Kathryn Radke

The SU and TM Envelope Protein Subunits of Bovine Leukemia Virus Are Linked by Disulfide Bonds, both in Cells and in Virions

ABSTRACT After the polyprotein precursor of retroviral envelope proteins is proteolytically cleaved, the surface (SU) and transmembrane (TM) subunits remain associated with each other by noncovalent interactions or by disulfide bonds. Disulfide linkages confer a relatively stable association between the SU and TM envelope protein subunits of Rous sarcoma virus and murine leukemia virus. In contrast, the noncovalent association between SU and TM of human immunodeficiency virus leads to significant shedding of SU from the surface of infected cells. The SU and TM proteins of bovine leukemia virus (BLV) initially were reported to be disulfide linked but later were concluded not to be, since TM is often lost during purification of SU protein. Here, we show that SU and TM of BLV do, indeed, associate through disulfide bonds, whether the envelope proteins are overexpressed in transfected cells, are produced in virus-infected cells, or are present in newly produced virions.

Envelope Proteins Containing Single Amino Acid Substitutions Support a Structural Model of the Receptor-Binding Domain of Bovine Leukemia Virus Surface Protein

ABSTRACT Functional domains of the strikingly conserved envelope (Env) glycoproteins of bovine leukemia virus (BLV) and its close relative, human T-cell leukemia virus type 1 (HTLV-1), are still being defined. We have used BLV Env protein variants to gain insights into the structure and function of this important determinant of viral infectivity. Each of 23 different single amino acid variants found in cDNA clones of env transcripts present after short-term culture of peripheral blood mononuclear cells from BLV-infected sheep was expressed in COS-1 cells and tested for the ability to mediate cell fusion and to be cleaved to surface (SU) and transmembrane (TM) protein subunits. Of 11 Env variants that failed to induce syncytia or did so poorly, 7 contained changes in amino acids identical or chemically conserved in the HTLV-1 Env protein. These seven included the four variants that showed aberrant proteolytic cleavage and poor cell surface expression, underscoring their importance for Env structure. Ten of 12 variants that retained wild-type syncytium-inducing ability clustered in the N-terminal half of BLV SU, which forms the putative receptor-binding domain (RBD). Several variants in the RBD showed evidence of subtle misfolding, as judged by reduced binding to monoclonal antibodies recognizing conformational epitopes F, G, and H formed by the N terminus of SU. We modeled the BLV RBD by aligning putative structural elements with known elements of the ecotropic Friend murine leukemia virus RBD monomer. All the variant RBD residues but one are exposed on the surface of this BLV model. These variants as well as function-altering, antibody-reactive residues defined by other investigators group on one face of the molecular model. They are strikingly absent from the opposite face, implying that it is likely to face inward in Env complexes. This surface might interact with the C-terminal domain of SU or with an adjacent monomer in the Env oligomer. This location suggests an orientation for the monomer of ecotropic Friend murine leukemia virus RBD.

Transcription of bovine leukemia virus in peripheral blood cells obtained during early infection in vivo.

Bovine leukemia virus (BLV) is transcriptionally silent in most circulating peripheral blood mononuclear cells (PBMCs) of animals with well-established infections. Using PBMCs from a newly infected sheep, we asked whether viral transcription proceeded differently during the initial months of infection, when the prevalence of BLV-infected cells and the hosts immunological response change markedly. Shortly after being injected with BLV, the animal displayed a characteristic, transient increase in PBMCs that transcribed BLV when cultured. Even when transcriptionally competent PBMCs were most prevalent (1.2%), only rare cells in the circulation (1 in 50,000) contained enough BLV transcripts to be identified readily by in situ hybridization. However, at one point several weeks later, some PBMCs appeared to contain small amounts of BLV RNA as soon as they had been purified from blood. Throughout this period, BLV-transcribing PBMCs greatly outnumbered virus-producing cells, which were counted using a new infectious centers assay. Its viscous medium reduced cell to cell contact among PBMCs, enabling increased detection of BLV-producing cells at a time when virus-specific killer cells might be active. Early infection was polyclonal, and most infected PBMCs transcribed BLV upon being cultured. By 2 months after infection, provirus-containing cells were as abundant as they had been earlier, but few cells transcribed BLV. These results suggest that BLV-infected cells are more easily stimulated to transcribe the provirus and produce infectious virus during the early months of a new infection.

Humoral immune response of experimentally infected sheep defines two early periods of bovine leukemia virus replication

We have correlated the virus-specific humoral immune response of sheep newly infected with bovine leukemia virus (BLV) with the appearance in their blood of cells that transcribe BLV RNA or produce virus in culture. Neutralizing antibodies and antibodies binding to the viral capsid protein were present in most animals early after infection, often before BLV-expressing cells were first detected in blood. Neutralizing antibodies increased rapidly during the period when the number of cells that expressed BLV was also increasing. However, the titers developed by individual animals were independent of the maximum number of BLV-expressing cells. Antibodies that bound to the viral surface glycoprotein on immunoblots became evident at the same time as large peaks in the numbers of BLV-expressing cells. Despite ensuing sharp drops in BLV-expressing cells, neutralizing titers remained relatively constant through the rest of the first 8 months after infection. Two early phases of BLV replication were thus defined: initial, low-level replication that induced neutralizing and capsid-specific antibodies followed by a second period of intense replication that induced sharp increases in antiviral antibodies and preceded the release of many infected cells into the blood.

Dileucine and YXXL Motifs in the Cytoplasmic Tail of the Bovine Leukemia Virus Transmembrane Envelope Protein Affect Protein Expression on the Cell Surface

ABSTRACT Several retroviruses downmodulate the cell surface expression of envelope (Env) proteins through peptide sequences located in the cytoplasmic tail of the transmembrane (TM) subunit. We investigated whether cell surface expression of a chimeric protein containing the cytoplasmic domain of the TM protein (CTM) of bovine leukemia virus (BLV) was regulated by two membrane-proximal dileucine motifs or by tyrosine Y487 or Y498 in YXXL motifs. A chimeric protein composed of the extracellular and membrane-spanning portions of human CD8-α plus a wild-type (wt) BLV CTM was detectable on the surface of only 40% of the cells in which it was transiently expressed. Replacement of either dileucine pair with alanines increased the level of surface display of chimeric proteins. Nearly all cells became surface positive when both dileucine motifs were altered simultaneously and when either an N-terminal segment containing both dileucine motifs or a C-terminal segment containing all YXXL motifs was deleted. In contrast, replacement of Y487 or Y498 with alanine or phenylalanine enabled only small increases in surface display compared with wt levels. Chimeric proteins had similar stabilities but were downmodulated from the cell surface at three different rates. Point mutants segregated into each of the three groups of proteins categorized according to these different rates. Interestingly, Y487 mutants were downmodulated less efficiently than Y498 mutants, which behaved like wt. CD8-CTM chimeric proteins were phosphorylated on serine residues, but the native BLV Env protein was not phosphorylated either in transfected cells or in a lymphoid cell line constitutively producing BLV. Thus, both dileucine and YXXL motifs within the BLV CTM contribute to downmodulation of a protein containing this domain. Interactions with other proteins may influence surface exposure of Env protein complexes in virus-infected cells, assisting in viral evasion of adaptive immunity.

Transient increases of blood mononuclear cells that could express bovine leukemia virus early after experimental infection of sheep.

To investigate the early spread of bovine leukemia virus (BLV) infection in vivo, we enumerated infected mononuclear cells that could express the BLV genome in vitro as they appeared in the peripheral blood of lambs newly injected with the virus. Cells that transcribed viral RNA within a few hours of isolation and cells that produced infectious virus in culture were first detected in very small numbers. Soon afterward, cells that expressed BLV transiently increased to represent 0.2 to 1.5% of the mononuclear cells. The increases occurred within leukocyte populations of normal size and cellular composition. Then, throughout the rest of the first 8 months, sharply reduced numbers of cells transcribed BLV or produced virus. All the infected animals tested by in situ hybridization displayed increased numbers of cells that transcribed BLV RNA, but only two-thirds had large increases of cells that produced infectious BLV in culture. In addition, BLV-transcribing cells exceeded virus-producing cells at most times after infection. These results demonstrate that transient increases of circulating, expression-competent cells characterize the first 3 to 4 months of BLV infection and that the extent of BLV genome expression by cultured mononuclear cells can differ among animals.