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Compared with the MHC of typical mammals, the chicken MHC is smaller and simpler, with only two class I genes found in the B12 haplotype. We make five points to show that there is a single-dominantly expressed class I molecule that can have a strong effect on MHC function. First, we find only one cDNA for two MHC haplotypes (B14 and B15) and cDNAs corresponding to two genes for the other six (B2, B4, B6, B12, B19, and B21). Second, we find, for the B4, B12, and B15 haplotypes, that one cDNA is at least 10-fold more abundant than the other. Third, we use 2D gel electrophoresis of class I molecules from pulse-labeled cells to show that there is only one heavy chain spot for the B4 and B15 haplotypes, and one major spot for the B12 haplotype. Fourth, we determine the peptide motifs for B4, B12, and B15 cells in detail, including pool sequences and individual peptides, and show that the motifs are consistent with the peptides binding to models of the class I molecule encoded by the abundant cDNA. Finally, having shown for three haplotypes that there is a single dominantly expressed class I molecule at the level of RNA, protein, and antigenic peptide, we show that the motifs can explain the striking MHC-determined resistance and susceptibility to Rous sarcoma virus. These results are consistent with the concept of a \"minimal essential MHC\" for chickens, in strong contrast to typical mammals.

The late assembly (L) domain of retrovirus Gag, required in the final steps of budding for efficient exit from the host cell, is thought to mediate its function through interaction with unknown cellular factors. Here, we report the identification of the Nedd4-1ike family of E3 ubiquitin protein ligases as proteins that specifically interact with the Rous sarcoma virus (RSV) L domain in vitro and in vivo. We screened a chicken embryo cDNA expression library by using a peptide derived from the RSV p2b sequence, isolating two unique partial cDNA clones. Neither clone interacted with a peptide containing mutations known to disrupt in vivo RSV L domain function or with human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus (EIAV) L domain-derived peptides. The WW domain region of one of the clones, late domain-interacting protein 1 (LDI-1), but not the C2 domain, bound RSV Gag and inhibited RSV Gag budding from human 293 cells in a dominant-negative manner, functionally implicating LDI-1 in RSV particle budding from cells. RSV Gag can be coimmune precipitated from cell extracts with an antisera directed at an exogenously expressed hemagglutinin (HA)-tagged LDI-1 or endogenous Nedd4 proteins. These findings mechanistically link the cellular ubiquitination pathway to retrovirus budding.

Disparate enveloped viruses initiate infection by fusing with endosomes. However, the highly diverse and dynamic nature of endosomes impairs mechanistic studies of fusion and identification of sub-cellular sites supporting the nucleocapsid release. We took advantage of the extreme stability of avian retrovirus-receptor complexes at neutral pH and of acid-dependence of virus-endosome fusion to isolate the latter step from preceding asynchronous internalization/trafficking steps. Viruses were trapped within endosomes in the presence of NH₄Cl. Removal of NH₄Cl resulted in a quick and uniform acidification of all subcellular compartments, thereby initiating synchronous viral fusion. Single virus imaging demonstrated that fusion was initiated within seconds after acidification and often culminated in the release of the viral core from an endosome. Comparative studies of cells expressing either the transmembrane or GPI-anchored receptor isoform revealed that the transmembrane receptor delivered the virus to more fusion-permissive compartments. Thus the identity of endosomal compartments, in addition to their acidity, appears to modulate viral fusion. A more striking manifestation of the virus delivery to distinct compartments in the presence of NH₄Cl was the viral core release into the cytosol of cells expressing the transmembrane receptor and into endosomes of cells expressing the GPI-anchored isoform. In the latter cells, the newly released cores exhibited restricted mobility and were exposed to a more acidic environment than the cytoplasm. These cores appear to enter into the cytosol after an additional slow temperature-dependent step. We conclude that the NH₄Cl block traps the virus within intralumenal vesicles of late endosomes in cells expressing the GPI-anchored receptor. Viruses surrounded by more than one endosomal membrane release their core into the cytoplasm in two steps--fusion with an intralumenal vesicle followed by a yet unknown temperature-dependent step that liberates the core from late endosomes.

Viral fusion proteins catalyze merger of viral and cell membranes through a series of steps that have not yet been well defined. To elucidate the mechanism of virus entry, we have imaged fusion between single virions bearing avian sarcoma and leukosis virus (ASLV) envelope glycoprotein (Env) and the cell membrane. Viral particles were labeled with a lipophilic dye and with palmitylated enhanced YFP that was incorporated into the inner leaflet of the viral membrane. When individual virions were bound to target cells expressing cognate receptors, they transferred their lipids and contents only when exposed to low, but not neutral, pH. These data are consistent with the proposed two-step mechanism of ASLV entry that involves receptor-priming followed by low pH activation. Most importantly, lipid mixing commonly occurred before formation of a small fusion pore that was quickly and sensitively detected by pH-dependent changes in palmitylated enhanced YFP fluorescence. Nascent fusion pores were metastable and irreversibly closed, remained small, or fully enlarged, permitting nucleocapsid delivery into the cytosol. These findings strongly imply that hemifusion and a small pore are the key intermediates of ASLV fusion. When added before low pH treatment, a peptide designed to prevent Env from folding into a final helical-bundle conformation abolished virus-cell fusion and infection. Therefore, we conclude that, after receptor-activation, Env undergoes low pH-dependent refolding into a six-helix bundle and, in doing so, sequentially catalyzes hemifusion, fusion pore opening, and enlargement.

The retroviral Gag polyprotein directs budding from the plasma membrane of infected cells. Until now, it was believed that Gag proteins of type C retroviruses, including the prototypic oncoretrovirus Rous sarcoma virus, were synthesized on cytosolic ribosomes and targeted directly to the plasma membrane. Here we reveal a previously unknown step in the subcellular trafficking of the Gag protein, that of transient nuclear localization. We have identified a targeting signal within the N-terminal matrix domain that facilitates active nuclear import of the Gag polyprotein. We also found that Gag is transported out of the nucleus through the CRM1 nuclear export pathway, based on observations that treatment of virus-expressing cells with leptomycin B resulted in the redistribution of Gag proteins from the cytoplasm to the nucleus. Internal deletion of the C-terminal portion of the Gag p10 region resulted in the nuclear sequestration of Gag and markedly diminished budding, suggesting that the nuclear export signal might reside within p10. Finally, we observed that a previously described matrix mutant, Myr1E, was insensitive to the effects of leptomycin B, apparently bypassing the nuclear compartment during virus assembly. Myr1E has a defect in genomic RNA packaging, implying that nuclear localization of Gag might be involved in viral RNA interactions. Taken together, these findings provide evidence that nuclear entry and egress of the Gag polyprotein are intrinsic components of the Rous sarcoma virus assembly pathway.

The molecular events underlying the immediate steps of retroviral uncoating, occurring after membrane fusion and leading to the formation of an active reverse transcription complex, are not known. To better understand these processes, we have developed a cell-free system that recapitualtes these early steps of retroviral replication by using avian sarcoma and leukosis virus as a model retrovirus. The substrates used in this system are viral particles that are trapped before completing membrane fusion. These virions are induced to fuse out of endosomes and the viral cores are released into solution where they are amenable to biochemical manipulation. This system revealed that membrane fusion is not sufficient to stimulate the formation of a reverse transcription complex. Instead, ATP hydrolysis and cellular factors >5 kDa in size are required. Furthermore, later steps of avian sarcoma and leukosis virus reverse transcription were stimulated by nuclear factors. The cell-free system should now allow for the definition of retroviral uncoating mechanisms and facilitate the identification and characterization of the cellular factors involved.

The fusion protein of avian sarcoma and leukosis virus is likely to fold into a six-helix bundle as part of its final configuration. A peptide, R99, inhibits fusion, probably by binding into the grooves of the triple-stranded coiled coil that becomes the central core of the six-helix bundle. The stages at which the envelope protein (Env) of avian sarcoma and leukosis virus subgroup A folds into a bundle during low pH-induced fusion were determined. Effector cells expressing Env were bound to target cells expressing the cognate receptor Tva, and intermediates of fusion were created. R99 was added and the extent of fusion inhibition was used to distinguish between a prebundle state with exposed grooves and a state in which the grooves were no longer exposed. The native conformation of Env was not sensitive to R99. But adding a soluble form of Tva to effector cells conferred sensitivity. Acidic pH applied at low temperature created an intermediate state of local hemifusion. Surprisingly, R99 caused these locally hemifused membranes to separate. This indicates that the grooves of Env were still exposed, that prebundle configurations of Env stabilized hemifused states, and that binding of R99 altered the conformation of Env. In the presence of an inhibitory lipid that blocks fusion before hemifusion, applying low pH at 37°C created an intermediate in which R99 was without effect. This suggests that the six-helix bundle can form before hemifusion and that subsequent conformational changes, such as formation of the trimeric hairpin, are responsible for pore formation and/or growth.

The oncogene function in primary epithelial cells is largely unclear. Recombination organ cultures in combination with the stable and transient gene transfer techniques by retrovirus and electroporation, respectively, enable us to transfer oncogenes specifically into primary epithelial cells of the developing avian glandular stomach (proventriculus). In this system, the epithelium and mesenchyme are mutually dependent on each other for their growth and differentiation. We report here that either stable or transient expression of v- src in the epithelium causes budding and migration of epithelial cells into mesenchyme. In response to the transient expression of v-Src or a constitutive active mutant of MEK, we observed immediate downregulation of the Sonic hedgehog gene and subsequent elimination of E-cadherine expression in migrating cells, suggesting the involvement of MAP kinase signaling pathway in these processes. v- src -expressing cells that were retained in the epithelium underwent apoptosis (anoikis) and detached from the culture. Continuous expression of v- src by, for example, Rous sarcoma virus (RSV) was required for the epithelial cells to acquire the ability to express type I collagen and fibronectin genes (mesenchymal markers), and finally to establish the epithelial–mesenchymal transition. These observations would partly explain why RSV does not apparently cause carcinoma formation, but induces sarcomas exclusively.

Two rat kidney cell lines transformed by two strains of ASV virus were investigated. It was demonstrated that these two lines (1) showed density-independent growth, (2) had a decreased requirement for serum in the culture medium, (3) had the ability to grow in a chemically defined medium (without serum), and the rate of this growth had increased with the increase in starting density of cells, and (4) had the ability of anchrage-independent growth, even without serum. These results confirmed autostimulation of growth of W12 and GCA cells. It was also shown that the crude conditioned media contained autocrine growth factors, which could be extracted with 1M acetic acid. The extracts (AEs) stimulated the growth of the parental cells and NRK-49F cells almost as well as 5% calf serum and the extraction resulted in several-fold purification of mitogenic substances. These substances were not only specific to parental lines, but also stimulated growth of other transformed lines and normal NRK-49F cells. Extracts from the conditioned media of W12 and GCA cells intensified the rate of anchorage-independent growth in the concentration-dependent manner. In AE-W12, two peaks of mitogenic activity were detected (F1, F2) and similarly in AE-GCA (F3, F4). Fractions F2 (approximately 8 kDa), F3 (approximately 25 kDa) and F4 (approximately 12 kDa) were thermostable but F1 (approximately 45 kDa) was thermolabile. All four fractions were sensitive to trypsin and DTT treatment, and were acid-stable. Using ELISA kit it was shown that W12 and GCA cells released TGFbeta1 and GCA cells released very small quantities of bFGF. These results confirmed the autocrine regulation of growth in both cell lines.

A tridecamer oligodeoxynucleotide, d(A-A-T-G-G-T-A-A-A-A-T-G-G), which is complementary to reiterated 3′- and 5′-terminal nucleotides of Rous sarcoma virus 35S RNA, is an efficient inhibitor of the translation of proteins specified by the viral RNA in the wheat embryo cell-free system. The inhibition specificity for oncornavirus RNA is greater than for rabbit reticulocyte mRNA or brome mosaic virus RNA. Other oligodeoxynucleotides of similar size have little or no specific effect on the RNA-directed translation. The tridecamer acts as a primer for the avian myeloblastosis virus DNA polymerase when Rous sarcoma virus heated 70S RNA is used as a template, offering evidence that it can hybridize to the RNA. The possible use of such an oligodeoxynucleotide hybridization competitor to inhibit Rous sarcoma virus replication is described in the preceding paper [Zamecnik, P. C. & Stephenson, M. L. (1978) Proc. Natl. Acad. Sci. USA. 75, 280-284].