Deletion of multiple immediate-early genes from herpes simplex virus reduces cytotoxicity and permits long-term gene expression in neurons.
Herpes simplex virus type 1 (HSV-1) has many attractive features that suggest its utility for gene transfer to neurons. However, viral cytotoxicity and transient transgene expression limit practical applications even in the absence of viral replication. Mutant viruses deleted for the immediate early (IE) gene, ICP4, an essential transcriptional transactivator, are toxic to many cell types in culture in which only the remaining IE genes are expressed. In order to test directly the toxicity of other IE gene products in neurons and develop a mutant background capable of longterm transgene expression, we generated mutants deleted for multiple IE genes in various combinations and tested their relative cytotoxicity in 9L rat gliosarcoma cells, Vero monkey kidney cells, and primary rat cortical and dorsal root neurons in culture. Viral mutants deleted simultaneously for the IE genes encoding ICP4, ICP22 and ICP27 showed substantially reduced cytotoxicity compared with viruses deleted for ICP4 alone or ICP4 in combination with either ICP22, ICP27 or ICP47. Infection of neurons in culture with these triple IE deletion mutants substantially enhanced cell survival and permitted transgene expression for over 21 days. Such mutants may prove useful for efficient gene transfer and extended transgene expression in neurons in vitro and in vivo. (+info)
Rubella virus-induced apoptosis varies among cell lines and is modulated by Bcl-XL and caspase inhibitors.
Rubella virus (RV) causes multisystem birth defects in the fetuses of infected women. To investigate the cellular basis of this pathology, we examined the cytopathic effect of RV in three permissive cell lines: Vero 76, RK13, and BHK21. Electron microscopy and the TUNEL assay showed that the cytopathic effect resulted from RV-induced programmed cell death (apoptosis) in all three cell lines, but the extent of apoptosis varied among these cells. At 48 h postinfection, the RK13 cell line showed the greatest number of apoptotic cells, the Vero 76 cell line was approximately 3-fold less, and BHK21 had very few. An increased multiplicity of infection and longer time postinfection were required for the BHK21 cell line to reach the level of apoptotic cells in Vero 76 at 48 h. Purified RV induced apoptosis in a dose-dependent fashion, but not UV-inactivated RV or virus-depleted culture supernatant. Specific inhibitors of the apoptosis-specific proteases caspases reduced RV-induced apoptosis and led to higher levels of RV components in infected cells. To address the role of regulatory proteins in RV-induced apoptosis, the antiapoptotic gene Bcl-2 or Bcl-XL was transfected into RK13 cells. Although a high level of Bcl-2 family proteins was expressed, no protection was observed from apoptosis induced by RV, Sindbis virus, or staurosporine in RK13 cells. In BHK21 cells, however, increased expression of Bcl-XL protected cells from apoptosis. The observed variability in apoptotic response to RV of these cell lines demonstrates that programmed cell death is dependent on the unique properties of each cell and may be indicative of how selective organ damage occurs in a congenital rubella syndrome fetus. (+info)
CLIP-170 highlights growing microtubule ends in vivo.
A chimera with the green fluorescent protein (GFP) has been constructed to visualize the dynamic properties of the endosome-microtubule linker protein CLIP170 (GFP-CLIP170). GFP-CLIP170 binds in stretches along a subset of microtubule ends. These fluorescent stretches appear to move with the growing tips of microtubules at 0.15-0.4 microm/s, comparable to microtubule elongation in vivo. Analysis of speckles along dynamic GFP-CLIP170 stretches suggests that CLIP170 treadmills on growing microtubule ends, rather than being continuously transported toward these ends. Drugs affecting microtubule dynamics rapidly inhibit movement of GFP-CLIP170 dashes. We propose that GFP-CLIP170 highlights growing microtubule ends by specifically recognizing the structure of a segment of newly polymerized tubulin. (+info)
Susceptibilities of Mycobacterium tuberculosis and Mycobacterium avium complex to lipophilic deazapteridine derivatives, inhibitors of dihydrofolate reductase.
Twelve lipophilic 2,4-diamino-5-methyl-5-deazapteridine derivatives and trimethoprim were evaluated for activity against Mycobacterium tuberculosis and Mycobacterium avium in vitro. Six of the compounds had MICs of < or =12.8 mg/L and < or =1.28 mg/L against M. tuberculosis and M. avium, respectively; trimethoprim MICs were >128 mg/L and >12.8 but < or =128 mg/L, respectively. Two compounds, with either a 2-methyl-5-methoxy phenyl or 2-methoxy-5-trifluoromethyl phenyl linked at the 6-position of the deazapteridine moiety by a CH2NH bridge, had MICs of < or =0.13 mg/L against M. avium; the two compounds also had apparent I50 values for dihydrofolate reductase of 2 and 8 nM, respectively, compared with an I50 of 400 nM with trimethoprim. Four of the compounds were selectively toxic to mycobacteria as compared with Vero cells. These results demonstrated that lipophilic antifolates can be synthesized which are more active against mycobacteria than trimethoprim and which possess selective toxicity. (+info)
Mutations in the retinoblastoma protein-binding LXCXE motif of rubella virus putative replicase affect virus replication.
The rubella virus (RV)-encoded protein NSP90, which contains the retinoblastoma protein (Rb)-binding motif LXCXE, interacts with Rb and RV replication is reduced in cells lacking Rb. Whether the LXCXE motif of RV NSP90 itself is essential for Rb binding and virus replication is not known. Therefore, in the present study, the functional role of this motif was investigated by site-directed mutagenesis in a plasmid from which infectious RV RNA can be produced. Three critical mutations in the motif, two substitutions at the conserved cysteine residue (C --> G and C --> R) and a deletion of the entire motif, were created. A cell-free translated NSP90 C terminus polypeptide containing the deletion did not bind to Rb and a polypeptide carrying the C --> R substitution had barely detectable binding affinity for Rb. Rb binding by the C --> G mutant was reduced significantly compared to that of wild-type protein. Correlating with the binding results, mutant viruses containing the LXRXE and LXGXE motifs had a reduction in replication to < 0.5% and 47% of the wild-type, respectively, while deletion of the motif was found to be lethal. By the first serial passage, replication of the LXRXE-carrying virus had increased from < 0.5% to 2% of the wild-type. Sequencing of the genome of this virus revealed a nucleotide change that altered the motif from LXRXE to LXSXE, which is a known Rb-binding motif in two protein phosphatase subunits. Thus, our results clearly demonstrate that the LXCXE motif is required for efficient RV replication. (+info)
Characterization of the L gene and 5' trailer region of Ebola virus.
The nucleotide sequences of the L gene and 5' trailer region of Ebola virus strain Mayinga (subtype Zaire) have been determined, thus completing the sequence of the Ebola virus genome. The putative transcription start signal of the L gene was identical to the determined 5' terminus of the L mRNA (5' GAGGAAGAUUAA) and showed a high degree of similarity to the corresponding regions of other Ebola virus genes. The 3' end of the L mRNA terminated with 5' AUUAUAAAAAA, a sequence which is distinct from the proposed transcription termination signals of other genes. The 5' trailer sequence of the Ebola virus genomic RNA consisted of 676 nt and revealed a self-complementary sequence at the extreme end which may play an important role in virus replication. The L gene contained a single ORF encoding a polypeptide of 2212 aa. The deduced amino acid sequence showed identities of about 73 and 44% to the L proteins of Ebola virus strain Maleo (subtype Sudan) and Marburg virus, respectively. Sequence comparison studies of the Ebola virus L proteins with several corresponding proteins of other non-segmented, negative-strand RNA viruses, including Marburg viruses, confirmed a close relationship between filoviruses and members of the Paramyxovirinae. The presence of several conserved linear domains commonly found within L proteins of other members of the order Mononegavirales identified this protein as the RNA-dependent RNA polymerase of Ebola virus. (+info)
The herpes simplex virus type 1 regulatory protein ICP27 is required for the prevention of apoptosis in infected human cells.
The herpes simplex virus type 1 (HSV-1) ICP27 protein is an immediate-early or alpha protein which is essential for the optimal expression of late genes as well as the synthesis of viral DNA in cultures of Vero cells. Our specific goal was to characterize the replication of a virus incapable of synthesizing ICP27 in cultured human cells. We found that infection with an HSV-1 ICP27 deletion virus of at least three separate strains of human cells did not produce immediate-early or late proteins at the levels observed following wild-type virus infections. Cell morphology, chromatin condensation, and genomic DNA fragmentation measurements demonstrated that the human cells died by apoptosis after infection with the ICP27 deletion virus. These features of the apoptosis were identical to those which occur during wild-type infections of human cells when total protein synthesis has been inhibited. Vero cells infected with the ICP27 deletion virus did not exhibit any of the features of apoptosis. Based on these results, we conclude that while HSV-1 infection likely induced apoptosis in all cells, viral evasion of the response differed among the cells tested in this study. (+info)
Glycoprotein gL-independent infectivity of pseudorabies virus is mediated by a gD-gH fusion protein.
Envelope glycoproteins gH and gL, which form a complex, are conserved throughout the family Herpesviridae. The gH-gL complex is essential for the fusion between the virion envelope and the cellular cytoplasmic membrane during penetration and is also required for direct viral cell-to-cell spread from infected to adjacent noninfected cells. It has been proposed for several herpesviruses that gL is required for proper folding, intracellular transport, and virion localization of gH. In pseudorabies virus (PrV), glycoprotein gL is necessary for infectivity but is dispensable for virion localization of gH. A virus mutant lacking gL, PrV-DeltagLbeta, is defective in entry into target cells, and direct cell-to-cell spread is drastically reduced, resulting in only single or small foci of infected cells (B. G. Klupp, W. Fuchs, E. Weiland, and T. C. Mettenleiter, J. Virol. 71:7687-7695, 1997). We used this limited cell-to-cell spreading ability of PrV-DeltagLbeta for serial passaging of cells infected with transcomplemented virus by coseeding with noninfected cells. After repeated passaging, plaque formation was restored and infectivity in the supernatant was observed. One single-plaque isolate, designated PrV-DeltagLPass, was further characterized. To identify the mutation leading to this gL-independent infectious phenotype, Southern and Western blot analyses, radioimmunoprecipitations, and DNA sequencing were performed. The results showed that rearrangement of a genomic region comprising part of the gH gene into a duplicated copy of part of the unique short region resulted in a fusion fragment predicted to encode a protein consisting of the N-terminal 271 amino acids of gD fused to the C-terminal 590 residues of gH. Western blotting and radioimmunoprecipitation with gD- and gH-specific antibodies verified the presence of a gDH fusion protein. To prove that this fusion protein mediates infectivity of PrV-DeltagLPass, cotransfection of PrV-DeltagLbeta DNA with the cloned fusion fragment was performed, and a cell line, Nde-67, carrying the fusion gene was established. After cotransfection, infectious gL-negative PrV was recovered, and propagation of PrV-DeltagLbeta on Nde-67 cells produced infectious virions. Thus, a gDH fusion polypeptide can compensate for function of the essential gL in entry and cell-to-cell spread of PrV. (+info)