CHAPTER 20

1. CHAPTER 20 Retrovirus

2. Definitions of the virus: • Retroviruses are a diverse group of RNA viruses that all replicate utilizing reverse transcriptase, a virus-encoded enzyme that synthesizes a DNA copy from the RNA viral genome. • All retroviruses contain reverse transcriptase and a diploid genome with two copies of single-stranded, positive-sense RNA. Virions are enveloped and typically form by budding from cell membranes.

3. Definitions of the virus: Retroviruses integrate into host genomes using unique virus-encoded enzyme, integrase. This allows them to acquire, but alter, host genetic sequences. The ability of retroviruses to integrate into the host cell genome can result in activation or inactivation of specific host cell genes near integration sites. The family Retroviridae is subdivided into two subfamilies (Orthoretrovirinae, Spumaretrovirinae) and seven genera (Alpharetrovirus, Betaretrovirus,

5. Definitions of the virus: Gammaretrovirus, Deltaretrovirus, Epsilonretrovirus, Lentivirus, and Spumavirus).

6. Definitions of the virus: Morphologically, four different types of particles were recognized, designated A, B, C, and D. Type C retrovirus morphogenesis involves the formation at the cell membrane of crescent-shaped nascent nucleocapsids. Oncogenic retroviruses include those with type B or type C particle morphology. Type A particles were intermediate forms of type B virions or found in cell lines as unique “intracisternal” A particles.

7. Definitions of the virus: Exogenous retroviruses are transmitted horizontally and transmitted via in-utero or germ-line infection. In contrast, endogenous retroviruses or retroviral elements are included in the genome of animals. These endogenous viruses or elements (retro-elements) are transmitted vertically as part of the genome of their host species and are passed from generation to generation as inherited genetic sequences.

8. The antigenic relationships among different retroviruses can be complex, as some envelope glycoprotein epitopes are retrovirus type-specific, but others are strain specific. Host-derived antibodies that neutralize viral infectivity are directed against surface envelope glycoproteins. Core protein epitopes by the gag gene are common to the retroviruses of particular animal they are group-specific antigens and referred to as “Gag.”

9. Retrovirus virions are enveloped, 80–100 nm in diameter, and have a unique three-layered structure. Innermost is the genome–nucleoprotein complex, which includes reverse transcriptase and has helical symmetry. This is enclosed within an icosahedral capsid, about 60 nm in diameter, which in turn is surrounded by an envelope derived from the host cell membrane, from which surface envelope glycoprotein spikes project.

10. The genomes of retroviruses are diploid, meaning two RNA copies are packaged in virions as an inverted dimer of two molecules of linear positive-sense, single stranded RNA. Each RNA genome copy is between 7 and 11 kb and has a 3’-polyadenylated tail and a 5’ cap. Virions are inactivated easily by lipid solvents or detergents and by heating. They are more resistant to ultraviolet and X-irradiation, because their diploid genomes can compensate for radiation-induced mutations during reverse transcription.

11. Retroviruses require reverse transcriptase for their replication. Reverse transcriptase serves as an RNA-dependent DNA polymerase, a DNA dependent DNA polymerase, and an RNase, with each distinctive function being carried out by partsof the molecule. Replication of retroviruses is dependent on host-cell RNA polymerases for transcription of the integrated DNA copy of the viral genome. The genome of replication competent (exogenous/non-defective) retroviruses contains three major genes, each encoding two or more proteins.

13. The gag gene encodes the virion core proteins [capsid (CA); nucleocapsid (NC); and matrix (MA)], the pol gene encodes reverse transcriptase (RT) and integrase (IN), and the env gene encodes the virion envelope proteins [surface (SU) and transmembrane (TM)]. Genome termini have several components, each of which is functionally important. For example, the R (repeat) and U (5’ and 3’ unique regions) are critical for reverse transcription, integration, and viral transcription after integration.

14. Retroviruses are classified as having acute transformingor chronic transforming biologic characteristics,based on the presence or absence of key transforminggenes in the viral genome. Those viruses that are capableof acute cellular transformation have been referred as rapidly or strongly transforming, because these retrovirusescontain viral oncogenes (v-onc) that, undercontrol of a viral promoter, increase the probabilityof expression of v-onc. The presence of the v-onc gene, which was originally acquired from a host genome is associated with deletions

15. elsewhere in the viral genomeas a result of “trade” of a viral gene for a host-cell gene (“onc”) during recombination. This trade of a viral gene for a cellular one reflects the packaging constraints during replication and the viral env gene is usually exchanged. Most v-onc-containing viruses are unable to synthesize a complete envelope, are replication defective and must associate with non-defective viruses that are replication competent that act as helper viruses to accomplish replication and spread to other hosts.

16. Rous sarcoma virus is an exception; its genome contains the viral oncogene, v-src, but it contains complete gag, pol, and env genes and is therefore replication competent. Chronic referred to as slowly transforming, retroviruses induce neoplasia by insertional mutagenesis through random integration into regions of the host genome that influence cell division, activating host oncogenes. In addition to encoding gag, pol, and env genes, complex retroviruses such as lentiviruses encode several other regulatory and “accessory” genes.

17. The regulatory genes of human immunodeficiency virus type 1 (HIV-1) include tat, which encodes a transactivator that enhances the efficiency of transcription by cellular RNA polymerase, by preventing premature termination of transcription, and rev, which encodes a protein that facilitates the export of non-spliced or singly spliced viral RNA from the nucleus to the cytoplasm. The HIV-1 accessory gene, nef, is not required for virus replication in cell culture, but is essential for replication in vivo. The Nef protein can downregulate expression of CD4 and may alter the activation of target cells in vivo.

18. The Vif protein of human and simian immunodeficiency viruses accumulates in the cytosol of infected cells and is incorporated in virions to enhance virus replication in lymphocytes. Vif promotes the degradation of a cell-derived cytidine deaminase, APOBEC 3G, blocking APOBEC 3G from inhibiting retrovirus replication by promoting the incorporation of deoxyuridine in the first minus-strand complementary DNA produced during reverse transcription.

19. The Vpr and Vpx virion-associated proteins contribute to nuclear localization of preinitiation complexes, influence cell-cycle regulation, and alter cell signaling within infected cells. Vpu is an amphipathic membrane protein of primate lentiviruses that promotes efficient release of virions from cells during budding. The accessory proteins can be important in the establishment and maintenance of retrovirus infections in their respective hosts.

21. The Vpr and Vpx virion-associated proteins contribute to nuclear localization of preinitiation complexes, influence cell-cycle regulation, and alter cell signaling within infected cells. Vpu is an amphipathic membrane protein of primate lentiviruses that promotes efficient release of virions from cells during budding. The accessory proteins can be important in the establishment and maintenance of retrovirus infections in their respective hosts.

22. The retrovirus replication begins when virion envelope glycoproteins bind to cellular receptors. The cellular receptors for virus attachment are unique to each retrovirus genus, so retroviruses are species restricted in their host range. After attachment, the viral envelope and the cell membrane fuse, allowing the virion core to enter the cytoplasm; less commonly, entry involves receptor-mediated endocytosis. Cells infected with a particular retrovirus are often resistant to superinfection by another closely related retrovirus.

23. Strains of avian leukosis virus have distinct interference patterns that reflect their individual effects on receptor expression. A double-stranded DNA copy of the retrovirus genomic single-stranded RNA is synthesized within the viral capsid by the virion associated reverse transcriptase. The reverse transcriptase enzyme contains a domain for polymerase activity, as well as an RNase H that degrades the RNA moiety of RNA–DNA hybrids.

24. Reverse transcription is dependent upon unique features of the reverse transcriptase enzyme such as its ability to “jump” between template strands. In the reverse transcription, some 300 to 1300 base pairs are added to the ends of each genomic RNA molecule. These termini, called long-terminal repeats (LTRs), have a complex secondary structure and are central in the replication strategy of all retroviruses. Host-cell-derived transfer RNAs (tRNAs) unique to each genera of retroviruses bind viral 5’-LTR sequences and act to prime the

25. reverse transcriptase. During the process of creating a DNA copy of its RNA genome, the RNase H portion of the reverse transcriptase molecule degrades the RNA strand to create short single strand DNA, which then hybridizes to the R region of the same or different genomic RNA molecule to complete synthesis of first-strand DNA. The digestion of viral RNA occurs, except short regions used as second DNA strand primers. Second strand DNA synthesis occurs by elongation to create the dsDNA intermediate and, ultimately, a linear dsDNA

26. copy of the viral RNA genome that is then transported to the nucleus before integration into the host chromosomal DNA using the virus-encoded integrase. Most retroviruses rely upon cell division for efficient passage into the nucleus and for integration to occur. Once these DNA forms are integrated into the host cell chromosome, the retroviral genomic copies are as “proviruses.” Proviruses are templates for transcription, including the transcription of genomic RNA and spliced messenger RNAs. Transcription by cellular RNA polymerase,

27. polymerase, initiated in the 5-LTR and ending in the 3-LTR, generates new virion RNA.

28. Retrovirus genomic integration mediated by integrase does not specifically particular host cell sequences. Local chromatin structure such as DNA bending or open chromatin that are transcriptionally active are favored sites. These cellular integration tendencies may explain carcinogenesis, as retroviruses tend to integrate near, and activate, cellular oncogenes. After the provirus is integrated, it may remain latent or be transcriptionally active. The viral LTR promotes and initiates transcription of different RNA species that are processed

29. similarly to host cell RNA. After transport to the cytoplasm, the mRNA forms two pools: one genomic RNA that is subsequently packaged in virions as genomic RNA, and another that includes mRNAs encoding Gag, Pro, Env, and Pol species. A major function of the LTR is to provide signals to initiate RNA synthesis and control the rate of transcription, in concert with both viral and cellular factors. Thus the LTR may determine disease progression by controlling replication of the retrovirus in specific cell types. Because the LTR is repeated in the

30. proviral forms of retroviruses, they must suppress the 3’-LTR from initiation of transcription. This is accomplished by adjacent gag signals that allow the 5’-LTR to be used for initiation at the 3’-LTR. All subgenomic RNAs have a splice start at the common 5’ donor. Most retroviruses rely upon the cellular splicing mechanisms to obtain splice messages. Bovine leukemia virus produce Rex that promotes genomic RNA or single-splice RNA species to be transported from the nucleus to the cytoplasm.

31. Replication of retroviruses is accompanied by a high mutation frequency due to the lack of a 3’ to 5’ exonuclease proofreading mechanism by reverse transcriptase. But many loci can tolerate mutation, others in genes encoding either enzymes or structural proteins may not tolerate high mutation rates if they result in defects that block replication or virus assembly. Gag and pol genes are more conserved, as are certain critical portions of env, whereas other regions of env, particularly those regions to which antibody binds, are highly variable.

32. The ability for the portions of env to tolerate variation without adversely impacting virus viability facilitates the evolution of new virus strains, including those that can escape immune control. There is a high frequency of recombination and gene rearrangements between retrovirus genomes in cells infected with more than one virus. Deletions, duplications, and inversions are relatively common, but most are probably lethal for the virus. These events occur during reverse transcription, during which there are a number of chances for the reverse

33. transcriptase enzyme to jump templates, produce duplications, or lead to mispriming. Retroviruses have a high rate of recombination. Retroviral protein synthesis occurs in the cytoplasm. Spliced mRNAs are used for translation of envelope proteins and accessory proteins. Full-length, non-spliced RNA serves as genomic RNA (packaged in virion) or as mRNA for Gag-Pro-Pol proteins. Translation occurs by ribosomal scanning of mRNAs with frameshifts and read-through translation occurring to produce proteins.

34. The precursor protein transcribed from 35S mRNAs includes structural and enzymatic proteins and is associated with free polyribosomes, but the 22–24S mRNAs encode the envelope proteins associated with membrane bound ribosomes. The Env protein is glycosylated and processed in the endoplasmic reticulum and Golgi complex before trafficking to the plasma membrane as a result of targeting modifications such as myristoylation.

35. Together with viral RNA, Gag and Gag-Pol precursors begin to assemble nucleocapsids on the inner side of the plasma membrane. Budding proceeds with nucleocapsids binding to Env proteins already fixed in the plasma membrane. Virus particle assembly is initiated with interaction of the NC domain of Gag precursor protein with packaging signals in genomic RNA. Proteolytic processing of structural precursor proteins is initiated during budding of virus particles, and continues in newly released

36. virus particles when the virions are infectious. Studies indicate that key retroviral late- budding (L) domains in Gag are required for the efficient release of nascent virions in concert with cellular co-factors involved in protein trafficking and sorting. Retroviruses replicate predominantly in dividing cells, without cytopathology or dramatic alteration of the metabolism of the cells. But the lentiviruses, cause cell death including syncytium formation and apoptosis. Infected cells may divide while producing large numbers of virions.

37. Cellular “oncogenes” (c-onc or proto-oncogenes) are responsible for normal cell growth and differentiation. During retrovirus replication, viral oncogenes (v-onc) can be created from c-onc through processes such as read-through transcription. Viral oncogenes include features that result in the loss of cellular control of v-onc activity. Viral oncogenes affect cell growth control by influencing or acting as growth factors, receptors, intracellular signal transducers, or transcription factors.

38. Oncogene capture is a feature of retrovirus replication and is considered an “illegitimate” recombination event. It is a non-homologous recombination event during reverse transcription. This occurs by two mechanisms each occurring when retroviruses integrate within or adjacent to a proto-oncogene; specifically, packaging of deleted and wild-type genomes can be followed by recombination, resulting in additional sequences “captured” by the virus. Alternatively, packaging of read-through transcripts can occur as RNA polymerases transcribe the provirus, and subsequent recombination results in the

39. incorporation of additional sequences into the new viral genome. Many events may block replication, but those that lead to a transformation event may be expressed as cancer. Retrovirus infections of chickens fall into two distinct groups: (1) the avian leukosis, myeloblastosis, and sarcoma viruses, which belong to the genus Alpharetrovirus; (2) the avian reticuloendotheliosis viruses, which belong to the genus Gammaretrovirus.

41. Avian retroviruses include (1) endogenous; (2) exogenous replication competent; (3) exogenous replication defective. Endogenous avian leukosis viruses occur in the genome of chicken as DNA proviruses. These endogenous retroviruses are not expressed or pathogenic unless recombination events create viruses associated with glioma. Exogenous avian leukosis viruses are replication competent and have gag, pol, and env genes. These avian retroviruses are generally non-pathogenic, but a small percentage of infected birds develop leukemia

42. or lymphoma. Some exogenous viruses acquire an oncogene (v-onc) from a cellular onc (c-onc) gene and then can induce malignant tumors rapidly. Such rapidly transforming (oncogenic) viruses lose part of their genome but acquire their oncogene, so that they become replication defective and dependent on the helper activity of a replication-competent virus. Rous sarcoma virus, have a full complement of viral genes plus a v-onc gene—they are rapidly oncogenic and capable of replication without a helper virus.

45. Oncogenic retroviruses are transmitted from one chicken to another horizontally or vertically, and vertical transmission occurs through infectious virus (complete virions) or through provirus integrated into the DNA of the germ cells. If chickens are infected horizontally when more than 5 or 6 days of age they are unlikely to develop leukemia; instead they develop a transient viremia and produce neutralizing antibody. If virus is transmitted congenitally via the egg or within the first few days of life, the chicken develops a viremia that persists for life

46. because of the induction of immunological tolerance. Such birds appear to grow normally, but subsequently develop leukemia and associated diseases and are a major source of exogenous virus. Avian sarcoma and leukosis viruses are classified into at 10 different subgroups (designated A–J) based on the cellular receptors used to infect the cell. Six subgroups occur in chickens (A, B, C, D, E, and J). The subgroups are associated with distinct patterns of disease.

47. In general, avian leukosis viruses are widespread in layer flocks of chickens, and most chickens have been infected within a few months. If rapidly transforming viruses are not present, disease occurs sporadically in birds more than 14 weeks of age, with an incidence of 1–2%. The incidence may be 20%, as occurred in Leukosis J in broiler breeding birds. The occurrence of avian leukosis in the field is generally from horizontal transmission of the virus.

48. In chickens infected with exogenous non-defective viruses, tumors occur when infection occurs congenitally and there is persistent viremia. Over the life of the chicken, proviral DNA is integrated into cells in a location where the activity of a c-onc gene is disturbed in such a way as to initiate tumor production. Various neoplasms are generated by replication defective leukemia viruses propagated by co-infection with a non-defective helper, which is usually an exogenous avian leukemia virus. These defective viruses arise as a rare event in bird.

49. The different pathogenic potential of the viruses is attributable to the different v-onc genes they carry, and is reflected in the different tumors that they induce. Lymphoid leukosis is the most common form of avian leukosis and occurs in chickens 14– 30 weeks of age. Clinical signs are non-specific, but the comb may be pale, shriveled, and occasionally cyanotic, and affected birds exhibit inappetence, emaciation, weakness, and abdominal swelling. Tumors are present for time before clinical illness is recognized, although the course may be rapid from the onset of disease.

50. Tumors, usually as discrete, nodular lesions, occur first in the cloacal bursa, with subsequent metastasis to liver, spleen, and other internal organs. These tumors consist of aggregates of lymphoblasts that express B lymphocyte markers. These cells secrete large amounts of immunoglobulin M, but their capacity to differentiate into IgG-, IgA- or IgE-producing cells is arrested. Tumor induction is caused by virus activation and overexpression of the c-myc oncogene, with contributions from Blym-1 and c-bis.