kurkkuun — Kustaa lähtee punaisten puolelle.
Hetkiseksi Laurin kasvot synkkenivät.
— Oletko koettanut estää?
— Olen, mutta mikään ei auta. Sepä minua nyt niin painaakin.
Onhan sinun näin ollen mahdotonta minua ajatellakaan. Unohda pois. Ja tee kaikki mitä voit. Minun ajatukseni seuraavat siunaten sinua joka paikassa. Hyvästi nyt vain.
Tyttö ojensi kätensä Laurille.
— Näin sinä et saa mennä. Veljesi ei pääse enää lähtemään, jos ei hän tänä yönä ole lähtenyt. Eikä se meidän välejämme voi rikkoa. Minä tulen takaisin ja otan omani vaikka punaisten pääpesästä! Nyt minun täytyy mennä.
Lauri epäröi hetkisen, mutta astui sitten reippaasti suksilleen.
— Näinkö sinä menet? kysyi tyttö tuskin kuultavasti.
Lauri hyppäsi suksiltaan ja sulki tytön syliinsä. Tyttö painoi huulensa hänen otsalleen, suulleen ja silmilleen, horjahti kuin juopunut ja pyrähti suksilleen. Lauri jäi kuin typertyneenä seisomaan, nousi vihdoin ja lähti voimainsa takaa hiihtämään tavoittaakseen toiset.
Miehet olivat jo saapuneet määräpaikkaansa. Viisikymmentä vahvaa nuorukaista reppuineen ja aseineen. Toisilla vanha luodikko tai haulikko, joihin kiireesti valettiin kuulia ja täytettiin panoksia. Tikareita tahkottiin ja vöitä viimeisteltiin. Miesten jokainen liike osoitti teräksistä reippautta.
— Tulipahan nuijasota vielä kerran.
— Saadaanpahan tosiaankin tapella. Kunpa nyt olisi tässä ryssiä alkajaisiksi!
Ja puhuja heilutteli ilmassa välkkyvätä asettaan.
— Tässä on ryssien varalle!
Muuan mies heitti sylyksen pamppuja lattialle.
— Selkä ensin pehmeäksi tavaritseilta ja sitten rajan taakse!
— Punikit samaan matkaan!
— Kukapa jouti ja kehtasi ryssiä kuljettaa. Auetkoon vain maa ja nielköön heidät!
— Suomen multa on liian hyvää ryssien peitteeksi. Rajan taa, sanon minä!
— Ja viekööt naisensa mukanaan!
— Taitaisi kertyä niitä yhtä paljon kuin ryssiäkin. Hyi helvetti moista häpeää! Suomen naiset ryssien hutsuina.
— Hirsipuun ansaitsisi jokainen!
Aseet ja selkäreput olivat kunnossa, rivit järjestyivät lähtemään. Kuului komennus.
— Asentoon! Rivit kaksintakaa! Eteenpäin mars!
Portaissa törmäsi vahtijoukkueen johtaja vastaan ja ilmoitti, että punaiset piirittävät taloa parhaillaan. Joku urkkija oli saattanut heille sanan suojeluskunnan kokoontumisesta, ja punikit olivat kerääntyneet talon riihen taakse, josta hyökkäsivät piirittämään taloa.
