The NYCBH vaccinia virus deleted for the innate immune evasion gene, E3L, protects rabbits against lethal challenge by rabbitpox virus.

Vaccinia virus deleted for the innate immune evasion gene, E3L, has been shown to be highly attenuated and yet induces a protective immune response against challenge by homologous virus in a mouse model. In this manuscript the NYCBH vaccinia virus vaccine strain was compared to NYCBH vaccinia virus deleted for E3L (NYCBHΔE3L) in a rabbitpox virus (RPV) challenge model. Upon scarification, both vaccines produced a desired skin lesion, although the lesion produced by NYCBHΔE3L was smaller. Both vaccines fully protected rabbits against lethal challenge by escalating doses of RPV, from 10LD(50) to 1000LD(50). A single dose of NYCBHΔE3L protected rabbits from weight loss, fever, and clinical symptoms following the lowest dose challenge of 10LD(50), however it allowed a moderate level of RPV replication at the challenge site, some spread to external skin and mucosal surfaces, and increased numbers of secondary lesions as compared to vaccination with NYCBH. Alternately, two doses of NYCBHΔE3L fully protected rabbits from weight loss, fever, and clinical symptoms, following challenge with 100-1000LD(50) RPV, and it prevented development of secondary lesions similar to protection seen with NYCBH. Finally, vaccination with either one or two doses of NYCBHΔE3L resulted in similar neutralizing antibody titers following RPV challenge as compared to titers obtained by vaccination with NYCBH. These results support the efficacy of the attenuated NYCBHΔE3L in protection against an orthologous poxvirus challenge.

The role of the cowpox virus crmA gene during intratracheal and intradermal infection of C57BL/6 mice.

Intratracheal (i.t.) infection of mice with cowpox virus (CPXV), is lethal at a lower dose than intranasal (i.n.) inoculation. CPXV deleted for cytokine response modifier A (CPXVDeltacrmA) was attenuated compared to CPXV after i.t. inoculation. This attenuation could not be attributed to differences in virus replication, immunomodulators, or cells infiltrating the lungs. Deletion of crmA also caused attenuation during intradermal (i.d.) infection. In contrast to i.t.-inoculated virus, deletion of crmA reduced virus replication at the site of infection. This difference correlated to increased numbers of CD3(+) cells in CPXVDeltacrmA-associated dermal lesions. Thus, crmA is a virulence factor in mice during either pulmonary or dermal cowpox infection; however the influence of crmA is more evident during i.d. inoculation. This suggests that the host immune response differs in the two routes of infection and emphasizes the need to consider the effect of route of infection when examining functions of virulence factors in vivo.

Rabbitpox virus and vaccinia virus infection of rabbits as a model for human smallpox.

The threat of smallpox release and use as a bioweapon has encouraged the search for new vaccines and antiviral drugs, as well as development of new small-animal models in which their efficacy can be determined. Here, we reinvestigate a rabbit model in which the intradermal infection of rabbits with very low doses of either rabbitpox virus (RPV) or vaccinia virus Western Reserve (VV-WR) recapitulates many of the clinical features of human smallpox. Following intradermal inoculation with RPV, rabbits develop systemic disease characterized by extensive viremia, numerous secondary lesions on the skin and mucocutaneous tissues, severe respiratory disease, death by 9 days postinfection, and, importantly, natural aerosol transmission between animals. Contrary to previous reports, intradermal infection with VV-WR also resulted in a very similar lethal systemic disease in rabbits, again with natural aerosol transmission between animals. When sentinel and index animals were cohoused, transmission rates approached 100% with either virus, with sentinel animals exhibiting a similar, severe disease. Lower rates of transmission were observed when index and sentinel animals were housed in separate cages. Sentinel animals infected with RPV with one exception succumbed to the disease. However, the majority of VV-WR-infected sentinel animals, while becoming seriously ill, survived. Finally, we tested the efficacy of the drug 1-O-hexadecyloxypropyl-cidofovir in the RPV/rabbit model and found that an oral dose of 5 mg/kg twice a day for 5 days beginning 1 day before infection was able to completely protect rabbits from lethal disease.

Amsacta moorei entomopoxvirus expresses an active superoxide dismutase.

The entomopoxvirus from Amsacta moorei serves as the prototype of the group B entomopoxviruses. One of the interesting genes found in Amsacta moorei entomopoxvirus (AmEPV) is a superoxide dismutase (sod) (open reading frame AMV255). Superoxide dismutases (SODs) catalyze the conversion of superoxide radicals to hydrogen peroxide and oxygen. Many vertebrate poxviruses contain a sod gene, but to date, none have been demonstrated to be active. There are three families of SODs, characterized by their metal ion-binding partners, Fe, Mn, or Cu and Zn. Poxvirus enzymes belong to the Cu-Zn SOD family. Unlike inactive vertebrate poxvirus SODs, AMVSOD contains all the amino acids necessary for function. We expressed and purified a 6X-His-tagged version of the AMVSOD in Escherichia coli. The recombinant AMVSOD demonstrates superoxide dismutase activity both in an in situ gel assay and by stopped flow spectrophotometry. The k(cat)/K(m) for AMVSOD is 4 x 10(7) M(-1)s(-1). In infected cells, the AMVSOD protein behaves as a dimer and is catalytically active; however, disruption of the gene in AMEPV has little or no effect on growth of the virus in cell culture. An analysis of mRNA expression indicates that AMVsod is expressed late during infection of Lymantria dispar (Ld652) cells and produces a discrete nonpolydisperse transcript. Characterization of protein expression with a monoclonal antibody generated against AMVSOD confirms that the AMVSOD protein can be classified as a late, postreplicative gene. Therefore, AMVSOD is the first example of an active poxvirus SOD.

Melatonin in mice: rhythms, response to light, adrenergic stimulation, and metabolism.

There has been relatively little research conducted on pineal melatonin production in laboratory mice, in part, due to the lack of appropriate assays. We studied the pineal and plasma rhythm, response to light, adrenergic stimulation, and metabolism of melatonin in CBA mice. With the use of a sensitive and specific melatonin RIA, melatonin was detected in the pineal glands at all times of the day >21 fmol/gland in CBA mice but not in C57Bl mice. Both plasma and pineal melatonin levels peaked 2 h before dawn in a 12:12-h light-dark photoperiod (162 +/- 31 pM and 1,804 +/- 514 fmol/gland, respectively). A brief light pulse (200 lx/15 min), 2 h before lights on, suppressed both plasma and pineal melatonin to near basal levels within 30 min. Exposure to light pulses 4 h after lights off or 2 h before lights on resulted in delays and advances, respectively, in the early morning decline of plasma and pineal melatonin on the next cycle. Administration of the beta-adrenergic agonist isoproterenol (20 mg/kg) 2 and 4 h after lights on in the morning resulted in a fivefold increase in plasma and pineal melatonin 2.5 to 3 h after the first injection. In the mouse, unlike the rat, melatonin was shown to be metabolized almost exclusively to 6-glucuronylmelatonin rather than 6-sulphatoxymelatonin. These studies have shown that the appropriate methodological tools are now available for studying melatonin rhythms in mice.

NAD+-dependent DNA ligase encoded by a eukaryotic virus.

We report the production, purification, and characterization of an NAD(+)-dependent DNA ligase encoded by the Amsacta moorei entomopoxvirus (AmEPV), the first example of an NAD(+) ligase from a source other than eubacteria. AmEPV ligase lacks the zinc-binding tetracysteine domain and the BRCT domain that are present in all eubacterial NAD(+) ligases. Nonetheless, the monomeric 532-amino acid AmEPV ligase catalyzed strand joining on a singly nicked DNA in the presence of a divalent cation and NAD(+). Neither ATP, dATP, nor any other nucleoside triphosphate could substitute for NAD(+). Structure probing by limited proteolysis showed that AmEPV ligase is punctuated by a surface-accessible loop between the nucleotidyltransferase domain, which is common to all ligases, and the N-terminal domain Ia, which is unique to the NAD(+) ligases. Deletion of domain Ia of AmEPV ligase abolished the sealing of 3'-OH/5'-PO(4) nicks and the reaction with NAD(+) to form ligase-adenylate, but had no effect on phosphodiester formation at a pre-adenylated nick. Alanine substitutions at residues within domain Ia either reduced (Tyr(39), Tyr(40), Asp(48), and Asp(52)) or abolished (Tyr(51)) sealing of a 5'-PO(4) nick and adenylyl transfer from NAD(+) without affecting ligation of DNA-adenylate. We conclude that: (i) NAD(+)-dependent ligases exist in the eukaryotic domain of the phylogenetic tree; and (ii) ligase structural domain Ia is a determinant of cofactor specificity and is likely to interact directly with the nicotinamide mononucleotide moiety of NAD(+).

Serotonin, excitatory amino acids and the photic control of melatonin rhythms and SCN c-FOS in the rat.

There is a growing acceptance that serotonergic pathways to the suprachiasmatic nucleus play an important role in the mediation and modulation of light entrainment of rhythms. In this study administration of the 5-HT(2A/2C) agonist (+/-)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane (DOI, 0.5 mg/kg) at mid dark caused a phase shift in the onset of the urinary excretion of 6-sulphatoxymelatonin in rats that was sustained for at least 8 days and was blocked by the specific 5-HT(2C) antagonist SB-242084. Administration of DOI (2 mg/kg) across the night resulted in the appearance of c-FOS in the nucleus of cells in the suprachiasmatic nucleus during subjective darkness, but did not cause induction at the time of expected lights on (CT0). By contrast light exposure induced c-fos throughout the night including CT0. Administration of the NMDA receptor antagonist MK-801 (3 mg/kg) prior to light pulses had no effect on c-fos in the first part of the night, but towards the expected time of lights on, became progressively more potent, such that by CT0, light induction of c-fos was almost completely inhibited. These results provide further evidence that serotonin plays a role in the mediation of light effects on rhythms in the rat.

The effects of the conserved extreme 3' end sequence of hepatitis C virus (HCV) RNA on the in vitro stabilization and translation of the HCV RNA genome.

BACKGROUND/AIMS: The discovery of an additional 98-base in the extreme 3' end of the hepatitis C virus (HCV) has fueled much speculation as to the role of this sequence on the behavior of the virus. It is now known that this additional 98-base sequence is present and conserved amongst HCV genotypes. This sequence is capable of forming complex and stable high-order structures that may be important in stabilizing the RNA to degradation, facilitating translation and regulating replication of the virus. We have examined the possible role of the HCV extreme 3' end sequence in stabilizing the HCV RNA genome and regulating translation in vitro. METHODS: The extreme 3' end sequence was cloned to downstream of two pre-existing two HCV clones: HCV1 (genotype 1a) and HCV-BK (genotype 1b). The reconstructed full-length clones were then tested in vitro for their stability and translation efficiency. RESULTS: We showed that the addition of the conserved 3' end sequence greatly enhanced the stability of HCV1 RNA but had only minimal effect on HCV-BK RNA in mammalian cytoplasmic extracts, suggesting that the requirements for HCV RNA stability vary amongst isolates. Following the optimization of in vitro translation conditions, it was demonstrated that the addition of this 3' end sequence did not affect the translation level from either HCV clone. CONCLUSIONS: The conserved 3' end of the HCV genome confers differential stabilizing effects on two HCV genotype 1 isolates and has no obvious role in the in vitro translation of either clone.

Complete genomic sequence of the Amsacta moorei entomopoxvirus: analysis and comparison with other poxviruses.

The genome of the genus B entomopoxvirus from Amsacta moorei (AmEPV) was sequenced and found to contain 232,392 bases with 279 unique open reading frames (ORFs) of greater than 60 amino acids. The central core of the viral chromosome is flanked by 9.4-kb inverted terminal repeats (ITRs), each of which contains 13 ORFs, raising the total number of ORFs within the viral chromosome to 292. ORFs with no known homology to other poxvirus genes were shown to constitute 33.6% of the viral genome. Approximately 28.6% of the AmEPV genome encodes homologs of the mammalian poxvirus colinear core genes, which are found dispersed throughout the AmEPV chromosome. There is also no significant gene order conservation between AmEPV and the orthopteran genus B poxvirus of Melanoplus sanguinipes (MsEPV). Novel AmEPV genes include those encoding a putative ABC transporter and a Kunitz-motif protease inhibitor. The most unusual feature of the AmEPV genome relates to the viral encoded poly(A) polymerase. In all other poxviruses this heterodimeric enzyme consists of a single large and a single small subunit. However, AmEPV appears to encode one large and two distinct small poly(A) polymerase subunits. AmEPV is one of the few entomopoxviruses which can be grown and manipulated in cell culture. The complete genomic sequence of AmEPV paves the way for an understanding and comparison of the molecular properties and pathogenesis between the entomopoxviruses of insects and the more intensively studied vertebrate poxviruses.

The cowpox virus serpin SPI-3 complexes with and inhibits urokinase-type and tissue-type plasminogen activators and plasmin.

The orthopoxvirus serpin SPI-3 is N-glycosylated and suppresses fusion between infected cells. Although SPI-3 contains motifs conserved in inhibitory serpins, no proteinase inhibition by SPI-3 has been demonstrated, and mutations within the serpin reactive center loop (RCL) do not affect the ability to regulate cell fusion. We demonstrate here that SPI-3 protein expressed by transcription/translation in vitro is able to form SDS-stable complexes with the serine proteinases plasmin, urokinase-type plasminogen activator (uPA), and tissue-type plasminogen activator (tPA), consistent with inhibitory activity of the serpin. Weaker complexes were noted with factor Xa and thrombin. Mutation of Arg-340/Ser-341 at the predicted P1/P1' sites within the RCL prevented the formation of complexes between SPI-3 and plasmin, uPA, or tPA, suggesting that the arginine at the P1 position was required for complex formation. SPI-3 protein lacking the N-terminal signal peptide was purified by means of an N-terminal His(10)-tag and gave complete inhibition in vitro of plasmin, uPA, and tPA and partial inhibition of factor Xa. SPI-3 is therefore a bifunctional protein that acts as a proteinase inhibitor and suppresses infected cell-cell fusion. As a proteinase inhibitor, SPI-3 has similar specificity to the leporipoxvirus SERP1 protein of myxoma virus, although the two serpins are less than 30% identical overall. The inhibition constants of SPI-3 for plasmin, uPA, and tPA were determined to be 0.64, 0.51, and 1.9 nM, respectively, very similar to the corresponding K(i) values of SERP1.

The cowpox virus SPI-3 and myxoma virus SERP1 serpins are not functionally interchangeable despite their similar proteinase inhibition profiles in vitro.

The myxoma virus (MYX) serpin SERP1 is a secreted glycoprotein with anti-inflammatory activity that is required for full MYX virulence in vivo. The cowpox virus (CPV) serpin SPI-3 (vaccinia virus ORF K2L) is a nonsecreted glycoprotein that blocks cell-cell fusion, independent of serpin activity, and is not required for virulence of vaccinia virus or CPV in mice. Although SPI-3 has only 29% overall identity to SERP1, both serpins have arginine at the P1 position in the reactive center loop, and SPI-3 has a proteinase inhibitory profile strikingly similar to that of SERP1 [Turner, P. C., Baquero, M. T., Yuan, S., Thoennes, S. R., and Moyer, R. W. (2000) Virology 272, 267-280]. To determine whether SPI-3 and SERP1 were functionally equivalent, a CPV variant was constructed where the SPI-3 gene was deleted and replaced with the SERP1 gene regulated by the SPI-3 promoter. Cells infected with CPVDeltaSPI-3::SERP1 secrete SERP1 and show extensive fusion, suggesting that SERP1 is unable to functionally substitute for SPI-3 in fusion inhibition. In the reciprocal experiment, both copies of SERP1 were deleted from MYX and replaced with SPI-3 under the control of the SERP1 promoter. Cells infected with the MYXDeltaSERP1::SPI-3 recombinant unexpectedly secreted SPI-3, suggesting either that the cellular secretory pathway is enhanced by MYX or that CPV encodes a protein that prevents SPI-3 secretion. MYXDeltaSERP1::SPI-3 was as attenuated in rabbits as MYXDeltaSERP1::lacZ, indicating that SPI-3 cannot substitute for SERP1 in MYX pathogenesis.

Serotonin depletion decreases light induced c-fos in the rat suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) is the locus of the biological clock in mammals. Daily light cycles entrain the endogenous circadian rhythms in mammals through direct and indirect neural pathways from the retinae to the suprachiasmatic nucleus. We have studied the effect of serotonin depletion on the photic induction of the early response gene c-fas in the SCN of rats. Serotonin depletion, verified by immunohistochemistry, produced a significant decrease (42%) in the number of c-FOS positive cells in the ventrolateral portion of the SCN. These results support the involvement of serotonin as a mediator of photic information to the SCN through the retinal projection to the dorsal raphe nucleus.

Granzyme B short-circuits the need for caspase 8 activity during granule-mediated cytotoxic T-lymphocyte killing by directly cleaving Bid.

Cytotoxic T lymphocytes (CTL) can trigger an apoptotic signal through the Fas receptor or by the exocytosis of granzyme B and perforin. Caspase activation is an important component of both pathways. Granzyme B, a serine proteinase contained in granules, has been shown to proteolytically process and activate members of the caspase family in vitro. In order to gain an understanding of the contributions of caspases 8 and 3 during granule-induced apoptosis in intact cells, we have used target cells that either stably express the rabbitpox virus-encoded caspase inhibitor SPI-2 or are devoid of caspase 3. The overexpression of SPI-2 in target cells significantly inhibited DNA fragmentation, phosphatidylserine externalization, and mitochondrial disruption during Fas-mediated cell death. In contrast, SPI-2 expression in target cells provided no protection against granzyme-mediated apoptosis, mitochondrial collapse, or cytolysis, leading us to conclude that SPI-2-inhibited caspases are not an essential requirement for the granzyme pathway. Caspase 3-deficient MCF-7 cells were found to be resistant to CTL-mediated DNA fragmentation but not to CTL-mediated cytolysis and loss of the mitochondrial inner membrane potential. Furthermore, we demonstrate that granzyme B directly cleaves the proapoptotic molecule Bid, bypassing the need for caspase 8 activation of Bid. These results provide evidence for a two-pronged strategy for mediating target cell destruction and provide evidence of a direct link between granzyme B activity, Bid cleavage, and caspase 3 activation in whole cells.

Thermocyclic entrainment of lizard blood plasma melatonin rhythms in constant and cyclic photic environments.

We assessed how chronic exposure to 6-h cryophase temperatures of 15 degrees C in an otherwise 33 degrees C environment entrains the rhythm of blood plasma melatonin rhythms in lizards (Tiliqua rugosa) subjected to constant dark (DD), constant light (LL), and to 12:12-h light-dark cycles (12L:12D). The peak of the melatonin rhythm was entrained by the cryophase temperature of the thermocycle in DD and LL, irrespective of the time at which the cryophase temperature was applied. Comparable thermocycles of 6 h at 15 degrees C imposed on a 12L:12D photocycle, however, affected the amplitude and phase of the melatonin rhythm, depending on the phase relationship between light and temperature. Cold pulses in the early light period and at midday resulted, respectively, either in low amplitude or nonexistent melatonin rhythms, whereas those centered in or around the dark phase elicited rhythms of high amplitude. Supplementary experiments in 12L:12D using two intermittent 6-h 15 degrees C cryophases, one delivered in the midscotophase and another in the midphotophase, elicited melatonin rhythms comparable to those in lizards subjected to constant 33 degrees C and 12L:12D. In contrast, lizards subjected to 12L:12D and a 33 degrees C:15 degrees C thermocycle, whose thermophase was aligned with the photophase, produced a threefold increase in the amplitude of the melatonin rhythm. Taken together, these results support the notion that there is an interaction between the external light and temperature cycle and a circadian clock in determining melatonin rhythms in Tiliqua rugosa.

Immunohistochemical localization of serotonin receptors in the rat suprachiasmatic nucleus.

Serotonin (5-HT) has been implicated in the regulation of circadian rhythms through its actions on the suprachiasmatic nucleus (SCN). Recent data suggests that, along with excitatory amino acids, serotonin may be important in the neural pathway that mediates the transmission of photic information to the circadian system. The present study uses immunohistochemistry to examine the presence of three different 5-HT receptor subtypes in the suprachiasmatic nucleus (5-HT2a, 5-HT2c and 5-HT7) in male albino Wistar rats. In the SCN, there was a considerable amount of 5-HT2c-receptor-like immunoreactivity, a lesser amount of 5-HT2a positive fibres and no staining with antiserum against the 5-HT7 receptor subtype. These results are compatible with previous pharmacological evidence obtained in our laboratory showing that serotonin acting through the 5-HT2c receptor subtype may be important in the phase shifting effects of light on the circadian system.

SPI-1-dependent host range of rabbitpox virus and complex formation with cathepsin G is associated with serpin motifs.

Serpins are a superfamily of serine proteinase inhibitors which function to regulate a number of key biological processes including fibrinolysis, inflammation, and cell migration. Poxviruses are the only viruses known to encode functional serpins. While some poxvirus serpins regulate inflammation (myxoma virus SERP1 and cowpox virus [CPV] crmA/SPI-2) or apoptosis (myxoma virus SERP2 and CPV crmA/SPI-2), the function of other poxvirus serpins remains unknown. The rabbitpox virus (RPV) SPI-1 protein is 47% identical to crmA and shares all of the serpin structural motifs. However, no serpin-like activity has been demonstrated for SPI-1 to date. Earlier we showed that RPV with the SPI-1 gene deleted, unlike wild-type virus, fails to grow on A549 or PK15 cells (A. Ali, P. C. Turner, M. A. Brooks, and R. W. Moyer, Virology 202:306-314, 1994). Here we demonstrate that in the absence of a functional SPI-1 protein, infected nonpermissive cells which exhibit the morphological features of apoptosis fail to activate terminal caspases or cleave the death substrates PARP or lamin A. We show that SPI-1 forms a stable complex in vitro with cathepsin G, a member of the chymotrypsin family of serine proteinases, consistent with serpin activity. SPI-1 reactive-site loop (RSL) mutations of the critical P1 and P14 residues abolish this activity. Viruses containing the SPI-1 RSL P1 or P14 mutations also fail to grow on A549 or PK15 cells. These results suggest that the full virus host range depends on the serpin activity of SPI-1 and that in restrictive cells SPI-1 inhibits a proteinase with chymotrypsin-like activity and may function to inhibit a caspase-independent pathway of apoptosis.

MK-801 administration blocks the effects of a 5-HT(2A/2C) agonist on melatonin rhythmicity and c-fos induction in the suprachiasmatic nucleus.

Both excitatory amino acids and serotonin have been implicated in the photic control of rhythms, but they have rarely been considered to interact. This study investigated the effects of the NMDA receptor antagonist, MK-801 on the phase shift of the melatonin rhythm and the induction of c-fos in the rat suprachiasmatic nucleus (SCN) provoked by the administration of the serotonin agonist DOI ((+/-)-1-(4-Iodo-2,5-dimethoxyphenyl)-2-aminopropane hydrochloride). The urinary excretion rate rhythm of the melatonin metabolite, 6-sulphatoxymelatonin was delayed by administration of DOI (0.5 mg/kg) at CT18 (6 h after subjective darkness onset) as previously reported by our group. Administration of MK-801 (3 mg/kg) 30 min before DOI blocked the shift in the onset of excretion of the melatonin metabolite on the following nights. Pre-treatment with MK-801 also inhibited by approximately 90% the induction of c-fos in the SCN by DOI at ZT18 (6 h after actual darkness onset) as determined by immunohistochemistry. These results provide evidence for a role of excitatory amino acids in the photomimetic effects of serotonin 5-HT(2C) agonists in the rat.

Myxoma virus Serp2 is a weak inhibitor of granzyme B and interleukin-1beta-converting enzyme in vitro and unlike CrmA cannot block apoptosis in cowpox virus-infected cells.

The Serp2 protein encoded by the leporipoxvirus myxoma virus is essential for full virulence (F. Messud-Petit, J. Gelfi, M. Delverdier, M. F. Amardeilh, R. Py, G. Sutter, and S. Bertagnoli, J. Virol. 72:7830-7839, 1998) and, like crmA of cowpox virus (CPV), is reported to inhibit the interleukin-1beta-converting enzyme (ICE, caspase-1) (F. Petit, S. Bertagnoli, J. Gelfi, F. Fassy, C. Boucraut-Baralon, and A. Milon, J. Virol. 70:5860-5866, 1996). Serp2 and CrmA both contain Asp at the P1 position within the serpin reactive site loop and yet are only 35% identical overall. Serp2 protein was cleaved by ICE but, unlike CrmA, did not form a stable complex with ICE that was detectable by native gel electrophoresis. Attempts to covalently cross-link ICE-serpin inhibitory complexes were successful with CrmA, but no complex between ICE and Serp2 was visible after cross-linking. Purified His10-tagged Serp2 protein was a relatively poor inhibitor of ICE, with a Ki of 80 nM compared to 4 pM for CrmA. Serp2 protein resembled CrmA in that a stable complex with the serine proteinase granzyme B was detectable after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, Serp2 was less effective at inhibiting granzyme B activity (Ki = 420 nM) than CrmA (Ki = 100 nM). Finally, Serp2 was tested for the ability to replace CrmA and inhibit apoptosis in LLC-PK1 cells infected with a CPV recombinant deleted for CrmA but expressing Serp2. Unlike wild-type-CPV-infected cells, apoptosis was readily observed in cells infected with the recombinant virus, as indicated by the induction of both nuclear fragmentation and caspase-mediated cleavage of DEVD-AMC [acetyl-Asp-Glu-Val-Asp-(amino-4-methyl coumarin)]. These results indicate that Serp2 is unable to functionally substitute for CrmA within the context of CPV and that the inhibition spectra for Serp2 and CrmA are distinct.

Poxvirus-encoded serpins do not prevent cytolytic T cell-mediated recovery from primary infections.

Previous observations that the highly conserved poxvirus-encoded serpins inhibit cytotoxic activities of alloreactive CTL via granule and/or Fas-mediated pathways was taken to indicate their involvement in immune evasion by poxviruses. We now show that interference with 51Cr release from target cells by ectromelia and cowpoxvirus is limited to alloreactive but not MHC-restricted CTL. The data are in support of the paramount importance of CTL and its effector molecule perforin in the recovery from primary ectromelia virus infection and question the role of serpins in the evasion of poxviruses from killing by CTL. Further analysis of poxvirus interference with target cell lysis by alloreactive CTL revealed that suppression primarily affects the Fas-mediated, and to a lesser extent, the granule exocytosis pathway. Serpin-2 is the main contributor to suppression for both killing pathways. In addition, inhibition of lysis was shown to be both target cell type- and MHC allotype-dependent. We hypothesize that differences in TCR affinities and/or state of activation between alloreactive and MHC-restricted CTL as well as the quality (origin) of target cells are responsible for the observed phenomenon.