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Lymphoproliferative disease virus (LPDV) is an exogenous oncogenic retrovirus that induces lymphoid tumors in some galliform species of birds. Historically, outbreaks of LPDV have been reported from Europe and Israel. Although the virus has previously never been detected in North America, herein we describe the widespread distribution, genetic diversity, pathogenesis, and evolution of LPDV in the United States. Characterization of the provirus genome of the index LPDV case from North America demonstrated an 88% nucleotide identity to the Israeli prototype strain. Although phylogenetic analysis indicated that the majority of viruses fell into a single North American lineage, a small subset of viruses from South Carolina were most closely related to the Israeli prototype. These results suggest that LPDV was transferred between continents to initiate outbreaks of disease. However, the direction (New World to Old World or vice versa), mechanism, and time frame of the transcontinental spread currently remain unknown., (© 2013 Published by Elsevier Inc.)

Integrase (IN) is the enzyme responsible for provirus integration of retroviruses into the host cell genome. We used an Avian Sarcoma and Leukemia Viruses (ASLV) integration assay to investigate the way in which IN integrates substrates mutated or devoid of one or both IN recognition sequences. We found that replacing U5 by non-viral sequences (U5del) or U3 by a mutated sequence (pseudoU3) resulted in two and three fold reduction of two-ended integration (integration of the two ends from a donor DNA) respectively, but had a slight effect on concerted integration (integration of both ends at the same site of target DNA). Further, IN was still able to integrate the viral ends of the double mutant (pseudoU3/U5del) in a two-ended and concerted integration reaction. However, efficiency and accuracy (i.e. fidelity of size duplication and of end cleavage) of integration were reduced.

During replicative cycle of retroviruses, the reverse-transcribed viral DNA is integrated into the cell DNA by the viral integrase (IN) enzyme. The central core domain of IN contains the catalytic site of the enzyme and is involved in binding viral ends and cell DNA as well as dimerization. We previously performed single amino acid substitutions in the core domain of an Avian Leukemia and Sarcoma Virus (ALSV) IN [Arch. Virol. 147 (2002) 1761]. Here, we modeled the resulting IN mutants and analyzed the ability of these mutants to mediate concerted DNA integration in an in vitro assay, and to form dimers by protein-protein cross-linking and size exclusion chromatography. The N197C mutation resulted in the inability of the mutant to perform concerted integration that was concomitant with a loss of IN dimerization. Surprisingly, mutations Q102G and A106V at the dimer interface resulted in mutants with higher efficiencies than the wild-type IN in performing two-ended concerted integration of viral DNA ends. The G139D and A195V mutants had a trend to perform one-ended DNA integration of viral ends instead of two-ended integration. More drastically, the I88L and L135G mutants preferentially mediated nonconcerted DNA integration although the proteins form dimers. Therefore, these mutations may alter the formation of IN complexes of higher molecular size than a dimer that would be required for concerted integration. This study points to the important role of core domain residues in the concerted integration of viral DNA ends as well as in the oligomerization of the enzyme.

The lack of a well-behaved permanent, adherent, nontransformed chicken cell line has made some experiments with avian leukosis-sarcoma viruses (ASLV) and vectors considerably more difficult. The EV-O-derived line, DF-1, supports the efficient replication of subgroups (A), (B), and (C) ASLV, as well as amphotrophic murine leukemia virus and an ASLV-derived vector that has its env gene derived from the env gene from an amphotrophic murine leukemia virus. The cell line responds appropriately to the expression of a transforming oncogene (v-myc) to a growth suppressor gene [p21(waf1)] and can be sorted (using FACS) if infected by an ASLV vector that expresses GFP., (Copyright 1998 Academic Press.)

We have characterized the structure and infectivity of an avian retrovirus, myeloblastosis associated virus (MAV), containing a genetically altered proteinase (PR). A site-directed mutant of MAV-PR that shows an increased proteolytic activity in vitro (about 20 times higher kcat/Km) as a consequence of substituting five amino acids from the substrate-binding pocket with those corresponding to the HIV-1 PR was cloned into a full-sized MAV plasmid. In particular, the wild-type MAV-PR gene was replaced with the mutant one. Despite encoding for an enzyme with increased PR activity, mutant plasmid-transfected turkey fibroblasts displayed an unimpaired virus production in cell cultures. Further, the mutant progeny virus was infectious and its pattern of gag processing products appeared identical to that of wild-type virus. However, by electron microscopy we found that the predominant morphology of mutant viral particles was altered. Instead of a centrally collapsed avian retroviral core, a more diffuse core was visualized for wild-type mutant virions, similar to that observed in mammalian C-type retroviruses.

We previously isolated an avian erythroblastosis virus, AEV-GEE35, in which the complete extracellular and transmembrane domains of the v-erbB oncoprotein were replaced with sequences from the gag and env proteins. The GEE35 virus was capable of transforming both fibroblasts and erythroblasts as efficiently as wild-type v-erbB. Analysis of the v-erbB proteins encoded by GEE35 revealed two proteins of similar molecular weights of approximately 130,000 Da. One of these proteins was an N-linked glycosylated membrane protein, whereas the other was a cytoplasmic protein. Biochemical characterization of these two proteins revealed that the transmembrane protein has the v-erbB domain outside the cell, such that it no longer had access to its tyrosine kinase substrates. This implies that it is the cytoplasmically located v-erbB-encoded protein that is responsible for the efficient transforming ability of this virus.

Avian erythroblastosis virus (AEV) is an acutely transforming retrovirus whose putative oncogenes (v-erb-A and v-erb-B) encode the proteins P74gag-erb-A and P61-68erb-B. The existence of these two gene products has prompted the question of whether one or both proteins are required in the transformation of erythroblasts and fibroblasts by AEV. In the accompanying manuscript, we describe the use of site-specific mutagenesis to generate mutants of AEV unable to synthesize P61-68erb-B. Here we present our analysis of the oncogenic potential of an AEV mutant unable to synthesize P74gag-erb-A due to a large deletion encompassing both gag and v-erb-A sequences. The erb-A-mutant retrovirus propagated quite poorly on fibroblasts in culture; however, fibroblasts harboring the erb-A mutant genome were transformed in the absence of P74gag-erb-A expression. The mutant virus failed to induce erythroleukemias in chickens, but the validity of this finding is compromised by the poor replicative capacity of the mutant. The results presented in this and the preceding manuscript indicate that P61-68erb-B is both necessary and sufficient for neoplastic transformation of fibroblasts by AEV; by contrast, a role for p74gag-erb-A in leukemogenesis by AEV has not yet been rigorously excluded.

The response to infection of chicken hemopoietic cells derived from the early stages of embryogenesis by avian myeloblastosis virus (AMV) and avian erythroblastosis virus (AEV) was investigated. It was found that erythroid progenitor cells were present in the blastoderm at a higher frequency than that of myeloid progenitor cells. These results correlate with the observation that target cells for AEV were found to be more numerous than those for AMV. Therefore, blastoderm cells are of potential value in understanding the mechanisms of oncogenesis at the level of the target cells.

The H strain of avian erythroblastosis virus (AEV-H) was recently isolated from the liver emulsion of a chicken that suffered from erythroblastosis after being inoculated with subgroup A leukosis virus. AEV-H induced erythroblastosis or sarcoma when inoculated into chickens and transformed chick embryo fibroblasts (CEF) in vitro. Analysis of viral proteins synthesized in cells, which were named HNP, transformed by AEV-H but not producing transforming virus revealed tha the genome of AEV-H directed the synthesis of the gag gene products, Pr76gag and Pr180gag-pol, which was the precursor of active reverse transcriptase. Thus the HNP produced virions that were not infectious due to a defect of the env gene. Studies on the viral RNA showed that a 35 S RNA, estimated to be 8000 nucleotides long, was the genomic RNA of AEV-H and probably carried one transforming gene, which is most likely erbB gene. The gene organization of AEV-H was suggested to be 5'-gag-pol-onc-3'. These data imply that the single oncogene is responsible for both erythroblastosis and sarcoma.

RSV transforms several nonhematopoietic cell types and as reported here also has the capacity to transform hematopoietic cells of the erythroid lineage. In vitro, the three RSV isolates tested induced erythroblast-like colonies in infected bone marrow cells that were distinguishable by size and cell arrangement from those induced by avian erythroblastosis virus (AEV). Also in contrast to AEV-transformed erythroblast cultures, isolated cell colonies induced by RSV required complex growth conditions in liquid medium similar to the in vitro conditions necessary for erythroblasts transformed by the acute leukemia virus E26. Temperature-shift experiments using temperature-sensitive (ts) NY68 RSV revealed that when grown at the nonpermissive temperature (42 degrees), mutant-infected cells became benzidine positive and partially differentiated into erythrocytes. Wild-type (wt) RSV-transformed cells did not undergo similar changes. However, both wt RSV-, and to a greater extent, ts RSV-transformed cultures at the permissive temperature (37 degrees) did contain populations of spontaneously differentiating erythroid cells signifying that the transforming activity of the virus did not fully arrest erythroid maturation. In addition, the RSV-transformed cells did express tyrosine kinase activity. When injected intravenously into birds, RSV induced an erythroblastosis-like disease similar to AEV but also caused fibrosarcomas and leg paralysis. These results show that RSV can alter the pattern of erythroid differentiation in a manner similar to, but distinct from, AEV and indicate that the tyrosine-specific pp60src kinase is involved in erythroid cell transformation. Since the src and erb B proteins share a significant amino acid homology, these data suggest that both may also share a common functional homology.

The avian leukemia sarcoma virus S13 transforms chicken and Japanese quail embryo fibroblasts and chicken erythroid cells in tissue culture. S13-induced erythroid transformation requires culture conditions suitable for the growth of normal erythroid precursors (H. Beug and M. J. Hayman (1984), Cell 36, 963-972). S13-transformed erythroid colonies contain a high percentage of cells that differentiate in absence of erythropoietin. S13 is defective in pol and env functions but can code for a complete set of gag proteins. Nonproducer cell clones transformed by S13 release a noninfectious viral particle containing gag but no functional env or pol proteins. They also synthesize a transformation-specific protein of 155,000 molecular weight. This protein reacts with antibody to viral envelope glycoproteins and appears to represent onc as well as env sequences. The 155,000-molecular weight env-linked protein does not cross react immunologically with an antiserum against the v-erb A and v-erb B gene products.