Uveitis and desquamating rash of the palms and soles.

Syphilis, a sexually transmitted disease caused by the spirochete Treponema pallidum, can affect nearly every organ system in the body. In particular, skin manifestations of secondary syphilis are common but nonspecific and can be a true masquerader of other skin disorders. Concomitant infection with HIV has been increasing and may cause even more unusual skin presentations. We present a patient with the atypical combination of palmoplantar keratoderma and ocular symptoms that closely resembled reactive arthritis (or Reiter's syndrome). When evaluating patients with HIV infection, clinicians should maintain a high level of suspicion for syphilis to accurately diagnose and treat this curable but potentially fatal disease.

Structure and mechanism of IFN-gamma antagonism by an orthopoxvirus IFN-gamma-binding protein.

Ectromelia virus (ECTV) encodes an IFN-gamma-binding protein (IFN-gammaBP(ECTV)) that disrupts IFN-gamma signaling and its ability to induce an antiviral state within cells. IFN-gammaBP(ECTV) is an important virulence factor that is highly conserved (>90%) in all orthopoxviruses, including variola virus, the causative agent of smallpox. The 2.2-A crystal structure of the IFN-gammaBP(ECTV)/IFN-gamma complex reveals IFN-gammaBP(ECTV) consists of an IFN-gammaR1 ligand-binding domain and a 57-aa helix-turn-helix (HTH) motif that is structurally related to the transcription factor TFIIA. The HTH motif forms a tetramerization domain that results in an IFN-gammaBP(ECTV)/IFN-gamma complex containing four IFN-gammaBP(ECTV) chains and two IFN-gamma dimers. The structure, combined with biochemical and cell-based assays, demonstrates that IFN-gammaBP(ECTV) tetramers are required for efficient IFN-gamma antagonism.

Poxvirus-encoded gamma interferon binding protein dampens the host immune response to infection.

Ectromelia virus (ECTV), a natural mouse pathogen and the causative agent of mousepox, is closely related to variola virus (VARV), which causes smallpox in humans. Mousepox is an excellent surrogate small-animal model for smallpox. Both ECTV and VARV encode a multitude of host response modifiers that target components of the immune system and that are thought to contribute to the high mortality rates associated with infection. Like VARV, ECTV encodes a protein homologous to the ectodomain of the host gamma interferon (IFN-gamma) receptor 1. We generated an IFN-gamma binding protein (IFN-gammabp) deletion mutant of ECTV to study the role of viral IFN-gammabp (vIFN-gammabp) in host-virus interaction and also to elucidate the contribution of this molecule to the outcome of infection. Our data show that the absence of vIFN-gammabp does not affect virus replication per se but does have a profound effect on virus replication and pathogenesis in mice. BALB/c mice, which are normally susceptible to infection with ECTV, were able to control replication of the mutant virus and survive infection. Absence of vIFN-gammabp from ECTV allowed the generation of an effective host immune response that was otherwise diminished by this viral protein. Mice infected with a vIFN-gammabp deletion mutant virus, designated ECTV-IFN-gammabp(Delta), produced increased levels of IFN-gamma and generated robust cell-mediated and antibody responses. Using several strains of mice that exhibit differential degrees of resistance to mousepox, we show that recovery or death from ECTV infection is determined by a balance between the host's ability to produce IFN-gamma and the virus' ability to dampen its effects.

Duplex strand joining reactions catalyzed by vaccinia virus DNA polymerase.

Vaccinia virus DNA polymerase catalyzes duplex-by-duplex DNA joining reactions in vitro and many features of these recombination reactions are reprised in vivo. This can explain the intimate linkage between virus replication and genetic recombination. However, it is unclear why these apparently ordinary polymerases exhibit this unusual catalytic capacity. In this study, we have used different substrates to perform a detailed investigation of the mechanism of duplex-by-duplex recombination catalyzed by vaccinia DNA polymerase. When homologous, blunt-ended linear duplex substrates are incubated with vaccinia polymerase, in the presence of Mg2+ and dNTPs, the appearance of joint molecules is preceded by the exposure of complementary single-stranded sequences by the proofreading exonuclease. These intermediates anneal to form a population of joint molecules containing hybrid regions flanked by nicks, 1-5 nt gaps, and/or short overhangs. The products are relatively resistant to exonuclease (and polymerase) activity and thus accumulate in joining reactions. Surface plasmon resonance (SPR) measurements showed the enzyme has a relative binding affinity favoring blunt-ended duplexes over molecules bearing 3'-recessed gaps. Recombinant duplexes are the least favored ligands. These data suggest that a particular combination of otherwise ordinary enzymatic and DNA-binding properties, enable poxvirus DNA polymerases to promote duplex joining reactions.

Identification of residues in the ectromelia virus gamma interferon-binding protein involved in expanded species specificity.

Gamma interferon (IFN-gamma) production is important in the host response to, and recovery from, infection with Ectromelia virus (ECTV) and Vaccinia virus (VACV). The orthopoxviruses have evolved several mechanisms to subvert the IFN-gamma response. IFN-gamma-binding protein (IFN-gammaBP) is a virally encoded homologue of the host IFN-gamma receptor that blocks the effects of IFN-gamma in the infected host. Unlike the cellular receptors, whose ligand specificity is restricted to their own species, the orthopoxvirus IFN-gammaBPs bind IFN-gamma from several species. The reason for this relaxed specificity has yet to be explained. ECTV, a mouse pathogen, encodes an IFN-gammaBP that has been shown to inhibit the activity of both human and murine IFN-gamma (hIFN-gamma and mIFN-gamma, respectively). In contrast, the IFN-gammaBP from VACV is unable to inhibit mIFN-gamma, but retains activity against hIFN-gamma. To determine which region(s) in the ECTV sequence is responsible for its ability to bind to mIFN-gamma with high affinity, a series of chimeric IFN-gammaBPs, as well as individual point mutants in the ECTV sequence corresponding to the amino acid changes from the VACV sequence, were constructed. The affinities of the chimeric and point mutant IFN-gammaBPs for mIFN-gamma were tested by using surface plasmon resonance and bioassay. By using this strategy, several key residues in the ligand-binding domains of the ECTV sequence have been identified that are responsible for high-affinity binding to mIFN-gamma. Substitution of the ECTV residue at these positions in VACV resulted in a dramatic increase in the affinity of the VACV IFN-gammaBP for mIFN-gamma.

The unique C termini of orthopoxvirus gamma interferon binding proteins are essential for ligand binding.

The orthopoxviruses ectromelia virus (ECTV) and vaccinia virus (VACV) express secreted gamma interferon binding proteins (IFN-gammaBPs) with homology to the ligand binding domains of the host's IFN-gamma receptor (IFN-gammaR1). Homology between these proteins is limited to the extracellular portions of the IFN-gammaR1 and the first approximately 200 amino acids of the IFN-gammaBPs. The remaining 60 amino acids at the C termini of the IFN-gammaBPs contain a single cysteine residue shown to be important in covalent dimerization of the secreted proteins. The function of the remaining C-terminal domain (CTD) has remained elusive, yet this region is conserved within all orthopoxvirus IFN-gammaBPs. Using a series of C-terminal deletion constructs, we have determined that the CTD is essential for IFN-gamma binding despite having no predicted homology to the IFN-gammaR1. Truncation of the ECTV IFN-gammaBP by more than two amino acid residues results in a complete loss of binding activity for both murine IFN-gamma and human IFN-gamma (hIFN-gamma), as measured by surface plasmon resonance (SPR) and bioassay. Equivalent truncation of the VACV IFN-gammaBP resulted in comparable loss of hIFN-gamma binding activity by SPR. Full-length IFN-gammaBPs were observed to form higher-ordered structures larger than the previously reported dimers. Mutants that were unable to bind IFN-gamma with high affinity in SPR experiments failed to assemble into these higher-ordered structures and migrated as dimers. We conclude that the unique CTD of orthopoxvirus IFN-gammaBPs is important for the assembly of covalent homodimers as well as the assembly of higher-ordered structures essential for IFN-gamma binding.

Application of immunoproteomics to rapid cytokine detection.

Cytokines are pivotal to a balanced innate or cell-mediated immune response, and can be indicative of disease progression and/or resolution. Methods to measure key cytokines rapidly with accuracy, precision, and sensitivity are consequently important. The current assay technologies, which are based on RT-PCR, immunoassays, or bioassays, are limited in their use in the clinic, in particular because of the long time (1-3 h) required to carry out the assays. An alternative, semi-quantitative approach described here, uses an immunological capture step and a mass spectral readout. The goal of the assay is speed rather than sensitivity or precision.

Biosynthesis of the IFN-gamma binding protein of ectromelia virus, the causative agent of mousepox.

Ectromelia virus (ECTV), the causative agent of mousepox, expresses an extracellular interferon-gamma binding protein (IFN-gammaBP) with homology to the ligand binding domains of the IFN-gamma high affinity receptor (IFN-gammaR1). Unlike the cellular receptor, the IFN-gammaBP binds IFN-gamma from several species. The IFN-gammaBP is synthesized early after infection, accumulating in the extracellular milieu as dimers composed of two protein species with Mr of 34.6 or 33.0 kDa. Homodimers are covalently linked by an interchain disulphide bond at position 216. The IFN-gammaBP has complex N-linked oligosaccharides at positions 41 and 149 as determined by site-directed mutagenesis and glycosidase treatment. Glycosylation at position 41 is required for secretion from mammalian cells and may play a role in the activity of the IFN-gammaBP. Glycosylation at position 149 is not required for secretion, and the lack of glycosylation at this site does not diminish ligand binding as measured by surface plasmon resonance (SPR) and ELISA.

Interleukin-18 and glycosaminoglycan binding by a protein encoded by Variola virus.

Poxvirus interleukin (IL)-18 binding proteins (IL-18BPs) are soluble decoys that inhibit the activity of IL-18. The aim of this study was to demonstrate IL-18 binding activity of the Variola virus protein D7L. D7L effectively inhibited the biological activity of IL-18 in a bioassay. We compared the affinity and kinetics of D7L and the Ectromelia virus IL-18BP, p13, for human and murine IL-18 using surface plasmon resonance and no differences were detected, indicating that the differences in amino acid sequence did not affect binding or species specificity. Both proteins had higher affinity for murine than human IL-18. This was similar to human IL-18BP and the Molluscum contagiosum virus IL-18BP, which also demonstrated higher affinity for human IL-18. The host range of Variola virus is limited to humans and thus the affinity of D7L for IL-18 does not correlate with its host range. Furthermore, we demonstrated that D7L is capable of interacting with glycosaminoglycans (GAGs) via the C terminus, while p13 is not. Importantly, D7L interacted with both GAG and IL-18 simultaneously, indicating that the binding sites were distinct.