Grace Hommel-Berrey

Requirement of caspase-3 for efficient apoptosis induction and caspase-7 activation but not viral replication or cell rounding in cells infected with vesicular stomatitis virus

Infection with vesicular stomatitis virus (VSV), a rhabdovirus and economically significant animal pathogen, was previously demonstrated to induce apoptosis. The mechanism of induction and the role of apoptosis in the VSV-host response have not been completely elucidated. Previous data from our laboratory have suggested that caspase-3 is required for the induction of apoptosis but not viral replication in VSV-infected cells. However, these studies used inhibitors that are selective but not specific for caspase-3. To circumvent this difficulty, we infected both MCF-7 cells which do not express caspases-3 (null), and stable transfectants which express caspase-3 (C3+). When caspase-3 null cells were infected, significant PARP cleavage did not occur, but when C3+ cells were infected, PARP cleavage did occur efficiently. Studies in null and C3+ also suggest that: (1) caspases-3 and -7 are activated sequentially after VSV infection; (2) cell shrinkage and detachment are caspase-3 dependent, but cell rounding is not; and (3) the viral titers were similar between caspase-3 null and C3+ cells suggesting that activation of caspases-3 and -7 are not required for viral replication. Taken together, these results strongly support that the activation of caspase-3 by VSV infection is required for efficient apoptosis induction but not viral replication in vitro. Apoptosis mediated by caspase-3, then, is likely either a host cell response to viral replication or perhaps may be required for in vivo viral replication and spread.

Caspase-3-like proteases are activated by infection but are not required for replication of vesicular stomatitis virus.

Infection with vesicular stomatitis virus (VSV), the prototype rhabdovirus, causes apoptotic DNA fragmentation, but the role of apoptosis in the VSV-host interaction remains unclear. Apoptosis is the gene-regulated mechanism triggered by a wide variety of stimuli that lead to cell death in a choreographed manner. In the present study, infection of the Jurkat T cell line with VSV led to activation of caspase-3 and caspase-7, with subsequent apoptotic events involving poly (ADP ribose) polymerase (PARP) cleavage, DNA fragmentation, and membrane damage. Caspase activation was correlated with viral protein expression suggesting a link between viral replication and apoptosis. We hypothesized that VSV replication might depend on apoptosis and that the inhibition of apoptosis would lead to significant decreases in viral titers. When various inhibitors of apoptosis in VSV-infected cells were used, PARP cleavage and DNA fragmentation were inhibited but the production of infectious progeny was not affected. In addition, we demonstrated that the activation of caspase-3-like proteases is required for VSV-induced apoptosis but not in vitro viral replication. Apoptosis following VSV infection is likely to be either a host-cell attempt to control viral replication or may be a ploy used by the virus to facilitate its in vivo replication and spread.

Fas-mediated cytotoxicity induces degradation of vesicular stomatitis virus RNA transcripts and reduces viral titer

Several investigators have recently examined the effect of Fas (CD95)-mediated apoptotic cell death on target cells (TC). The effect of Fas-mediated death on viral RNA within the TC, however, has not been explored. In this study, we investigated the ability of the Fas pathway to mediate pre-lytic degradation of vesicular stomatitis virus (VSV) RNA and TC RNA. We show that engagement of Fas antigen on VSV-infected Jurkat cells induces pre-lytic degradation of VSV RNA transcripts, whereas full-length VSV genome RNA, known to be tightly associated with viral proteins, is not degraded. Cellular RNA, including beta-actin and glyceraldehyde-3-phosphate-dehydrogenase mRNAs, is also degraded by Fas-mediated cytotoxicity. In addition, Fas-mediated cytotoxicity reduced the yield of VSV plaque-forming units (PFU) from Jurkat by an average of 82.0%. An anti-Fas blocking Ab inhibited the RNA degradation and restored the number of VSV PFU to near control levels. These data indicate that the Fas lytic pathway could play a role in the elimination of viruses through degradation of intracellular viral RNA. reserved

Relevance of soluble cytotoxic factors generated by mycoplasma-contaminated targets to natural killer cell-mediated killing

The possible involvement of soluble cytotoxic factors (SCF) in the lytic mechanism of natural killer (NK) cell-mediated cytotoxicity (CMC) was investigated. Tumor target cells (TC) were examined for their ability to stimulate the release of SCF from human peripheral blood lymphocytes (PBL). SCF were detected when effector cells (EC) were stimulated with mycoplasma-contaminated (MC)-TC and with concanavalin A (Con A), but mycoplasma-free (MF)-TC were unable to induce SCF. Pretreatment of EC with beta-interferon augmented the production of SCF with MC-K562 and Con A, but not with MF-K562. However, MF-K562 once infected with the culture supernatant from MC-K562, as well as mycoplasmas concentrated from culture supernatant, were able to induce SCF. Only Con A was capable of generating cytotoxic supernatants from EC that had been inactivated with MF-K562 and from EC depleted of Leu 11b positive cells. Moreover, when EC were evaluated for NK activity after coculture with MF-K562, their ability to kill fresh MF-K562 in the standard NK-CMC assay was greatly reduced. This loss of lytic potential was not accompanied by SCF release. Collectively, these data suggest that mycoplasma organisms play a crucial role in the induction of human SCF, and the implication that SCF, including NK cytotoxic factors (NKCF), are the lytic mediators in NK-CMC needs to be re-evaluated.

Identification of a novel HLA-DQB1 allele, DQB1*05:19, in an African American family by sequence-based typing.

The novel allele, DQB1*05:19, differs from the closest matching allele, DQB1*05:02:01, by one nucleotide substitution.

Identification of a novel HLA‐DRB1 allele, DRB1*09:20, by sequence‐based typing in an unrelated bone marrow donor

The novel allele HLA-DRB1*09:20 differs from HLA-DRB1*09:01:02 by one nucleotide substitution at codon 9 (AAG->CAG) and differs from HLA-DRB1*09:07 by one nucleotide substitution at codon 13 (TAT->TTT).

Target cell-directed degradation of perforin mRNA in CTL: Lack of correlation with loss of protein and lytic ability

We have previously shown that CTL and NK cells rapidly down regulate perforin mRNA and become functionally inactive within 4-6 hr after exposure to sensitive target cells (TC). We report here for the first time that CTL also down regulate perforin mRNA upon exposure to resistant, but binding, TC. When three separate human MHC-restricted CTL lines were exposed to resistant TC, perforin mRNA was rapidly degraded. Removal of both extracellular Ca++ and Mg++ prevented perforin message down regulation, whereas removal of Ca++ alone did not, indicating that CTL:TC binding was required. Unlike the response of CTL exposed to sensitive TC, resistant TC did not trigger serine esterase (SE) release, suggesting distinct signalling pathways for perforin mRNA down regulation and granule exocytosis. Moreover, using western analysis, we showed that there was limited (< 10%) perforin protein release after CTL:TC interaction, suggesting that CTL loss of lytic activity after exposure to sensitive TC is not due to massive depletion of perforin. Treatment of CTL with mAb to CD2, CD3, CD2 + CD3, CD8, Class I and LFA-1 did not induce perforin mRNA down regulation. Furthermore, mAb to CD2, CD3, CD8, Class I, Class II, CD54 and LFA-1 did not block TC-mediated perforin mRNA down regulation although lysis of TC was inhibited.

Inhibition of Banzi Viral RNA Synthesis in BHK-21 Cells by Banzi Virion Matrix Protein

Treatment of BHK-21 cells with purified matrix (M) protein isolated from Banzi virions resulted in decreased production of Banzi virus and in reduced levels of virus-specific positive-strand and negative-strand RNA. The M protein had no effect on the uncoating of the viral genome. This inhibition was virus-specific since the replication of unrelated togaviruses in BHK-21 cells was not affected. Purified M protein inhibited the in vitro activity of the Banzi viral RNA polymerase; prior treatment of the M protein with either trypsin or antiviral serum blocked this inhibition.