Hepatitis B virus (see Chapter 35), a member of the Hepadnaviridae family, is characterized by 42-nm spherical virions with a circular genome of double-stranded DNA (3.2 kbp). One strand of the DNA is incomplete and variable in length. Studies of the virus are hampered because it has not been grown in cell culture.
In addition to causing hepatitis, hepatitis B virus is a risk factor in the development of liver cancer in humans. Epidemiologic and laboratory studies have proved persistent infection with hepatitis B virus to be an important cause of chronic liver disease and the development of hepatocellular carcinoma. Hepatitis B virus infections occurring in adults are usually resolved, but primary infections in neonates and young children tend to become chronic in up to 90% of cases. It is these persistent hepatitis B virus infections established early in life that carry the highest risk of hepatocellular carcinoma later in life. The mechanism of oncogenesis remains obscure. Persistent viral infection leads to necrosis, inflammation, and liver regeneration that, over time, results in cirrhosis; hepatocellular carcinoma usually arises out of this background. The hepatitis B virus transactivator protein, X protein, is a potential viral oncoprotein. A dietary carcinogen, aflatoxin, may be a cofactor for hepatocellular carcinoma, especially in Africa and China.
The advent of an effective hepatitis B vaccine for the prevention of primary infection raised the possibility of prevention of hepatocellular carcinoma, particularly in areas of the world where infection with hepatitis B virus is hyperendemic (eg, Africa, China, Southeast Asia). Twenty years after the initiation of a universal hepatitis B vaccination program in Taiwan, chronic hepatitis B virus infection rates and liver cancer incidence rates were markedly reduced.
Woodchucks are an excellent model for hepatitis B virus infections of humans. A similar virus, woodchuck hepatitis virus, establishes chronic infections in both newborn and adult woodchucks, many of which develop hepatocellular carcinomas within a 3-year period.
Hepatitis C virus (see Chapter 35), a member of the Flaviviridae family, contains a genome of single-stranded RNA 9.4 kb in size. It appears that the majority of infections become persistent, even in adults. Chronic infection with hepatitis C virus leads to chronic inflammation and cirrhosis, and is also considered to be a causative factor in hepatocellular carcinoma. The development of hepatocellular carcinoma is likely mediated by a combination of virus and host specific mechanisms. There are over 170 million chronic carriers of hepatitis C virus, with 1-5% of these going on to develop hepatocellular carcinoma.
There are currently over 350 million people worldwide persistently infected with hepatitis B virus leading to over 500,000 deaths from hepatocellular cancer annually.
Retroviruses contain an RNA genome and an RNA-directed DNA polymerase (reverse transcriptase). RNA tumor viruses in this family mainly cause tumors of the reticuloendothelial and hematopoietic systems (leukemias, lymphomas) or of connective tissue (sarcomas).
Important properties of the retroviruses are listed in Table 43-3.
TABLE 43-3Important Properties of Retroviruses |Favorite Table|Download (.pdf) TABLE 43-3 Important Properties of Retroviruses
|Virion: Spherical, 80–110 nm in diameter, helical nucleoprotein within icosahedral capsid |
|Composition: RNA (2%), protein (about 60%), lipid (about 35%), carbohydrate (about 3%) |
|Genome: Single-stranded RNA, linear, positive-sense, 7–11 kb, diploid; may be defective; may carry oncogene |
|Proteins: Reverse transcriptase enzyme contained inside virions |
|Envelope: Present |
|Replication: Reverse transcriptase makes DNA copy from genomic RNA; DNA (provirus) integrates into cellular chromosome; provirus is template for viral RNA |
|Maturation: Virions bud from plasma membrane |
Infections do not kill cells
May transduce cellular oncogenes; may activate expression of cell genes
Proviruses remain permanently associated with cells and are frequently not expressed
Many members are tumor viruses
Structure and Composition
The retrovirus genome consists of two identical subunits of single-stranded, positive-sense RNA, each 7–11 kb in size. The reverse transcriptase contained in virus particles is essential for viral replication.
Retrovirus particles contain the helical ribonucleoprotein within an icosahedral capsid that is surrounded by an outer membrane (envelope) containing glycoprotein and lipid. Type- or subgroup-specific antigens are associated with the glycoproteins in the viral envelope, which are encoded by the env gene; group-specific antigens are associated with the virion core, which are encoded by the gag gene.
Three morphologic classes of extracellular retrovirus particles—as well as an intracellular form—are recognized, based on electron microscopy. They reflect slightly different processes of morphogenesis by different retroviruses. Examples of each are shown in Figure 43-1.
Comparative morphology of types A, B, C, and D retroviruses. A: Intracytoplasmic type A particles (representing immature precursor of budding type B virus). B: Budding type B virus. C: Mature, extracellular type B virus. D: Lack of morphologically recognizable intracytoplasmic form for type C virus. E: Budding type C virus. F: Mature, extracellular type C virus. G: Intracytoplasmic type A particle (representing immature precursor form of type D virus). H: Budding type D virus. I: Mature, extracellular type D virus. All micrographs are approximately 87,000×. Thin sections were double-stained with uranyl acetate and lead citrate. (Courtesy of D Fine and M Gonda.)
Type A particles occur only intracellularly and appear to be noninfectious. Intracytoplasmic type A particles, 75 nm in diameter, are precursors of extracellular type B viruses, whereas intracisternal type A particles, 60–90 nm in diameter, are unknown entities. Type B viruses are 100–130 nm in diameter and contain an eccentric nucleoid. The prototype of this group is the mouse mammary tumor virus, which occurs in “high mammary cancer” strains of inbred mice and is found in particularly large amounts in lactating mammary tissue and milk. It is readily transferred to suckling mice, in whom the incidence of subsequent development of adenocarcinoma of the breast is high. The type C viruses represent the largest group of retroviruses. The particles are 90–110 nm in diameter, and the electron-dense nucleoids are centrally located. The type C viruses may exist as exogenous or endogenous entities (see as follows). The lentiviruses are also type C viruses. Finally, the type D retroviruses are poorly characterized. The particles are 100–120 nm in diameter, contain an eccentric nucleoid, and exhibit surface spikes shorter than those on type B particles.
The Retroviridae family is divided into seven genera: Alpharetrovirus (which contains avian leukosis and sarcoma viruses), Betaretrovirus (mouse mammary tumor virus), Gammaretrovirus (mammalian leukemia and sarcoma viruses), Deltaretrovirus (human T-lymphotropic viruses [HTLV] and bovine leukemia virus), Epsilonretrovirus (fish viruses), Spumavirus (which contains viruses able to cause “foamy” degeneration of inoculated cells but which are not associated with any known disease process), and Lentivirus (which encompasses agents able to cause chronic infections with slowly progressive neurologic impairment, including HIV; see Chapter 44).
Retroviruses can be organized in various ways depending on their morphologic, biologic, and genetic properties. Differences in genome sequences and natural host range are frequently used, but antigenic properties are not. Retroviruses may be grouped morphologically (types B, C, and D); the vast majority of isolates display type C characteristics.
Retroviruses have been isolated from virtually all vertebrate species. Natural infections by a given virus are usually limited to a single species, though infections across species barriers may occur. Group-specific antigenic determinants on the major internal (core) protein are shared by viruses from the same host species. All mammalian viruses are more closely related to one another than to those from avian species.
The RNA tumor viruses most widely studied experimentally are the sarcoma viruses of chickens and mice and the leukemia viruses of mice, cats, chickens, and humans.
C. Exogenous or Endogenous
Exogenous retroviruses are spread horizontally and behave as typical infectious agents. They initiate infection and transformation only after contact. In contrast to endogenous viruses, which are found in all cells of all individuals of a given species, gene sequences of exogenous viruses are found only in infected cells. The pathogenic retroviruses all appear to be exogenous viruses.
Retroviruses may also be transmitted vertically through the germ line. Viral genetic information that is a constant part of the genetic constitution of an organism is designated as “endogenous.” An integrated retroviral provirus behaves like a cluster of cellular genes and is subject to regulatory control by the cell. This cellular control usually results in partial or complete repression of viral gene expression. Its location in the cellular genome and the presence of appropriate cellular transcription factors determine to a great extent whether (and when) viral expression will be activated. It is not uncommon for normal cells to maintain the endogenous viral infection in a quiescent form for extended periods of time.
Many vertebrates, including humans, possess multiple copies of endogenous RNA viral sequences. The endogenous viral sequences are of no apparent benefit to the animal. However, endogenous proviruses of mammary tumor virus carried by inbred strains of mice express superantigen activities that influence the T-cell repertoires of the animals.
Endogenous viruses are usually not pathogenic for their host animals. They do not produce any disease and cannot transform cells in culture. (There are examples of disease caused by replication of endogenous viruses in inbred strains of mice.)
Important features of endogenous viruses are as follows: (1) DNA copies of RNA tumor virus genomes are covalently linked to cellular DNA and are present in all somatic and germ cells in the host; (2) endogenous viral genomes are transmitted genetically from parent to offspring; (3) the integrated state subjects the endogenous viral genomes to host genetic control; and (4) the endogenous virus may be induced to replicate either spontaneously or by treatment with extrinsic (chemical) factors.
The presence or absence of an appropriate cell surface receptor is a major determinant of the host range of a retrovirus. Infection is initiated by an interaction between the viral envelope glycoprotein and a cell surface receptor. Ecotropic viruses infect and replicate only in cells from animals of the original host species. Amphotropic viruses exhibit a broad host range (able to infect cells not only of the natural host but of heterologous species as well) because they recognize a receptor that is widely distributed. Xenotropic viruses can replicate in some heterologous (foreign) cells but not in cultured cells from the natural host. Many endogenous viruses have xenotropic host ranges.
Retroviruses have a simple genetic content, but there is some variation in the number and type of genes contained. The genetic makeup of a virus influences its biologic properties. Genomic structure is a useful way of categorizing RNA tumor viruses (Figure 43-2).
Genetic organization of representative retroviruses. A: Nondefective, replication-competent viruses. Examples of retroviruses with simple and complex genomes are shown. An open rectangle shows the open reading frame for the indicated gene. If the rectangles are offset vertically, their reading frames are different. Horizontal lines connecting two rectangles indicate that this segment is spliced out. Simple genomes: ALV, avian leukosis virus (Alpharetrovirus); MLV, murine leukemia virus (Gammaretrovirus); MMTV, mouse mammary tumor virus (Betaretrovirus). Complex genomes: HIV, human immunodeficiency virus type 1 (Lentivirus); HTLV, human T-lymphotropic virus (Deltaretrovirus). B: Viruses carrying oncogenes. Several examples are shown, with the oncogene shaded; all are defective except RSV. Ab-MLV, Abelson murine leukemia virus (abl oncogene) (Gammaretrovirus); Ha-MSV, Harvey murine sarcoma virus (ras oncogene) (Gammaretrovirus); MC29, avian myelocytomatosis virus (myc oncogene) (Alpharetrovirus); Mo-MSV, Moloney murine sarcoma virus (mos oncogene) (Gammaretrovirus); RSV, Rous sarcoma virus (src oncogene) (Alpharetrovirus). The scale for genome sizes is shown at the bottom of each panel. (Modified with permission from Vogt VM: Retroviral virions and genomes. In Coffin JM, Hughes SH, Varmus HE [editors]. Retroviruses. Cold Spring Harbor Laboratory Press, 1997.)
The standard leukemia viruses (Alpharetrovirus and Gammaretrovirus) contain genes required for viral replication: gag, which encodes the core proteins (group-specific antigens); pro, which encodes a protease enzyme; pol, which encodes the reverse transcriptase enzyme (polymerase); and env, which encodes the glycoproteins that form projections on the envelope of the particle. The gene order in all retroviruses is 5′-gag-pro-pol-env-3′.
Some viruses, exemplified by the human retroviruses (Deltaretrovirus and Lentivirus), contain additional genes downstream from the env gene. One is a transactivating regulatory gene (tax or tat) that encodes a nonstructural protein that alters the transcription or translational efficiency of other viral genes. The lentiviruses, including HIV, have a more complex genome and contain several additional accessory genes (see Chapter 44).
Retroviruses with either of these two genomic structures will be replication-competent (in appropriate cells). Because they lack a transforming (onc) gene, they cannot transform cells in tissue culture. However, they may have the ability to transform precursor cells in blood-forming tissues in vivo.
The directly transforming retroviruses carry an onc gene. The transforming genes carried by various RNA tumor viruses represent cellular genes that have been appropriated by those viruses at some time in the distant past and incorporated into their genomes (see Figure 43-2).
Such viruses are highly oncogenic in appropriate host animals and can transform cells in culture. With very few exceptions, the addition of the cellular DNA results in the loss of portions of the viral genome. Consequently, the sarcoma viruses usually are replication-defective; progeny virus is produced only in the presence of helper viruses. The helper viruses are generally other retroviruses (leukemia viruses), which may recombine in various ways with the defective viruses. These defective transforming retroviruses have been the source of many of the recognized cellular oncogenes.
The retroviruses that contain oncogenes are highly oncogenic. They are sometimes referred to as “acute transforming” agents because they induce tumors in vivo after very short latent periods and rapidly induce morphologic transformation of cells in vitro. The viruses that do not carry an oncogene have a much lower oncogenic potential. Disease (usually of blood cells) appears after a long latent period (ie, “slow-transforming”); cultured cells are not transformed.
Briefly, neoplastic transformation by retroviruses is the result of a cellular gene that is normally expressed at low, carefully regulated levels becoming activated and expressed constitutively. In the case of the acute transforming viruses, a cellular gene has been inserted by recombination into the viral genome and is expressed as a viral gene under the control of the viral promoter. In the case of the slow-transforming leukemia viruses, the viral promoter or enhancer element is inserted adjacent to or near the cellular gene in the cellular chromosome.
Replication of Retroviruses
A schematic outline of a typical retrovirus replication cycle, represented by HTLV, is shown in Figure 43-3. The pol gene encodes the unique polymerase (reverse transcriptase) protein that has four enzymatic activities (protease, polymerase, RNase H, and integrase). After virus particles have adsorbed to and penetrated host cells, the viral RNA serves as the template for the synthesis of viral DNA through the action of the viral enzyme reverse transcriptase, functioning as an RNA-dependent DNA polymerase. By a complex process, sequences from both ends of the viral RNA become duplicated, forming the long terminal repeat located at each end of the viral DNA (Figure 43-4). Long terminal repeats are present only in viral DNA. The newly formed viral DNA becomes integrated into the host cell DNA as a provirus. The structure of the provirus is constant, but its integration into host cell genomes can occur at different sites. The very precise orientation of the provirus after integration is achieved by specific sequences at the ends of both long terminal repeats.
Overview of human T-lymphotropic virus (HTLV) replication cycle. The virus particle attaches to a cell surface receptor, and the viral capsid enters the cell. The viral reverse transcriptase enzyme produces a DNA copy of the genome RNA within the capsid in the cytoplasm. The DNA enters the nucleus and is integrated at random into cell DNA, forming the provirus. The integrated provirus serves as template for the synthesis of viral transcripts, some of which are unspliced and will be encapsidated as genomic RNAs and others and some of which are spliced and will serve as mRNAs. Viral proteins are synthesized; the proteins and genome RNAs assemble; and particles bud from the cell. Capsid proteins are proteolytically processed by the viral protease producing mature, infectious virions, shown schematically as conversion from a square to an icosahedral core. (Courtesy of SJ Marriott.)
Comparison of structures of retrovirus RNA genome and integrated provirus DNA. A virus particle contains two identical copies of the single-stranded RNA genome. The 5′ terminal is capped, and the 3′ terminal is polyadenylated. A short sequence, R, is repeated at both ends; unique sequences are located near the 5′ (U5) and 3′ (U3) ends. U3 contains promoter and enhancer sequences. The integrated provirus DNA is flanked at each end by the long terminal repeat (LTR) structure generated during synthesis of the DNA copy by reverse transcription. Each LTR contains U3, R, and U5 sequences. The LTRs and coding regions of the retrovirus genome are not drawn to scale.
Progeny viral genomes may then be transcribed from the provirus DNA into viral RNA. The U3 sequence in the long terminal repeat contains both a promoter and an enhancer. The enhancer may help confer tissue specificity on viral expression. The proviral DNA is transcribed by the host enzyme, RNA polymerase II. Full-length transcripts (capped, polyadenylated) serve as genomic RNA for encapsidation in progeny virions. Some transcripts are spliced, and the subgenomic messenger RNAs (mRNAs) are translated to produce viral precursor proteins that are modified and cleaved to form the final protein products.
If the virus happens to contain a transforming gene, the oncogene plays no role in replication. This is in marked contrast to the DNA tumor viruses, in which the transforming genes are also essential viral replication genes.
Virus particles assemble and emerge from infected host cells by budding from plasma membranes. The viral protease then cleaves the Gag and Pol proteins from the precursor polyprotein, producing a mature infectious virion prepared for reverse transcription when the next cell is infected.
A salient feature of retroviruses is that they are not cytolytic; that is, they do not kill the cells in which they replicate. The exceptions are the lentiviruses, which may be cytolytic (see Chapter 44). The provirus remains integrated within the cellular DNA for the life of the cell. There is no known way to cure a cell of a chronic retrovirus infection.
A. Human T-Lymphotropic Viruses
Only a few retroviruses are linked to human tumors. The human T-lymphotropic virus (HTLV) group of retroviruses has probably existed in humans for thousands of years. HTLV-1 has been established as the causative agent of adult T-cell leukemia-lymphomas (ATL) as well as a nervous system degenerative disorder called tropical spastic paraparesis. It does not carry an oncogene. Three related human viruses, HTLV-2, HTLV-3, and HTLV-4, have been isolated but have not been conclusively associated with a specific disease. HTLV-1 and HTLV-2 share about 65% sequence homology and display significant serologic cross-reactivity.
The human lymphotropic viruses have a marked affinity for mature T cells. HTLV-1 is expressed at very low levels in infected individuals. It appears that the viral promoter-enhancer sequences in the long terminal repeat may be responsive to signals associated with the activation and proliferation of T cells. If so, the replication of the viruses may be linked to the replication of the host cells—a strategy that would ensure efficient propagation of the virus.
The human retroviruses are transregulating (see Figure 43-2). They carry a gene, tax, whose product alters the expression of other viral genes. Transactivating regulatory genes are believed to be necessary for viral replication in vivo and may contribute to oncogenesis by also modulating cellular genes that regulate cell growth.
There are several genetic subtypes of HTLV-1, with the major ones being subtypes A, B, and C (these do not represent distinct serotypes).
The virus is distributed worldwide, with an estimated 20 million infected individuals. Clusters of HTLV-associated disease are found in certain geographic areas (southern Japan, Melanesia, the Caribbean, Central and South America, and parts of Africa) (Figure 43-5). Although less than 1% of people worldwide have HTLV-1 antibody, up to 5% of the population in endemic areas may be seropositive.
Subtypes of human T-lymphotropic virus type 1 are geographically distributed in endemic foci. A: Japan, India, the Caribbean, and the Andes; B: Japan and India; C: West Africa and the Caribbean; D: Central Africa; E: Papua New Guinea. (Courtesy of N Mueller.)
ATL is poorly responsive to therapy. The 5-year survival rate for patients with this cancer is less than 5%.
Transmission of HTLV-1 seems to involve cell-associated virus. Mother-to-child transmission via breastfeeding is an important mode. Efficiency of transmission from infected mother to child is estimated at 15–25%. Such early-life infections are associated with the greatest risk of ATL. Blood transfusion is an effective means of transmission, as are sharing blood-contaminated needles (drug abusers) and sexual intercourse.
Seroepidemiology has linked infection with HTLV-1 to a syndrome called HTLV-1–associated myelopathy/tropical spastic paraparesis (HAM/TSP). The primary clinical feature is development of progressive weakness of the legs and lower body. The patient’s mental faculties remain intact. HAM/TSP is described as being of the same magnitude and importance in the tropics as is multiple sclerosis in Western countries. Other HTLV-1–associated diseases include uveitis and infective dermatitis.
B. Human Immunodeficiency Viruses
A group of human retroviruses has been established as the cause of acquired immune deficiency syndrome (AIDS) (see Chapter 44). The HIV are cytolytic and nontransforming and are classified as lentiviruses. However, AIDS patients are at elevated risk of several types of cancer because of the immune suppression associated with HIV infection. These cancers include cervical cancer, Kaposi sarcoma, lymphomas, head and neck cancer, liver cancer, and oral cancer.
The simian foamy viruses from the Spumavirus genus are highly prevalent in captive nonhuman primates. Humans occupationally exposed to the primates can be infected with foamy viruses, but these infections have not resulted in any recognized disease.