Replication of viral RNA: RNA synthetase from Escherichia coli infected with phage MS2 or Qb

An RNA synthetase is formed in Escherichia coli after infection with bacteriophages containing RNA. Specific annealing techniques revealed that, from the very outset of the reaction in vitro, the partially purified enzyme-template complex synthesizes parental-type plus strands, namely, MS2-RNA when isolated from cells infected with MS2 phage and Q(beta)-RNA in the case of cells infected with Q(beta) phage. This is in contrast to the situation found with Q(beta) replicase primed with Q(beta)-RNA, where the initial product is the complementary strand.     

Detection of human cytomegalovirus in peripheral blood lymphocytes in a natural infection

In situ hybridization was used to detect human cytomegalovirus (HCMV) in the peripheral blood mononuclear cells of some naturally infected (seropositive) individuals. A subpopulation of cells hybridized specifically to a portion of the HCMV genome that is heavily transcribed during the immediate-early period of infection. The hybridization signal was markedly reduced by base hydrolysis and ribonuclease, and therefore the probe appears to be detecting viral RNA. A fluorescence-activated cell sorter was used to select lymphocytes bearing the OKT4 and OKT8 markers. Hybridization with the HCMV probe revealed a higher proportion of positive cells in the OKT4 than in the OKT8 subset. This observation specifically identifies lymphocytes as a cell population involved in natural HCMV infection and suggests that lymphocytes may be a reservoir for maintaining infection and may also serve as a vehicle for its spread by blood transfusion.     

Host immune system gene targeting by a viral miRNA

Virally encoded microRNAs (miRNAs) have recently been discovered in herpesviruses. However, their biological roles are mostly unknown. We developed an algorithm for the prediction of miRNA targets and applied it to human cytomegalovirus miRNAs, resulting in the identification of the major histocompatibility complex class I-related chain B (MICB) gene as a top candidate target of hcmv-miR-UL112. MICB is a stress-induced ligand of the natural killer (NK) cell activating receptor NKG2D and is critical for the NK cell killing of virus-infected cells and tumor cells. We show that hcmv-miR-UL112 specifically down-regulates MICB expression during viral infection, leading to decreased binding of NKG2D and reduced killing by NK cells. Our results reveal a miRNA-based immunoevasion mechanism that appears to be exploited by human cytomegalovirus.     

Surface expression of HLA-E, an inhibitor of natural killer cells, enhanced by human cytomegalovirus gpUL40

The nonclassical major histocompatibility complex (MHC) class I molecule HLA-E inhibits natural killer (NK) cell-mediated lysis by interacting with CD94/NKG2A receptors. Surface expression of HLA-E depends on binding of conserved peptides derived from MHC class I molecules. The same peptide is present in the leader sequence of the human cytomegalovirus (HCMV) glycoprotein UL40 (gpUL40). It is shown that, independently of the transporter associated with antigen processing, gpUL40 can up-regulate expression of HLA-E, which protects targets from NK cell lysis. While classical MHC class I molecules are down-regulated, HLA-E is up-regulated by HCMV. Induction of HLA-E surface expression by gpUL40 may represent an escape route for HCMV.     

Regulation of CD8+ T cell development by thymus-specific proteasomes

Proteasomes are responsible for generating peptides presented by the class I major histocompatibility complex (MHC) molecules of the immune system. Here, we report the identification of a previously unrecognized catalytic subunit called beta5t. beta5t is expressed exclusively in cortical thymic epithelial cells, which are responsible for the positive selection of developing thymocytes. Although the chymotrypsin-like activity of proteasomes is considered to be important for the production of peptides with high affinities for MHC class I clefts, incorporation of beta5t into proteasomes in place of beta5 or beta5i selectively reduces this activity. We also found that beta5t-deficient mice displayed defective development of CD8(+) T cells in the thymus. Our results suggest a key role for beta5t in generating the MHC class I-restricted CD8(+) T cell repertoire during thymic selection.     

Requirement for positive selection of gamma delta receptor-bearing T cells.

The alpha beta and gamma delta T cell receptors for antigen (TCR) delineate distinct T cell populations. TCR alpha beta-bearing thymocytes must be positively selected by binding of the TCR to major histocompatibility complex (MHC) molecules on thymic epithelium. To examine the requirement for positive selection of TCR gamma delta T cells, mice bearing a class I MHC-specific gamma delta transgene (Tg) were crossed to mice with disrupted beta 2 microglobulin (beta 2M) genes. The Tg+beta 2M- (class I MHC-) offspring had Tg+ thymocytes that did not proliferate to antigen or Tg-specific monoclonal antibody and few peripheral Tg+ cells. This is evidence for positive selection within the gamma delta T cell subset.     

The role of beta 2-microglobulin in peptide binding by class I molecules

Efficient transport of class I major histocompatibility complex molecules to the cell surface requires association of the class I heavy chain with endogenous peptide and the class I light chain, beta 2-microglobulin (beta 2M). A mutant cell line deficient in beta 2M transports low amounts of nonpeptide-associated heavy chains to the cell surface that can associate with exogenously provided beta 2M and synthetic peptide antigens. Normal beta 2M-sufficient cells grown in serum-free media devoid of beta 2M also require an exogenous source of beta 2M to efficiently bind synthetic peptide. Thus, class I molecules on normal cells do not spontaneously bind or exchange peptides.     

Human cytomegalovirus directly induces the antiviral protein viperin to enhance infectivity

Viperin is an interferon-inducible protein that is directly induced in cells by human cytomegalovirus (HCMV) infection. Why HCMV would induce viperin, which has antiviral activity, is unknown. We show that HCMV-induced viperin disrupts cellular metabolism to enhance the infectious process. Viperin interaction with the viral protein vMIA resulted in viperin relocalization from the endoplasmic reticulum to the mitochondria. There, viperin interacted with the mitochondrial trifunctional protein that mediates β-oxidation of fatty acids to generate adenosine triphosphate (ATP). This interaction with viperin, but not with a mutant lacking the viperin iron-sulfur cluster-binding motif, reduced cellular ATP generation, which resulted in actin cytoskeleton disruption and enhancement of infection. This function of viperin, which was previously attributed to vMIA, suggests that HCMV has coopted viperin to facilitate the infectious process.     

Crystal structure of Thermotoga maritima ribosome recycling factor: a tRNA mimic

Ribosome recycling factor (RRF), together with elongation factor G (EF-G), catalyzes recycling of ribosomes after one round of protein synthesis. The crystal structure of RRF was determined at 2.55 angstrom resolution. The protein has an unusual fold where domain I is a long three-helix bundle and domain II is a three-layer beta/alpha/beta sandwich. The molecule superimposes almost perfectly with a transfer RNA (tRNA) except that the amino acid-binding 3' end is missing. The mimicry suggests that RRF interacts with the posttermination ribosomal complex in a similar manner to a tRNA, leading to disassembly of the complex. The structural arrangement of this mimicry is entirely different from that of other cases of less pronounced mimicry of tRNA so far described.     

Systemic leukocyte-directed siRNA delivery revealing cyclin D1 as an anti-inflammatory target

Cyclin D1 (CyD1) is a pivotal cell cycle-regulatory molecule and a well-studied therapeutic target for cancer. Although CyD1 is also strongly up-regulated at sites of inflammation, its exact roles in this context remain uncharacterized. To address this question, we developed a strategy for selectively silencing CyD1 in leukocytes in vivo. Targeted stabilized nanoparticles (tsNPs) were loaded with CyD1-small interfering RNA (siRNA). Antibodies to beta(7) integrin (beta(7) I) were then used to target specific leukocyte subsets involved in gut inflammation. Systemic application of beta(7) I-tsNPs silenced CyD1 in leukocytes and reversed experimentally induced colitis in mice by suppressing leukocyte proliferation and T helper cell 1 cytokine expression. This study reveals CyD1 to be a potential anti-inflammatory target, and suggests that the application of similar modes of targeting by siRNA may be feasible in other therapeutic settings.     

Influenza virus effects on cell membrane proteins

During infection by influenza virus, viral proteins become firmly attached to (or part of) host cell plasma membrane, nuclear membranes, mitochondrial, and microsomal membranes. Purified virus particles contain less than I percent host cell protein. Virus envelope proteins completely replace host membrane proteins in those discrete spots on the plasma membrane from which progeny virions bud out.     

Direct recognition of cytomegalovirus by activating and inhibitory NK cell receptors

Natural killer (NK) cells express inhibitory receptors for major histocompatibility complex (MHC) class I antigens, preventing attack against healthy cells. Mouse cytomegalovirus (MCMV) encodes an MHC-like protein (m157) that binds to an inhibitory NK cell receptor in certain MCMV-susceptible mice. In MCMV-resistant mice, this viral protein engages a related activating receptor (Ly49H) and confers host protection. These activating and inhibitory receptors are highly homologous, suggesting the possibility that one evolved from the other in response to selective pressure imposed by the pathogen.     

Viral persistence in neurons explained by lack of major histocompatibility class I expression

Viruses frequently persist in neurons, suggesting that these cells can evade immune surveillance. In a mouse model, 5 x 10(6) cytotoxic T lymphocytes (CTLs), specific for lymphocytic choriomeningitis virus (LCMV), did not lyse infected neurons or cause immunopathologic injury. In contrast, intracerebral injection of less than 10(3) CTL caused disease and death when viral antigens were expressed on leptomeningeal and choroid plexus cells of the nervous system. The neuronal cell line OBL21 expresses little or no major histocompatibility (MHC) class I surface glycoproteins and when infected with LCMV, resisted lysis by virus-specific CTLs. Expression of MHC heavy chain messenger RNA was limited, but beta 2-microglobulin messenger RNA and protein was made normally. OBL21 cells were made sensitive to CTL lysis by transfection with a fusion gene encoding another MHC class I molecule. Hence, neuronal cells probably evade immune surveillance by failing to express MHC class I molecules.     

Activation of polyphosphoinositide hydrolysis in T cells by H-2 alloantigen but not MLS determinants

Murine minor lymphocyte-stimulating (Mls) determinants are cell surface antigens that stimulate strong primary T cell responses; the responding T cells display restricted T cell receptor (TCR) V beta gene usage. Interaction of T cells with mitogens or major histocompatibility complex (MHC) antigens activated the polyphosphoinositide (PI) signaling pathway, but this pathway was not triggered by Mls recognition. However, interleukin-2 (IL-2) secretion and proliferation to all three stimuli were comparable. Thus, although recognition of both allo-H-2 and Mls determinants is thought to be mediated by the TCR, these antigens appear to elicit biochemically distinct signal transduction pathways.     

Functional delivery of a cytosolic tRNA into mutant mitochondria of human cells

Many maternally inherited and incurable neuromyopathies are caused by mutations in mitochondrial (mt) transfer RNA (tRNA) genes. Kinetoplastid protozoa, including Leishmania, have evolved specialized systems for importing nucleus-encoded tRNAs into mitochondria. We found that the Leishmania RNA import complex (RIC) could enter human cells by a caveolin-1-dependent pathway, where it induced import of endogenous cytosolic tRNAs, including tRNA(Lys), and restored mitochondrial function in a cybrid harboring a mutant mt tRNA(Lys) (MT-TK) gene. The use of protein complexes to modulate mitochondrial function may help in the management of such genetic disorders.