A viral protein that selectively downregulates ICAM-1 and B7-2 and modulates T cell costimulation.

Kaposi's sarcoma-associated (KS-associated) herpesvirus (KSHV) is a B-lymphotropic agent linked to AIDS-related lymphoproliferative disorders and KS. We and others have earlier identified two viral genes, K3 and K5, that encode endoplasmic reticulum proteins that downregulate surface MHC-I chains by enhancing their endocytosis. Here we have examined the ability of these proteins to influence the disposition of other host surface proteins implicated in immune recognition and activation. We report that K5, but not K3, expression in BJAB cells dramatically reduces ICAM-1 and B7-2 surface expression; B7-1 expression is unaffected. This K5-induced reduction can be reversed by coexpression of a dominant negative mutant of dynamin, indicating that the loss of ICAM and B7-2 surface expression is due to their enhanced endocytosis. This downregulation is functionally significant, because K5-transfected B cells show substantial impairment in their ability to induce T cell activation. K5 is thus the first example of a viral modulator of immunological synapse formation and T cell costimulation. We propose that its expression reduces T cell responses to KSHV-infected B cells early in infection, thereby diminishing antiviral cytokine release and the production of stimulatory signals for CTL generation.

A novel class of herpesvirus-encoded membrane-bound E3 ubiquitin ligases regulates endocytosis of proteins involved in immune recognition.

Kaposi's sarcoma-associated herpesvirus encodes two transmembrane proteins (modulator of immune recognition [MIR]1 and MIR2) that downregulate cell surface molecules (MHC-I, B7.2, and ICAM-1) involved in the immune recognition of infected cells. This downregulation results from enhanced endocytosis and subsequent endolysosomal degradation of the target proteins. Here, we show that expression of MIR1 and MIR2 leads to ubiquitination of the cytosolic tail of their target proteins and that ubiquitination is essential for their removal from the cell surface. MIR1 and MIR2 both contain cytosolic zinc fingers of the PHD subfamily, and these structures are required for this activity. In vitro, addition of a MIR2-glutathione S-transferase (GST) fusion protein to purified E1 and E2 enzymes leads to transfer of ubiquitin (Ub) to GST-containing targets in an ATP- and E2-dependent fashion; this reaction is abolished by mutation of the Zn-coordinating residues of the PHD domain. Thus, MIR2 defines a novel class of membrane-bound E3 Ub ligases that modulates the trafficking of host cell membrane proteins.

Kaposi's sarcoma-associated herpesvirus encodes two proteins that block cell surface display of MHC class I chains by enhancing their endocytosis.

Down-regulation of the cell surface display of class I MHC proteins is an important mechanism of immune evasion by human and animal viruses. Herpesviruses in particular encode a variety of proteins that function to lower MHC I display by several mechanisms. These include binding and retention of MHC I chains in the endoplasmic reticulum, dislocation of class I chains from the ER, inhibition of the peptide transporter (TAP) involved in antigen presentation, and shunting of newly assembled chains to lysosomes. Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is a human herpesvirus strongly linked to the development of KS and to certain AIDS-associated lymphoproliferative disorders. Here we show that KSHV encodes two distinctive gene products that function to dramatically reduce cell surface MHC I expression. These viral proteins are localized predominantly to the ER. However, unlike previously described MHC I inhibitors, they do not interfere with the synthesis, translocation, or assembly of class I chains, nor do they retain them in the ER. Rather, they act to enhance endocytosis of MHC I from the cell surface; internalized class I chains are delivered to endolysosomal vesicles, where they undergo degradation. These KSHV proteins define a mechanism of class I down-regulation distinct from the mechanisms of other herpesviruses and are likely to contribute importantly to immune evasion during viral infection.

Molecular mechanism of tumorigenesis in mice transgenic for the human T cell leukemia virus Tax gene.

The infection by human T lymphotropic virus type I is associated with adult T cell leukemia and several inflammatory degenerative disorders, including tropical spastic paraparesis. To investigate the role of the Tax protein in the development of diseases linked to human T lymphotropic virus type I infection, we generated two lines of transgenic mice carrying the tax gene under the control of the viral promoter. The expression of the transgene was low in these mice and was restricted to the central nervous system and testis. Mice from both lines developed various types of tumors, including fibrosarcomas and adenocarcinomas. Tax was expressed at a high level in fibrosarcomas and in cell lines derived from these tumors. In tumor-derived cells, the expression of Tax led to an increased degradation of IkappaB alpha and IkappaB beta and caused stable nuclear translocation of nuclear factor-kappaB. This translocation was essential for cell proliferation, as shown by expressing a nondegradable form of IkappaBbeta in these cells. Therefore, Tax-induced cell transformation in mice correlates with the degradation of IkappaB alpha and IkappaB beta and with the constitutive activation of NF-kappaB.

Infection of choroid plexus cells by human T cell leukaemia virus type I.

Very little is known about the factors that determine the outcome of infection by human T cell leukaemia virus type I (HTLV-I) and the neurotropism of this virus is still a controversial point. In transgenic mice, the HTLV-I LTR is active mainly in the central nervous system (CNS), in parenchyma as well as in ependymal and choroid plexus cells. The latter are of particular interest and could represent the way of entry of the virus into the CNS. In this study we show that primary cultures of sheep choroid plexus can be infected with HTLV-I, leading to characteristic multinucleated syncytial cells containing virus RNA and proteins. HTLV-I p24 Gag protein was detected in the culture medium and the presence of virus particles was observed by electron microscopy 40 days after infection. At this time post-infection HTLV-I could be transmitted to human cord blood cells.

Analysis of the expression directed by two HTLV-I promoters in transgenic mice.

We generated several lines of mice transgenic for the lacZ reporter gene under the control of an HTLV-I LTR. Two different LTR were used; one was isolated from a case of Adult T-cell Leukemia (ATL), the other from a case of Tropical Spastic Paraparesis (TSP/HAM). These LTR differed at 18 nucleotide positions. The pattern of expression of the transgene, studied at the RNA level by RT-PCR, was the same regardless of the origin of the promoter. The beta-galactosidase activity was detected primarily in the central nervous system, in the parenchyma, the choroid plexus and the ependymal cells along the ventricles. In parenchyma, double labelling experiments showed that the cells expressing beta-galactosidase were neurons. These results show that choroid plexus cells and ependymal cells, as well as some neurons, are permissive for the activity of the HTLV-I promoter. The origin of the LTR had not influence on the pattern of expression of the reporter gene.

HTLV-I transgenic models: an overview.

Human T cell leukemia virus type I (HTLV-I) is the agent of a wide spectrum of human diseases. The mechanisms by which a single virus can cause neurodegenerative disorders as well as leukemia is still a matter of debate. Transgenic mice have been used to assess the contribution of different viral elements in viral tropism as well as on cell transformation in vivo. In particular, transgenic models were generated to study the tissue specificity of expression directed by the viral long terminal repeat and the pathological effects induced by the Tax protein of HTLV-I. These models have led to a description of the cell types able to support the viral expression in vivo, and the use of Tax-transgenic mice has demonstrated that this protein is oncogenic and able to induce muscular atrophy and arthropathies. Finally, these models could provide a useful system to study therapeutic approaches for HTLV-I-associated diseases.

A search for human T-cell leukemia virus type I in the lesions of patients with tropical spastic paraparesis and polymyositis.

We searched for the presence of human T-cell leukemia virus type I (HTLV-I) sequences in central nervous system and muscle lesions of 3 patients with tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM) and 3 patients with HTLV-I-associated polymyositis. Proviral DNA coding for the Tax protein was found by polymerase chain reaction amplification in DNA extracted from lesions of every patient with TSP/HAM or HTLV-I-associated polymyositis. In contrast, viral RNA was found occasionally by in situ hybridization in muscle lesions of some patients with polymyositis, but was never found in central nervous system lesions of TSP/HAM patients.

Roles of RNase E, RNase II and PNPase in the degradation of the rpsO transcripts of Escherichia coli: stabilizing function of RNase II and evidence for efficient degradation in an ams pnp rnb mutant.

The Escherichia coli rpsO gene gives rise to different mRNA species resulting either from termination of transcription or from processing of primary transcripts by RNase E and RNase III. The main degradation pathway of these transcripts involves a rate-limiting RNase E cleavage downstream of the structural gene which removes the 3' terminal stem-loop structure of the transcription terminator. This structure protects the message from the attack of 3'-5' exonucleases and its removal results in very rapid degradation of the transcript by polynucleotide phosphorylase and RNase II. Polynucleotide phosphorylase is also able to degrade slowly the mRNA harboring the 3' terminal hairpin of the terminator. In contrast, RNase II appears to protect the rpsO mRNA species which retains the 3' hairpin structure. Rapid degradation of the rpsO mRNA is observed after inactivation of RNase II even in a strain deficient for RNase E and polynucleotide phosphorylase. The enzyme(s) involved in this degradation pathway is not known. We detected an unstable elongated rpsO mRNA presumably resulting from the addition of nucleotides at the 3' end of the transcript.

Localization and biochemical characterization of alfalfa mosaic virus replication complexes.

Replication complexes were isolated from alfalfa mosaic virus-infected tobacco protoplasts. Most of the RNA-synthesizing activity appears to colocalize with the intact chloroplasts upon sucrose-gradient centrifugation of cellular homogenates. Further analysis of these replication complexes showed that the enzyme is strongly associated with the outside of the chloroplasts, the endogenous template being well protected against ribonuclease action. RNA polymerase activity is sensitive to protease treatment of intact chloroplast fraction showing that an essential part of the enzyme complex is facing the in vitro medium, and probably the cytosol in vivo.