Infection of APC by human cytomegalovirus controlled through recognition of endogenous nuclear immediate early protein 1 by specific CD4(+) T lymphocytes

Infections by human CMV are controlled by cellular immune responses. Professional APC such as monocytes and macrophages can be infected in vivo and are considered as a reservoir of virus. However, CMV-specific CD4(+) responses against infected APC have not been reported. To develop a model of CD4-infected APC interaction, we have transfected the U373MG astrocytoma cell line with the class II transactivator (CIITA). Confocal microscopy experiments showed that U373MG-CIITA cells expressed markers characteristic of APC. Functional assays demonstrated that infected U373MG-CIITA APC processed and presented both exogenous and endogenously neosynthesized nuclear immediate early (IE) protein 1 through the MHC class II pathway. More importantly, endogenous presentation of IE1 by infected APC lead to efficient control of CMV infection as revealed by decreased viral titer. Thus, these results describe the endogenous presentation of a nuclear viral protein by the MHC class II pathway and suggest that IE1-specific CD4(+) T cells may play an important role in CMV infection by directly acting against infected APC.     

Hepatitis C virus-like particle morphogenesis

Although much is known about the hepatitis C virus (HCV) genome, first cloned in 1989, little is known about HCV structure and assembly due to the lack of an efficient in vitro culture system for HCV. Using a recombinant Semliki forest virus replicon expressing genes encoding HCV structural proteins, we observed for the first time the assembly of these proteins into HCV-like particles in mammalian cells. This system opens up new possibilities for the investigation of viral morphogenesis and virus-host cell interactions.     

Competition between SsrA tagging and translational termination at weak stop codons in Escherichia coli

SsrA is a tmRNA involved in tagging polypeptides on stalled ribosomes. The resulting fusion proteins are then degraded. We purified endogenous SsrA-tagged proteins by means of a genetically engineered SsrA and identified some of them. Analysis of the proteins suggested that they are tagged at their C-terminal extremities. One of them, ribokinase, is expressed from a messenger with a poorly efficient stop codon, leading to translational recoding events. A change in the ribokinase coding sequence from a weak to a strong translational stop sequence (UGAc to UAAu) annihilated SsrA tagging. Translational termination by UGA recruits the translational release factor (RF) 2. We observed that SsrA tagging of ribokinase was inversely correlated with RF2 activity, revealing a dynamic competition between translational termination and SsrA tagging.     

The concerted conformational changes during human rhinovirus 2 uncoating

Delivery of the rhinovirus genome into the cytoplasm involves a cooperative structural modification of the viral capsid. We have studied this phenomenon for human rhinovirus serotype 2 (HRV2). The structure of the empty capsid has been determined to a resolution of better than 15 A by cryo-electron microscopy, and the atomic structure of native HRV2 was used to examine conformational changes of the capsid. The two proteins around the 5-fold axes make an iris type of movement to open a 10 A diameter channel which allows the RNA genome to exit, and the N terminus of VP1 exits the capsid at the pseudo 3-fold axis. A remarkable modification occurs at the 2-fold axes where the N-terminal loop of VP2 bends inward, probably to detach the RNA.     

Sex and the single (double-strand) break

It has been known for some time that DNA double-strand breaks (DSBs) initiate homologous recombination during meiosis. Two recent studies show that the fate of a single DSB in yeast is strongly influenced by the presence of other breaks in the genome, hinting that cell-wide or chromosome-regional mechanisms control the outcome of DSB repair.     

Oncogenic ras and p53 cooperate to induce cellular senescence

Oncogenic activation of the mitogen-activated protein (MAP) kinase cascade in murine fibroblasts initiates a senescence-like cell cycle arrest that depends on the ARF/p53 tumor suppressor pathway. To investigate whether p53 is sufficient to induce senescence, we introduced a conditional murine p53 allele (p53(val135)) into p53-null mouse embryonic fibroblasts and examined cell proliferation and senescence in cells expressing p53, oncogenic Ras, or both gene products. Conditional p53 activation efficiently induced a reversible cell cycle arrest but was unable to induce features of senescence. In contrast, coexpression of oncogenic ras or activated mek1 with p53 enhanced both p53 levels and activity relative to that observed for p53 alone and produced an irreversible cell cycle arrest that displayed features of cellular senescence. p19(ARF) was required for this effect, since p53(-/-) ARF(-/-) double-null cells were unable to undergo senescence following coexpression of oncogenic Ras and p53. Although the levels of exogenous p53 achieved in ARF-null cells were relatively low, the stabilizing effects of p19(ARF) on p53 could not explain the cooperation between oncogenic Ras and p53 in promoting senescence. Hence, enforced p53 expression without oncogenic ras in p53(-/-) mdm2(-/-) double-null cells produced extremely high p53 levels but did not induce senescence. Taken together, our results indicate that oncogenic activation of the MAP kinase pathway in murine fibroblasts converts p53 into a senescence inducer through both quantitative and qualitative mechanisms.     

Cgamma H2 of Met174 side chain is the site of covalent attachment of a substance P analog photoactivable in position 5

Analogs of substance P (H-RPKPQQFFGLM-NH(2)) incorporating a photoreactive para-benzoyl-l-phenylalanine (p-Bzl)Phe at position 4, 5, 6, 9, or 10 of the sequence have been synthesized and pharmacologically characterized previously as full NK-1 receptor agonists. In this study we show that all analogs, [BAPA(0), (p-Bzl)Phe(x), Met(O(2))(11)]SP also display high yields (40-70%) of NK-1 receptor photolabeling. To identify the site of photoinsertion in the receptor, covalent ligand/receptor complexes were digested with enzymes or chemically cleaved with cyanogen bromide and purified with streptavidin-coated magnetic beads before matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. Only the analog photoreactive at position 5 gave irreversible, reproducible, and unequivocal covalent linkage. Sequential digestions of the covalent complex, substance P analog photoreactive at position 5/NK-1 receptor, with trypsin, endo-GluC and carboxypeptidase Y, led to the identification of the tripeptide (173)TMP(175) in the second extracellular loop of the hNK-1 receptor as the site of photoinsertion. Reaction of cyanogen bromide on the pentapeptide TMPSR did not yield the expected cleavage on the carboxylic side of methionine. The high precision of mass spectrometry analysis on the mass measured led us to determine that C(gamma)H(2) of Met(174) was the site of covalent linkage of the photoreactive substance P analog. Such an insertion (photolinked ligand) on its C(gamma)H(2) renders methionine refractory to CNBr cleavage.