1. An Everlasting War Dance Between Retrotransposons and Their Metazoan Hosts

David E. Symer and Jef D. Boeke

Many classes of transposons and retrotransposons have invaded and shaped the genomes of their metazoan hosts. Class I transposons (i.e. retrotransposons) include both elements which contain long terminal repeats (LTRs) and many others lacking LTRs. Along with retroviruses, retrotransposons share a fundamental mechanism of mobilisation through reverse transcription of an RNA template, via enzymatic activity of a reverse transcriptase. However, unlike retroviruses, retrotransposons remain entirely intracellular during their life cycle. Several distinctive and overlapping genome defense mechanisms have been developed over evolutionary time, as host organisms have fought back against these mobile retroelements. The ongoing conflict between these myriad genomic parasites and their widespread host organisms has resulted in positive and deleterious consequences including exaptation, genomic deletions, certain diseases including cancers, and the generation of diversity probably including the formation of new species.

2. Endogenous Retroviruses

Joachim Denner

Endogenous retroviruses are genetic elements representing the result of retrovirus infections and integration of the proviruses into the germline of vertebrates including humans. Retroviruses use the enzyme reverse transcriptase (RT) to transcribe their RNA genome into cDNA and incorporate it into the cellular genome. Infections of germ cells result in the presence of these viruses in the genome all cells of the organism and transmission of these. sequneces to the offspring, Only some endogenous retroviruses are replication competent and produce infectious particles; most are defective. Although the role of endogenous retroviruses during tumour development and autoimmune diseases is still unclear, sufficient evidence has accumulated indicating that retroviruses play an important role in physiological processes. Endogenous retroviruses are involved in placental differentiation and immunosuppression during pregnancy, and retroviral long term repeats (LTR) regulate the expression of cellular genes. During evolution three main processes took place: First, an accumulation of defective proviral DNA ("junk DNA"), second a development of stronger restriction strategies by the host and third, an utilisation, "enslavement" of retroviral genes and LTRs. Since transspecies transmissions of retroviruses are very common, endogenous retrovirus may be important also for the health of other species. For example, pig cells can release porcine endogenous retroviruses that infect human cells and therefore represent a risk for xenotransplantations involving pig cells or organs.

3. Retroviral Particles, Proteins and Genomes

Norbert Bannert, Uwe Fiebig and Oliver Hohn

Retroviruses, a large group of enveloped viruses named for their typical reverse transcription and integration, comprise seven genera, all of which have the basic proviral genomic structure 5' LTR-gag-pro-pol-env-3' LTR. Despite these similarities however, retroviruses have major differences with regard to their genomic organisation, protein composition, and architecture. Genera for which differences are limited to the four invariant genes - gag (group specific antigen), pro (protease), pol (polymerase) and env (envelope) - are classified as simple retroviruses, whereas genera that encode accessory proteins are classified as complex retroviruses. Despite the retroviruses' matching sequences, the flanking long terminal repeats (LTRs) are dissimilar with regard to most of their functions. This chapter describes the general and the genera-specific facets of retroviral morphology and genomic organisation, and their consequences for transcription and protein expression.

4. Retroviral Entry and Uncoating

Walther Mothes and Pradeep D. Uchil

Retroviruses form small 100 nm particles of simple composition, yet are able to replicate, spread and cause severe diseases. This is possible, because throughout their replication cycle, retroviruses utilise host factors and hijack cellular pathways. In addition, retroviruses have to overcome a strong innate and adaptive immune response. As such, retroviral replication is the result of a complex co-evolution of viral biology, the cell biology of the host and immune evasion. This complex nature of retroviral infections also applies to the subject of this chapter, how retroviruses enter cells, uncoat to reverse transcribe and to deliver their genomes into the nucleus of the cell. We will discuss the viral aspects of entry, cover cell biological aspects of viral trafficking and deal with innate cellular factors targeting incoming viruses. Finally, we will review virus entry in the context of retroviral pathogenesis and discuss how virus entry and budding are coordinated at sites of cell-cell contact during a spreading infection.

5. Reverse Transcription and Integration

Alan Engelman

Retroviruses are unique among animal viruses in that their replication requires the recombination of their own genetic material with that of the infected host cell. Two virus-encapsulated enzymes, reverse transcriptase and integrase, are dedicated to provirus formation. Reverse transcriptase, using a packaged cellular tRNA primer to initiate DNA synthesis from the viral RNA template, generates linear double-stranded DNA within the context of the reverse transcription nucleoprotein complex. The integrase enzyme processes the neo-synthesised DNA ends as the preintegration complex moves toward the cell nucleus. After finding a suitable chromatin acceptor site, the integrase recombines the processed DNA ends with a cell chromosome. This chapter focuses on the mechanisms of viral DNA synthesis, its transport to the nucleus, and the resulting chromosomal DNA integration.

6. Transcription, splicing and transport of retroviral RNA

Tina Lenasi, Xavier Contreras, and B. Matija Peterlin

Studies of retroviruses have contributed greatly to our understanding of mechanisms that regulate eukaryotic gene expression. They include transcription, processing of nascent transcripts and transport of mRNA species from the nucleus to the cytoplasm. For example, analyses of viral promoters and enhancers revealed important aspects of initiation and elongation of transcription by RNA polymerase II. Sites of integration further emphasised contributions of chromatin and distal interactions between cis-acting sequences to the expression of viral genes and those of nearby oncogenes that lead to the transformation of target cells. At the level of DNA, they also introduced the concept of transcriptional interference for the silencing of viral 3' long terminal repeats, where their transcription terminates and nascent transcripts become polyadenylated. Next, studies of their complex splicing patterns revealed suboptimal splice donor and acceptor sites, splicing enhancers and silencers, as well as the competition between splicing and export of incompletely spliced retroviral mRNA species from the nucleus to the cytoplasm. There, they defined RNA and protein export mechanisms for cellular and viral macromolecules. Finally, current studies of the silencing of retroviral genomes promise to elucidate mechanism for turning on and off the expression of eukaryotic genes. Of all mammalian retroviruses, HIV has been studied the most and forms the basis of this chapter. However, lessons learned from this primate lentivirus inform all other retroviruses.

7. Assembly and release

Heinrich G. Göttlinger and Winfried Weissenhorn

Retroviral assembly and release are both mediated by the viral Gag polyprotein. Proteolytic processing of Gag within the immature virus particle yields the internal structural proteins of the mature virion, of which matrix (MA), capsid (CA), and nucleocapsid (NC) are common to all retroviruses. Within the context of the unprocessed polyprotein, the MA domain is primarily required for Gag membrane targeting and for the incorporation of the viral surface glycoproteins into progeny virions. The CA domain of Gag provides the major driving force for the assembly of immature particles. After rearranging into a different type of lattice subsequent to the proteolytic processing of Gag, CA forms the core of the mature virion. NC nucleates immature particle assembly through the concentration of Gag on RNA molecules, and also plays an essential role in the encapsidation of the viral RNA genome. Retroviral Gag proteins also harbour conserved motifs that co-opt a cellular budding machinery to promote the separation of the lipid envelope of the nascent virion from the cell surface, and thus the release of an extracellular virion. The distinct roles of the various Gag domains in virus morphogenesis are the subject of this review.

8. Transmission and Epidemiology

Hans Lutz, Gerhard Hunsmann, and Jörg Schüpbach

Although the prevalence of feline retroviruses has decreased significantly during the last 20 years, they still occur worldwide and in some areas they are still of veterinary importance. In Europe and the USA, EIAV infection has almost been eradicated. As BIV does not cause disease, it is not studied widely and little information is available on its prevalence. BLV occurs in many countries and is of considerable economic importance. The small ruminant retroviruses CAEV and VMV occur worldwide and in some areas at high frequency. Lentiviruses collectively called SIV affect both, non-primates and primates. They are naturally present in Africa but not in Asia, North and South America. In 2006, HIV-1 and HIV-2 infection was estimated by the United Nations Programme on HIV/AIDS to have affected 39.5 Mio people. The worldwide prevalence among adults was estimated to be 1%. Most affected by the AIDS epidemic is the population living in sub-Saharan Africa. FeLV, BIV, BLV and VMV are usually transmitted by direct or even indirect contact. FIV is predominantly transmitted by bites and via milk. EIAV and CAEV are transmitted via milk, by fomites and iatrogenically. SIV and HIV are transmitted through sexual contact and by contaminated needles and blood. Cross-species transmissions of FeLV and VMV occur occasionally. The HIV epidemic is the result of the zoonotic transmissions of SIV from chimpanzees.

9. Pathogenesis of Oncoviral Infections

Finn Skou Pedersen and Annette Balle Sørensen

Retroviruses cause cancer in natural or laboratory settings by a variety of mechanisms. The acutely transforming or transducing retroviruses induce tumours in animals within days to weeks. They harbour a host-cell derived gene, an oncogene, which infiltrate signalling cascades that regulate cell growth and survival. The oncoprotein encoded by the viral oncogene is activated to dominant signalling, either by deregulated expression or as a result of a modified protein structure that uncouples downstream signalling from upstream physiological signals. The cis-acting or non-acutely transforming viruses cause disease with latency periods of months. These viruses work as insertional mutagens to promote multi-step oncogenesis, and large-scale mapping of proviral insertions in tumour DNAs provides a rich source of candidate genes with a potential role in cancer of non-retroviral aetiology. Viral proteins may also stimulate target cells to proliferate. One example is the mouse mammary tumour virus which stimulates lymphocytes via a virus-encoded superantigen. Other examples are the mitogenic stimulation of erythrocyte precursor cells by the defective envelope protein of the mouse spleen focus-forming virus, and the direct oncogenic affect of the envelope protein of the Jaagsiekte sheep retrovirus.

10. Pathogenesis of Immunodeficiency Virus Infections

Guido Poli and Volker Erfle

Extensive analysis of naturally occurring simian immunodeficiency viruses (SIVs) and comparative phylogenetic studies with human immunodeficiency viruses (HIVs) suggests that the latter are close relatives of the SIVcpz viruses of chimpanzees (HIV-1) or the SIVsmm viruses of sooty mangabys (HIV-2). Crossing of species barriers resulted in adaptation to the human host and subsequent acquisition of a pathogenic phenotype. Naturally occurring T lymphocyte-tropic lentiviral infections are highly prevalent and productive but are not usually pathogenic for native hosts. Crossing species barriers may produce an abortive infection or, as in the case of the HIVs, may enhance virulence after several cycles of transmission. The large number of species carrying these viruses may suggest that infection confers an evolutionary advantage to the host. The virulent T-lymphocyte-tropic lentiviruses have a similar genomic structure and exhibit comparable replication strategies. Their major targets are lymphocytes populating lymphoid organs and tissues, and antigen-presenting cells (dendritic cells, mononuclear phagocytes). Within these targets the virus can replicate to very high titres and thereby exhaust CD4+ T cells, producing profound immunodeficiency. Although the infection of lymphoid organs and tissue is the pathologic hallmark of HIV infection, this virus also infects cells of the central nervous system. This chapter discusses various pathogenic mechanisms involved in immune activation and dysregulation, and summarises characteristics of HIV/SIV gene-host factor interaction in the immune and central nervous systems.

11. Retroviral Restriction Factors

Jeremy Luban

Over the course of the retrovirus replication cycle, viral cDNA is inserted into host chromosomal DNA to establish the provirus. This process results in a permanent insertion mutation in the host cell genome. Host cells have evolved intracellular factors that block the spread of retroviral infection. Some of these antiviral factors act prior to integration and therefore also block the mutagenic potential of infection. Several such factors have been identified, including Fv1, the APOBEC3 complex, and TRIM5. These host factors potently block HIV-1 and other retroviruses from establishment of the provirus. Here we will review current understanding of Fv1, APOBEC3, and TRIM5 proteins, with particular emphasis on TRIM5.

12. Molecular Vaccines and Correlates of Protection

Stephen Norley and Reinhard Kurth

The failure of 'classical' vaccines to induce protection to the most important of all retroviruses, HIV, has led to the development of a huge variety of 'molecular vaccines', i.e. vaccines produced using modern molecular biological techniques. Such vaccines range from simple plasmid DNA coding for the genes of choice, through recombinant viruses carrying such genes to engineered bacteria designed to deliver HIV genes to the mucosal immune system. Evaluation of such vaccines in animal models has resulted in sporadic successes and many failures and the few human clinical trials have been, at best, negative. However, the relative success of molecular vaccines in combating other retroviral infections and the continuing refinement of HIV/SIV vaccines showing some efficacy suggests that a molecular AIDS vaccine may be achievable. In the end, the HIV/AIDS pandemic will only be defeated by the development of an effective, stable, and inexpensive vaccine.

13. Gammaretroviral and Lentiviral Vectors for Gene Delivery

Michael D. Mühlebach, Silke Schüle, Nina Gerlach, Matthias Schweizer, Christian Buchholz, Christine Hohenadl and Klaus Chichutek

Gammaretroviral and lentiviral vectors for gene therapy have been developed that mediate stable genetic modification of treated cells by chromosomal integration of the transferred vector genomes. This is highly desired, not only for research use, but also for clinical gene therapy aiming at the long-term correction of genetic defects, e.g., in stem and progenitor cells. Retroviral vector particles with tropism for various target cells have been designed. Due to split genome vector design the risk of replication-competent retrovirus formation has been minimized. Gammaretroviral and lentiviral vectors have so far been used in more than 300 clinical trials, addressing treatment options for various diseases. In some cases these trials resulted in benefit for treated patients suffering from life threatening disease. However, insertional mutagenesis due to vector integration in or next to cellular proto-oncogenes was concluded to be necessary for the lymphoproliferative disease observed in some patients treated with gammaretrovirally modified haematopoietic stem cells for X-linked severe combined immunodeficiency disease. These findings prompted the design of gammaretroviral vectors harbouring self-inactivating (SIN) Long Terminal Repeats (LTRs), which current lentiviral vectors already have. SIN vectors may reduce the effect of insertional mutagenesis and proto-oncogene activation, thereby reducing the risk of oncogenesis. With a view to future clinical use, new developments such as cell entry targeting will further improve the safety and efficacy of retroviral vectors.

14. Non-primate Mammalian and Fish Retroviruses

Maribeth V. Eiden, Kathryn Radke, Joel Rovnak and Sandra L. Quackenbush

The pioneering phase of the study of retroviruses resulted in the identification of viruses associated with diseases in chickens, mice and cats. Retroviruses have since been isolated from many vertebrate species, and classified into seven genera that can be grouped into two general categories. Alpharetroviruses, betaretroviruses and gammaretroviruses are genetically simple, encoding only nucleoprotein, matrix, capsid, reverse transcriptase, integrase, protease and envelope proteins. Deltaretroviruses, epsilonretroviruses, lentiviruses and spumaviruses are considered complex because they encode in addition to the proteins listed above, a number of ancillary proteins that often play an important role in gene regulation. In this chapter we review recent findings of representative simple mammalian gammaretroviruses and the complex piscine epsilonretroviruses and bovine leukemia virus with the intent of illustrating how these viruses have shed light on the mechanisms of viral function, evolution and pathogenesis within the animal kingdom that hosts them.

15. Simian Exogenous Retroviruses

Jonathan Luke Heeney and Ernst J. Verschoor

Simians include diverse species of monkeys which are globally distributed predominantly in the southern hemisphere, and ape species that are restricted to the rainforests of central Africa and Southeast Asia. Types of simian retroviruses have been detected in almost all species of non-human primates which have been studied. As early identification and classification of primate retroviruses was largely possible due to the efforts to establish cell lines from various primate species, there were limitations. Firstly, the search for simian retroviruses was frequently restricted to only a few species cell lines. Secondly, not all retroviruses were permissive to the available cell lines due to species specific restriction or cell-type specific factors. The earliest discoveries of simian retroviruses were often based on a specific observations which arose from diagnostic workups of cases of then undefined illness or unusual cancers. This chapter focuses on the four main groups of exogenous simian retroviruses; type D simian retroviruses (SRV), simian foamy viruses (SFV) commonly known as spumaviruses, the simian T-cell lymphotropic viruses (STLV), and the expanding group of simian immunodeficiency viruses (SIV). Initially viruses in these subgroups of simian retroviruses were identified based on the diseases from which they were associated with. Retroperitoneal firbrosarcomas and chronic wasting disease led to the early identification of the SRVs, found to be associated with a spectrum of diseases in different species of macaques. Other simian retroviruses are frequently asymptomatic in their natural hosts and in some cases simply cause cytopathic effect (CPE) in cell culture (SFV). In the last two decades molecular techniques have provided us with much more insight into the phylogeny of the wide variety of diverse retroviruses, knowledge which has enriched the seroprevalence evidence of widespread retroviral infections in many different primate species.

16. HTLV and HIV

Marvin S. Reitz, Jr and Robert C. Gallo

For many years, retroviruses were known to be the cause of many kinds of animal leukemias and hematopoietic tumors. In spite of the high expectation that this would also be true for humans, very little evidence for retroviral involvement in any human diseases was forthcoming. In the late 1970s, however, due to the development of sensitive and specific molecular methods to identify retroviruses and to produce large scale cultures of T lymphocytes, HTLV-I was discovered and implicated as the cause of adult T cell leukemia, a particular and relatively infrequent leukemia prevalent in southern Japan and parts of the Caribbean, and tropical spastic paraparesis, a demyelinating neuropathy similar to multiple sclerosis. The discovery that HTLV-I can be transmitted by breast milk has led to a significant decline in HTLV-I infections in Japan. Although no retroviruses have been identified to date in other human leukemias or related diseases, the efforts that resulted in the discovery of HTLV-I were critical in isolating HIV-1 and identifying it as the cause of AIDS. The ability to grow the HIV-1 in quantity allowed the development of a blood test that has saved countless lives. The development of effective anti-retroviral drugs has made HIV-1 infection a somewhat manageable chronic condition rather than a certain death sentence. Although vaccine trials thus far have been rather disappointing, an effective vaccine is one of our most important needs.