Leporipoxvirus Cu,Zn-superoxide dismutase (SOD) homologs are catalytically inert decoy proteins that bind copper chaperone for SOD

Many Chordopoxviruses encode catalytically inactive homologs of cellular Cu-Zn superoxide dismutase (SOD). The biological function of these proteins is unknown, although the proteins encoded by Leporipoxviruses have been shown to promote a slow decline in the level of superoxide dismutase activity in virus-infected cells. To gain more insights into their function, we have further characterized the enzymatic and biochemical properties of a SOD homolog encoded by Shope fibroma virus. Shope fibroma virus SOD has retained the zinc binding properties of its cellular homolog, but cannot bind copper. Site-directed mutagenesis showed that it requires at least four amino acid substitutions to partially restore copper binding activity, but even these changes still did not restore catalytic activity. Reciprocal co-immunoprecipitation experiments showed that recombinant Shope fibroma virus SOD forms very stable complexes with cellular copper chaperones for SOD and these observations were confirmed using glutathione-S-transferase tagged proteins. Similar viral SOD/chaperone complexes were formed in cells infected with a closely related myxoma virus, where we also noted that some of the SOD antigen co-localizes with mitochondrial markers using confocal fluorescence microscopy. About 2% of the viral SOD was subsequently detected in gradient-purified mitochondria extracted from virus-infected cells. These poxviral SOD homologs do not form stable complexes with cellular Cu,Zn-SOD or affect its concentration. We suggest that Leporipoxvirus SOD homologs are catalytically inert decoy proteins that are designed to interfere in the proper metallation and activation of cellular Cu,Zn-SOD. This reaction might be advantageous for tumorigenic poxviruses, since higher levels of superoxide have been proposed to have anti-apoptotic and tumorigenic activity.     

A Human Rotavirus with Rearranged Genes 7 and 11 Encodes a Modified NSP3 Protein and Suggests an Additional Mechanism for Gene Rearrangement

A human rotavirus (isolate M) with an atypical electropherotype with 14 apparent bands of double-stranded RNA was isolated from a chronically infected immunodeficient child. MA-104 cell culture adaptation showed that the M isolate was a mixture of viruses containing standard genes (M0) or rearranged genes: M1 (containing a rearranged gene 7) and M2 (containing rearranged genes 7 and 11). The rearranged gene 7 of virus M1 (gene 7R) was very unusual because it contained two complete open reading frames (ORF). Moreover, serial propagation of virus M1 in cell culture indicated that gene 7R rapidly evolved, leading to a virus with a deleted gene 7R (gene 7RDelta). Gene 7RDelta coded for a modified NSP3 protein (NSP3m) of 599 amino acids (aa) containing a repetition of aa 8 to 296. The virus M3 (containing gene 7RDelta) was not defective in cell culture and actually produced NSP3m. The rearranged gene 11 (gene 11R) had a more usual pattern, with a partial duplication leading to a normal ORF followed by a long 3' untranslated region. The rearrangement in gene 11R was almost identical to some of those previously described, suggesting that there is a hot spot for gene rearrangements at a specific location on the sequence. It has been suggested that in some cases the existence of short direct repeats could favor the occurrence of rearrangement at a specific site. The computer modeling of gene 7 and 11 mRNAs led us to propose a new mechanism for gene rearrangements in which secondary structures, besides short direct repeats, might facilitate and direct the transfer of the RNA polymerase from the 5' to the 3' end of the plus-strand RNA template during the replication step.     

Decreased intracellular superoxide levels activate Sindbis virus-induced apoptosis

Infection of many cultured cell types with Sindbis virus (SV), an alphavirus, triggers apoptosis through a commonly utilized caspase activation pathway. However, the upstream signals by which SV activates downstream apoptotic effectors, including caspases, remain unclear. Here we report that in AT-3 prostate carcinoma cells, SV infection decreases superoxide (O-2) levels within minutes of infection as monitored by an aconitase activity assay. This SV-induced decrease in O-2 levels appears to activate or modulate cell death, as a recombinant SV expressing the O-2 scavenging enzyme, copper/zinc superoxide dismutase (SOD), potentiates SV-induced apoptosis. A recombinant SV expressing a mutant form of SOD, which has reduced SOD activity, has no effect. The potentiation of SV-induced apoptosis by wild type SOD is because of its ability to scavenge intracellular O-2 rather than its ability to promote the generation of hydrogen peroxide. Pyruvate, a peroxide scavenger, does not affect the ability of wild type SOD to potentiate cell death; and increasing the intracellular catalase activity via a recombinant SV vector has no effect on SV-induced apoptosis. Moreover, increasing intracellular O-2 by treatment of 3T3 cells with paraquat protects them from SV-induced death. Altogether, our results suggest that SV may activate apoptosis by reducing intracellular superoxide levels and define a novel redox signaling pathway by which viruses can trigger cell death.     

Mechanism of pock formation by Shope fibroma virus on monolayers of rabbit cells

Israeli, Ella (Rambam Hospital, Haifa, Israel). Mechanism of pock formation by Shope fibroma virus on monolayers of rabbit cells. J. Bacteriol. 92:727-732. 1966.-The mechanism of pock formation by the Shope fibroma virus (SFV) on rabbit cultures in vitro was studied with the use of p-fluorophenylalanine, 5-bromodeoxyuridine, and 5-iododeoxyuridine. The inhibitors were used to inhibit, and to initiate, virus replication at different times after infection. It was shown that pock formation required virus replication to a threshold value of 25 plaque-forming units per pock area, and that this amount of virus can be accumulated during a period about 3 days less than that required for pock formation. Inhibition of virus growth, and of cell multiplication, after this threshold has been reached, did not prevent pock development. A delay in the onset of virus growth required to reach the threshold virus content, caused an about equivalent delay in the time of pock formation. In the absence of inhibitors, pocks were not formed after infection of 84 rabbit embryo clones, or five mixtures of clones containing five to seven clones each. The results indicate that pock formation by SFV in vitro was the result of cell aggregation, and not of cell multiplication, in special types of cells.     

The Vaccinia Virus Superoxide Dismutase-Like Protein (A45R) Is a Virion Component That Is Nonessential for Virus Replication

A characterization of the A45R gene from vaccinia virus (VV) strain Western Reserve is presented. The open reading frame is predicted to encode a 125-amino-acid protein (M(r), of 13,600) with 39% amino acid identity to copper-zinc superoxide dismutase (Cu-Zn SOD). Sequencing of the A45R gene from other orthopoxviruses, here and by others, showed that the protein is highly conserved in all viruses sequenced, including 16 strains of VV, 2 strains of cowpox virus, camelpox virus, and 4 strains of variola virus. In all cases the protein lacks key residues involved in metal ion binding that are important for the catalytic activity. The A45R protein was expressed in Escherichia coli, purified, and tested for SOD activity, but neither enzymatic nor inhibitory SOD activity was detected. Additionally, no virus-encoded SOD activity was detected in infected cells or purified virions. A monoclonal antibody raised against the A45R protein expressed in E. coli identified the A45R gene product as a 13.5-kDa protein that is expressed late during VV infection. Confocal microscopy of VV-infected cells indicated that the A45R protein accumulated predominantly in cytoplasmic viral factories. Electron microscopy and biochemical analyses showed that the A45R protein is incorporated into the virion core. A deletion mutant lacking the majority of the A45R gene and a revertant virus in which the deleted gene was restored were constructed and characterized. The growth properties of the deletion mutant virus were indistinguishable from those of wild-type and revertant viruses in all cell lines tested, including macrophages. Additionally, the virulence and pathogenicity of the three viruses were also comparable in murine and rabbit models of infection. A45R is unusual in being the first VV core protein described that affects neither virus replication nor virulence.     

A27L Protein Mediates Vaccinia Virus Interaction with Cell Surface Heparan Sulfate

Vaccinia virus has a wide host range and infects mammalian cells of many different species. This suggests that the cell surface receptors for vaccinia virus are ubiquitously expressed and highly conserved. Alternatively, different receptors are used for vaccinia virus infection of different cell types. Here we report that vaccinia virus binds to heparan sulfate, a glycosaminoglycan (GAG) side chain of cell surface proteoglycans, during virus infection. Soluble heparin specifically inhibits vaccinia virus binding to cells, whereas other GAGs such as condroitin sulfate or dermantan sulfate have no effect. Heparin also blocks infections by cowpox virus, rabbitpox virus, myxoma virus, and Shope fibroma virus, suggesting that cell surface heparan sulfate could be a general mediator of the entry of poxviruses. The biochemical nature of the heparin-blocking effect was investigated. Heparin analogs that have acetyl groups instead of sulfate groups also abolish the inhibitory effect, suggesting that the negative charges on GAGs are important for virus infection. Furthermore, BSC40 cells treated with sodium chlorate to produce undersulfated GAGs are more refractory to vaccinia virus infection. Taken together, the data support the notion that cell surface heparan sulfate is important for vaccinia virus infection. Using heparin-Sepharose beads, we showed that vaccinia virus virions bind to heparin in vitro. In addition, we demonstrated that the recombinant A27L gene product binds to the heparin beads in vitro. This recombinant protein was further shown to bind to cells, and such interaction could be specifically inhibited by soluble heparin. All the data together indicated that A27L protein could be an attachment protein that mediates vaccinia virus binding to cell surface heparan sulfate during viral infection.     

Mapping and sequencing of a gene from myxoma virus that is related to those encoding epidermal growth factor and transforming growth factor alpha.

Myxoma virus, a Leporipoxvirus and agent of myxomatosis, was shown to possess a gene with the potential to encode an epidermal growth factorlike factor. Its relationship to other members of this family, including the poxvirus growth factors from Shope fibroma virus and vaccinia virus, was analyzed. Alignment of DNA sequences and related open reading frames of myxoma virus and Shope fibroma virus indicated colinearity of genes between these poxviruses.     

Deletion analysis of two tandemly arranged virulence genes in myxoma virus, M11L and myxoma growth factor.

Myxoma virus (MYX) is a leporipoxvirus of rabbits that induces a lethal syndrome characterized by disseminated tumorlike lesions, generalized immunosuppression, and secondary gram-negative bacterial infection. A MYX deletion mutant (vMYX-GF- delta M11L) was constructed to remove the entire myxoma growth factor (MGF) coding sequence and that for the C-terminal five amino acids of the partially overlapping upstream gene, M11L. Unexpectedly, this deletion completely abrogates the capacity of MYX to cause the characteristic disease symptoms of myxomatosis. Upon inoculation of rabbits with vMYX-GF- delta M11L, recipient animals developed only a benign, localized nodule reminiscent of a Shope fibroma virus-induced tumor in which a single primary lesion appeared at the site of injection and then completely regressed within 14 days, leaving the animals resistant to challenge with wild-type MYX. No evidence of the purulent conjunctivitis and rhinitis that always accompany wild-type MYX infection was observed. To ascertain whether the attenuation observed in vMYX-GF- delta M11L was due to a combined effect of the MGF deletion and alteration of the upstream M11L gene, two additional MYX recombinants were constructed: an MGF- virus (vMYX-GF-) containing an intact M11L gene and an M11L- virus (vMYX-M11L-) containing an intact MGF gene. Infection with vMYX-GF- resulted in moderated symptoms of myxomatosis, but all clinical stages of the disease were still detectable. In contrast, disruption of M11L alone dramatically reduced the virus virulence, resulting in a nonlethal syndrome whose clinical course was nevertheless distinct from that of vMYX-GF- delta M11L. Upon inoculation with vMYX-M11L-, rabbits developed primary and secondary tumors which were larger and more circumscribed than those of wild-type MYX recipients. Whereas wild-type MYX infection always includes severe, purulent conjunctivitis and rhinitis, vMYX-M11L- recipients remained healthy and displayed only minimal signs of respiratory distress. By about 30 days after infection, the tumors induced by vMYX-M11L- had completely regressed and these animals were immune to challenge with wild-type MYX. Histological analysis indicated that tumors induced by vMYX-M11L- are much more heavily infiltrated with macrophages and heterophils and that the sites of viral replication are more edematous and necrotic than those of wild-type infection, suggesting that the host was able to mount a more vigorous inflammatory response to vMYX-M11L- infection.(ABSTRACT TRUNCATED AT 400 WORDS)     

A rabbitpox virus serpin gene controls host range by inhibiting apoptosis in restrictive cells.

Poxviruses are unique among viruses in encoding members of the serine proteinase inhibitor (serpin) superfamily. Orthopoxviruses contain three serpins, designated SPI-1, SPI-2, and SPI-3. SPI-1 encodes a 40-kDa protein that is required for the replication of rabbitpox virus (RPV) in PK-15 or A549 cells in culture (A. N. Ali, P. C. Turner, M. A. Brooks, and R. W. Moyer, Virology 202:305-314, 1994). Examination of nonpermissive human A549 cells infected with an RPV mutant disrupted in the SPI-1 gene (RPV delta SPI-1) suggests there are no gross defects in protein or DNA synthesis. The proteolytic processing of late viral structural proteins, a feature of orthopoxvirus infections associated with the maturation of virus particles, also appears relatively normal. However, very few mature virus particles of any kind are produced compared with the level found in infections with wild-type RPV. Morphological examination of RPV delta SPI-1-infected A549 cells, together with an observed fragmentation of cellular DNA, suggests that the host range defect is associated with the onset of apoptosis. Apoptosis is seen only in RPV delta SPI-1 infection of nonpermissive (A549 or PK-15) cells and is absent in all wild-type RPV infections and RPV delta SPI-2 mutant infections examined to date. Although the SPI-1 gene is expressed early, before DNA replication, the triggering apoptotic event occurs late in the infection, as RPV delta SPI-1-infected A549 cells do not undergo apoptosis when infections are carried out in the presence of cytosine arabinoside. While the SPI-2 (crmA) gene, when transfected into cells, has been shown to inhibit apoptosis, our experiments provide the first indication that a poxvirus serpin protein can inhibit apoptosis during a poxvirus infection.     

The genome of Shope fibroma virus, a tumorigenic poxvirus, contains a growth factor gene with sequence similarity to those encoding epidermal growth factor and transforming growth factor alpha.

Degenerate oligonucleotide probes corresponding to a highly conserved region common to epidermal growth factor, transforming growth factor alpha, and vaccinia growth factor were used to identify a novel growth factor gene in the Shope fibroma virus genome. Sequence analysis indicates that the Shope fibroma growth factor is a distinct new member of this family of growth factors.     

Comparison of Cytocidal and Noncytocidal Strains of Shope Rabbit Fibroma Virus

Seven strains of Shope fibroma virus were compared for their effect on rabbit cells in vitro. All but one of the naturally occurring strains examined in this study produced a similar response in the infected cultures. This consisted of continued cell multiplication together with changes in cell morphology and growth pattern. In contrast, a recently isolated strain of fibroma virus, the M1 strain, was found to produce a gradual cell destruction under the same cultural conditions. A comparison of the cytocidal M1 strain with a representative noncytocidal strain in vitro showed no differences in the rate of multiplication, plaque type, antigenic composition, or heat lability. Only minor differences were found in the tumors produced in rabbits by these strains.     

Ubiquitin-dependent down-regulation of the neurokinin-1 receptor

Transient stimulation with substance P (SP) induces endocytosis and recycling of the neurokinin-1 receptor (NK(1)R). The effects of sustained stimulation by high concentrations of SP on NK(1)R trafficking and Ca(2+) signaling, as may occur during chronic inflammation and pain, are unknown. Chronic exposure to SP (100 nm, 3 h) completely desensitized Ca(2+) signaling by wild-type NK(1)R (NK(1)Rwt). Resensitization occurred after 16 h, and cycloheximide prevented resensitization, implicating new receptor synthesis. Lysine ubiquitination of G-protein-coupled receptors is a signal for their trafficking and degradation. Lysine-deficient mutant receptors (NK(1)RDelta5K/R, C-terminal tail lysines; and NK(1)RDelta10K/R, all intracellular lysines) were expressed at the plasma membrane and were functional because they responded to SP by endocytosis and by mobilization of Ca(2+) ions. SP desensitized NK(1)Rwt, NK(1)RDelta5K/R, and NK(1)RDelta10K/R. However, NK(1)RDelta5K/R and NK(1)RDelta10K/R resensitized 4-8-fold faster than NK(1)Rwt by cycloheximide-independent mechanisms. NK(1)RDelta325 (a naturally occurring truncated variant) showed incomplete desensitization, followed by a marked sensitization of signaling. Upon labeling receptors in living cells using antibodies to extracellular epitopes, we observed that SP induced endocytosis of NK(1)Rwt, NK(1)RDelta5K/R, and NK(1)RDelta10K/R. After 4 h in SP-free medium, NK(1)RDelta5K/R and NK(1)RDelta10K/R recycled to the plasma membrane, whereas NK(1)Rwt remained internalized. SP induced ubiquitination of NK(1)Rwt and NK(1)RDelta5K/R as determined by immunoprecipitation under nondenaturing and denaturing conditions and detected with antibodies for mono- and polyubiquitin. NK(1)RDelta10K/R was not ubiquitinated. Whereas SP induced degradation of NK(1)Rwt, NK(1)RDelta5K/R and NK(1)RDelta10K/R showed approximately 50% diminished degradation. Thus, chronic stimulation with SP induces ubiquitination of the NK(1)R, which mediates its degradation and down-regulation.     

Induction and prevention of apoptosis in human HEp-2 cells by herpes simplex virus type 1

Cultured human epithelial cells infected with an ICP27 deletion strain of herpes simplex virus type 1 (HSV-1) show characteristic features of apoptotic cells including cell shrinkage, nuclear condensation, and DNA fragmentation. These cells do not show such apoptotic features when infected with a wild-type virus unless the infections are performed in the presence of a protein synthesis inhibitor. Thus, both types of virus induce apoptosis, but the ICP27-null virus is unable to prevent this process from killing the cells. In this report, we show that this ICP27-deficient virus induced apoptosis in human HEp-2 cells through a pathway which involved the activation of caspase-3 and the processing of the death substrates DNA fragmentation factor and poly(ADP-ribose) polymerase. The induction of apoptosis by wild-type HSV-1 occurred prior to 6 h postinfection (hpi), and de novo viral protein synthesis was not required to induce the process. The ability of the virus to inhibit apoptosis was shown to be effective between 3 to 6 hpi. Wild-type HSV-1 infection was also able to block the apoptosis induced in cells by the addition of cycloheximide, staurosporine, and sorbitol. While U(S)3- and ICP22-deficient viruses showed a partial prevention of apoptosis, deletion of either the U(L)13 or vhs gene products did not affect the ability of HSV-1 to prevent apoptosis in infected cells. Finally, we demonstrate that in UV-inactivated viruses, viral binding and entry were not sufficient to induce apoptosis. Taken together, these results suggest that either gene expression or another RNA metabolic event likely plays a role in the induction of apoptosis in HSV-1-infected human cells.     

Virus-induced loss of class I MHC antigens from the surface of cells infected with myxoma virus and malignant rabbit fibroma virus.

Shope fibroma virus (SFV) is a leporipoxvirus that causes localized benign fibromas in immunocompetent adult rabbits that spontaneously regress due, in part, to a cell-mediated immune response. Myxoma virus (MYX) and malignant rabbit fibroma virus (MRV) are related leporipoxviruses that induce rapidly lethal generalized infections accompanied by tumors and immunosuppression. Because only these latter two viruses are known to compromise cell-mediated antiviral responses, cell surface levels of class I MHC molecules in SFV-, MRV-, and MYX-infected cells were investigated by fluorescent activated cell sorting analysis using a variety of different anti-HLA mAb. After infection with MYX or MRV there is a rapid decrease in the levels of detectable surface class I epitopes as detected by each antibody and by 24 h postinfection class I MHC Ag levels at the cell surface approach the level of background fluorescence observed with control antibodies. In contrast, only a moderate class I decrease is seen during infection with either SFV or vaccinia virus, an orthopoxvirus that is neither tumorigenic nor immunosuppressive. Surface class I marker loss induced by MYX and MRV is not simply due to nonspecific inhibition of total cellular protein synthesis by the viruses because class I levels decrease much further than the extent measured by estimating surface marker turnover in the presence of the protein synthesis inhibitor cycloheximide. Thus the loss of cellular surface class I molecules greatly exceeds the drop in level caused by complete blockage of host cell gene expression, and must involve removal or masking of preexisting class I epitopes from the cell surface by MRV/MYX. Cell surface levels of the transferrin receptor are unaffected by MYX and MRV infection, suggesting the observed class I decrease is not a nonspecific effect on total cell surface glycoproteins. Analysis of cells infected with MRV/MYX in the presence of cycloheximide or of cytosine arabinoside, an inhibitor of poxviral DNA replication, indicates that the class I marker loss is mediated in part by one or more viral late gene products. A probable explanation is that MRV/MYX late protein(s) interact with the class I MHC complex to either physically sequester these away from the cell surface and inhibit their recycling or else induce a conformational change that precludes recognition by all class I antibodies tested. In either event, we propose that such a major perturbation of the class I MHC complex would likely downregulate the class I-mediated presentation of viral Ag required to initiate cell-mediated immunity to these viruses.     

Matrix Protein and Another Viral Component Contribute to Induction of Apoptosis in Cells Infected with Vesicular Stomatitis Virus

The induction of apoptosis in host cells is a prominent cytopathic effect of vesicular stomatitis virus (VSV) infection. The viral matrix (M) protein is responsible for several important cytopathic effects, including the inhibition of host gene expression and the induction of cell rounding in VSV-infected cells. This raises the question of whether M protein is also involved in the induction of apoptosis. HeLa or BHK cells were transfected with M mRNA to determine whether M protein induces apoptosis when expressed in the absence of other viral components. Expression of M protein induced apoptotic morphological changes and activated caspase-3 in both cell types, indicating that M protein induces apoptosis in the absence of other viral components. An M protein containing a point mutation that renders it defective in the inhibition of host gene expression (M51R mutation) activated little, if any, caspase-3, while a deletion mutant lacking amino acids 4 to 21 that is defective in the virus assembly function but fully functional in the inhibition of host gene expression was as effective as wild-type (wt) M protein in activating caspase-3. To determine whether M protein influences the induction of apoptosis in the context of a virus infection, the M51R M protein mutation was incorporated onto a wt background by using a recombinant infectious cDNA clone (rM51R-M virus). The timing of the induction of apoptosis by rM51R-M virus was compared to that by the corresponding recombinant wt (rwt) virus and to that by tsO82 virus, the mutant virus in which the M51R mutation was originally identified. In HeLa cells, rwt virus induced apoptosis faster than did rM51R-M virus, demonstrating a role for M protein in the induction of apoptosis. In contrast to the results obtained with HeLa cells, rwt virus induced apoptosis more slowly than did rM51R-M virus in BHK cells. This indicates that a viral component other than M protein contributes to induction of apoptosis in BHK cells and that wt M protein acts to delay induction of apoptosis by the other viral component. tsO82 virus induced apoptosis more rapidly than did rM51R-M virus in both HeLa and BHK cells. These two viruses contain the same point mutation in their M proteins, suggesting that sequence differences in genes other than that for M protein affect their rates of induction of apoptosis.     

Impairment of hepatitis B virus virion secretion by single-amino-acid substitutions in the small envelope protein and rescue by a novel glycosylation site

Mutations in the S region of the hepatitis B virus (HBV) envelope gene are associated with immune escape, occult infection, and resistance to therapy. We previously identified naturally occurring mutations in the S gene that alter HBV virion secretion. Here we used transcomplementation assay to confirm that the I110M, G119E, and R169P mutations in the S domain of viral envelope proteins impair virion secretion and that an M133T mutation rescues virion secretion of the I110M and G119E mutants. The G119E mutation impaired detection of secreted hepatitis B surface antigen (HBsAg), suggesting immune escape. The R169P mutant protein is defective in HBsAg secretion as well and has a dominant negative effect when it is coexpressed with wild-type envelope proteins. Although the S domain is present in all three envelope proteins, the I110M, G119E, and R169P mutations impair virion secretion through the small envelope protein. Conversely, coexpression of just the small envelope protein of the M133T mutant could rescue virion secretion. The M133T mutation could also overcome the secretion defect caused by the G145R immune-escape mutation or mutation at N146, the site of N-linked glycosylation. In fact, the M133T mutation creates a novel N-linked glycosylation site ((131)NST(133)). Destroying this site by N131Q/T mutation or preventing glycosylation by tunicamycin treatment of transfected cells abrogated the effect of the M133T mutation. Our findings demonstrate that N-linked glycosylation of HBV envelope proteins is critical for virion secretion and that the secretion defect caused by mutations in the S protein can be rescued by an extra glycosylation site.     

Contrasting effects of matrix protein on apoptosis in HeLa and BHK cells infected with vesicular stomatitis virus are due to inhibition of host gene expression

Vesicular stomatitis virus (VSV) is a potent inducer of apoptosis in host cells. Recently, it has been shown that two VSV products are involved in the induction of apoptosis, the matrix (M) protein, and another viral product that has yet to be identified (S. A. Kopecky et. al., J. Virol. 75:12169-12181, 2001). Comparison of recombinant viruses containing wild-type (wt) or mutant M proteins showed that wt M protein accelerates VSV-induced apoptosis in HeLa cells, while wt M protein delays apoptosis in VSV-infected BHK cells. Our hypothesis to explain these results is that both effects of M protein are due to the ability of M protein to inhibit host gene expression. This hypothesis was tested by infecting cells with an M protein mutant virus defective in the inhibition of host gene expression (rM51R-M virus) in the presence or absence of actinomycin D, another inhibitor of host gene expression. Actinomycin D accelerated induction of apoptosis of HeLa cells infected with rM51R-M virus and delayed apoptosis in BHK cells infected with rM51R-M virus, similar to the effects of wt M protein. The idea that the induction of apoptosis by M protein in HeLa cells is due to its ability to inhibit host gene expression was further tested by comparing the activation of upstream caspase pathways by M protein versus that by actinomycin D or 5,6-dichlorobenzimidazole riboside (DRB). Expression of M protein activated both caspase-8 and caspase-9-like enzymes, as did treatment with actinomycin D or DRB. Induction of apoptosis by M protein, actinomycin D, and DRB was inhibited in stably transfected HeLa cell lines that overexpress Bcl-2, an antiapoptotic protein that inhibits the caspase-9 pathway. A synthetic inhibitor of caspase-8, Z-IETD-FMK, did not inhibit induction of apoptosis by M protein, actinomycin D, or DRB. Taken together, our data support the hypothesis that the induction of apoptosis by M protein is caused by the inhibition of host gene expression and that the caspase-9 pathway is more important than the caspase-8 pathway for the induction of apoptosis by M protein and other inhibitors of host gene expression.     

An RNAi strategy for treatment of amyotrophic lateral sclerosis caused by mutant Cu,Zn superoxide dismutase

Amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease) is a neurodegenerative disease characterized by motor neuron degeneration, paralysis and death. One cause of this disease is mutations in the Cu,Zn superoxide dismutase (SOD1) gene. As mutant SOD1 acquires a toxic property that kills motor neurons, by reducing the mutant protein the disease progression may be slowed or prevented. While mutant SOD1 is toxic, the wild-type SOD1 is indispensable for motor neuron health. Therefore, the ideal therapeutic strategy would be to inhibit selectively the mutant protein expression. Previously we have demonstrated that RNA interference (RNAi) can selectively inhibit some mutant SOD1 expression. However, more than 100 SOD1 mutants can cause ALS and all mutants cannot be inhibited selectively by RNAi. To overcome this obstacle, we have designed a replacement RNAi strategy. Using this strategy, all mutants and wild-type genes are inhibited by RNAi. The wild-type SOD1 function is then replaced by designed wild-type SOD1 genes that are resistant to the RNAi. Here we demonstrate the concept of this strategy.     

Vesicular stomatitis viruses expressing wild-type or mutant M proteins activate apoptosis through distinct pathways

Vesicular stomatitis virus (VSV) induces apoptosis by at least two mechanisms. The viral matrix (M) protein induces apoptosis via the mitochondrial pathway due to the inhibition of host gene expression. However, in some cell types, the inhibition of host gene expression by VSV expressing wild-type (wt) M protein delays VSV-induced apoptosis, indicating that another mechanism is involved. In support of this, the recombinant M51R-M (rM51R-M) virus, expressing a mutant M protein that is defective in its ability to inhibit host gene expression, induces apoptosis much more rapidly in L929 cells than do viruses expressing wt M protein. Here, we determine the caspase pathways by which the rM51R-M virus induces apoptosis. An analysis of caspase activity, using fluorometric caspase assays and Western blots, indicated that each of the main initiator caspases, caspase-8, caspase-9, and caspase-12, were activated during infection with the rM51R-M virus. The overexpression of Bcl-2, an inhibitor of the mitochondrial pathway, or MAGE-3, an inhibitor of caspase-12 activation, did not delay apoptosis induction in rM51R-M virus-infected L929 cells. However, an inhibitor of caspase-8 activity significantly delayed apoptosis induction. Furthermore, the inhibition of caspase-8 activity prevented the activation of caspase-9, suggesting that caspase-9 is activated by cross talk with caspase-8. These data indicate that VSV expressing the mutant M protein induces apoptosis via the death receptor apoptotic pathway, a mechanism distinct from that induced by VSV expressing the wt M protein.