Karl W. Boehme

Human Cytomegalovirus Activates Inflammatory Cytokine Responses via CD14 and Toll-Like Receptor 2

ABSTRACT Human cytomegalovirus (CMV) is a ubiquitous opportunistic pathogen that causes significant morbidity and mortality in immuncompromised people. An understanding of how CMV induces and circumvents host immunity is of critical importance in efforts to design effective therapeutics. It was recently discovered that mere cell contact by CMV particles leads to profound modulation of cellular gene expression, including induction of inflammatory cytokines and interferon-stimulated genes characteristic of innate immune detection. These findings suggest that a membrane receptor recognizes a CMV envelope protein(s), leading to innate immune activation. Here, we show that the pattern recognition receptors Toll-like receptor 2 (TLR2) and CD14 recognize CMV virions and trigger inflammatory cytokine production. Induction of inflammatory cytokines is mediated via TLR2-dependent activation of NF-κB. Since many of the pathological processes associated with CMV disease are facilitated or directly mediated by inflammatory cytokines, identification of the host membrane detection machinery may ultimately lead to improved therapeutics.

Human Cytomegalovirus Envelope Glycoproteins B and H Are Necessary for TLR2 Activation in Permissive Cells

Human CMV (HCMV) is a ubiquitous member of the Herpesviridae family and an opportunistic pathogen that poses significant health risks for immunocompromised patients. HCMV pathogenesis is intimately tied to the immune status of the host, thus characterization of the innate immune response to HCMV infection is critical for understanding disease progression. Previously, we identified TLR2 as a host factor that detects and initiates inflammatory cytokine secretion in response to HCMV independent of viral replication. In this study, we show that two entry-mediating envelope gp, gp B (gB) and gp H (gH), display determinants recognized by TLR2. Neutralizing Abs against TLR2, gB and gH inhibit inflammatory cytokine responses to HCMV infection, suggesting that inflammatory cytokine stimulation by HCMV is mediated by interactions between these envelope gp and TLR2. Furthermore, both gB and gH coimmunoprecipitate with TLR2 and TLR1, indicating that these envelope gp directly interact with TLR2 and that a TLR2/TLR1 heterodimer is a functional sensor for HCMV. Because our previous studies were conducted in model cell lines, we also show that TLR2 is expressed by HCMV permissive human fibroblast cell strains, and that TLR2 is a functional sensor in these cells. This study further elucidates the importance and potency of envelope gp as a class of molecules displaying pathogen-associated molecular patterns that are recognized with immediate kinetics by TLRs in permissive cells.

Human Cytomegalovirus Elicits a Coordinated Cellular Antiviral Response via Envelope Glycoprotein B

ABSTRACT Previous studies have shown that human cytomegalovirus (CMV) is a potent elicitor of interferon-stimulated gene (ISG) expression. Induction of the interferon pathway does not require replication-competent virus, and envelope glycoprotein B (gB) from CMV is a viral structural component that can directly induce transcription of ISGs. Here we extend these earlier findings by defining the consequences of inducing the interferon pathway. We found that cells respond to CMV or soluble gB by establishing a functional antiviral state within cell types critical in CMV biology, such as fibroblasts and endothelial cells. We have also discovered new insights into the mechanism by which the pathway is initiated. Interferon regulatory factor 3 (IRF3), a key transcriptional regulator of cellular interferon responses, is activated by CMV virions and soluble gB. Thus, IRF3 becomes activated via “outside-in” signal transduction events. This is a novel mechanism of activation of this key transcription factor by viruses. In comparison to soluble gB (gB1-750), which comprises the entire ectodomain of gB, a truncation mutant encompassing only the amino-terminal region of gB (gB1-460) was markedly less effective at inducing antiviral responses. This indicates that the region of gB from residues 461 to 750 is important for initiation of the antiviral response. In addition, CMV and gB establish an antiviral state in alpha/beta interferon null cells, illustrating that primary induction of ISGs by CMV and gB is sufficient to establish the antiviral response and that interferon secretion is not necessary for the antiviral effect. Taken together, our findings reveal that CMV initiates a coordinated antiviral response through contact between gB and an as-yet-unidentified cell surface receptor(s).

Differential initiation of innate immune responses induced by human cytomegalovirus entry into fibroblast cells.

Infection of permissive fibroblasts with human CMV (HCMV, AD169) is accompanied by a robust activation of innate immune defense. In this study, we show that inflammatory cytokine (IC) secretion and activation of the type I IFN pathway (αβ IFN) are initiated through distinct mechanisms. HCMV is recognized by TLR2 leading to the NF-κB activation and IC secretion. However, the IFN response to HCMV is not a TLR2-dependent process, as a dominant negative TLR2 does not affect the antiviral response to infection. Additionally, bafilomycin, an endosomal acidification inhibitor, has no effect on HCMV-induced IFN responses suggesting that IFN signaling is independent of endosomal resident TLRs. By contrast, disruption of lipid rafts by depletion of cellular cholesterol inhibits both HCMV entry as well as IFN responses. Cholesterol depletion had no effect on the induction of ICs by HCMV, illustrating a biological distinction at the cellular level with the initiation of innate immune pathways. Furthermore, HCMV entry inhibitors block IFN responses but not IC signaling. In particular, blocking the interaction of HCMV with β1 integrin diminished IFN signaling, suggesting that this virus-cell interaction or subsequent downstream steps in the entry pathway are critical for downstream signal transduction events. These data show that HCMV entry and IFN signaling are coordinated processes that require cholesterol-rich microdomains, whereas IC signaling is activated through outright sensing via TLR2. These findings further highlight the complexity and sophistication of innate immune responses at the earliest points in HCMV infection.

Reovirus infection triggers inflammatory responses to dietary antigens and development of celiac disease

A nonpathogenic virus can promote inflammatory immunity to dietary antigens and may be linked to the development of celiac disease. Viruses compound dietary pathology Reoviruses commonly infect humans and mice asymptomatically. Bouziat et al. found that immune responses to two gut-infecting reoviruses take different paths in mice (see the Perspective by Verdu and Caminero). Both reoviruses invoked protective immune responses, but for one reovirus, when infection happened in the presence of a dietary antigen (such as gluten or ovalbumin), tolerance to the dietary antigen was lost. This was because this strain prevented the formation of tolerogenic T cells. Instead, it promoted T helper 1 immunity to the dietary antigen through interferon regulatory factor 1 signaling. Celiac disease patients also exhibited elevated levels of antibodies against reovirus. Science, this issue p. 44; see also p. 29 Viral infections have been proposed to elicit pathological processes leading to the initiation of T helper 1 (TH1) immunity against dietary gluten and celiac disease (CeD). To test this hypothesis and gain insights into mechanisms underlying virus-induced loss of tolerance to dietary antigens, we developed a viral infection model that makes use of two reovirus strains that infect the intestine but differ in their immunopathological outcomes. Reovirus is an avirulent pathogen that elicits protective immunity, but we discovered that it can nonetheless disrupt intestinal immune homeostasis at inductive and effector sites of oral tolerance by suppressing peripheral regulatory T cell (pTreg) conversion and promoting TH1 immunity to dietary antigen. Initiation of TH1 immunity to dietary antigen was dependent on interferon regulatory factor 1 and dissociated from suppression of pTreg conversion, which was mediated by type-1 interferon. Last, our study in humans supports a role for infection with reovirus, a seemingly innocuous virus, in triggering the development of CeD.

Dual selection mechanisms drive efficient single-gene reverse genetics for rotavirus

Current methods for engineering the segmented double-stranded RNA genome of rotavirus (RV) are limited by inefficient recovery of the recombinant virus. In an effort to expand the utility of RV reverse genetics, we developed a method to recover recombinant viruses in which independent selection strategies are used to engineer single-gene replacements. We coupled a mutant SA11 RV encoding a temperature-sensitive (ts) defect in the NSP2 protein with RNAi-mediated degradation of NSP2 mRNAs to isolate a virus containing a single recombinant gene that evades both selection mechanisms. Recovery is rapid and simple; after two rounds of selective passage the recombinant virus reaches titers of ≥104 pfu/mL. We used this reverse genetics method to generate a panel of viruses with chimeric NSP2 genes. For one of the chimeric viruses, the introduced NSP2 sequence was obtained from a pathogenic, noncultivated human RV isolate, demonstrating that this reverse genetics system can be used to study the molecular biology of circulating RVs. Combining characterized RV ts mutants and validated siRNA targets should permit the extension of this “two-hit” reverse genetics methodology to other RV genes. Furthermore, application of a dual selection strategy to previously reported reverse genetics methods for RV may enhance the efficiency of recombinant virus recovery.

Reovirus nonstructural protein σ1s is required for establishment of viremia and systemic dissemination

Serotype-specific patterns of reovirus disease in the CNS of newborn mice segregate with the viral S1 gene segment, which encodes attachment protein σ1 and nonstructural protein σ1s. The importance of receptor recognition in target cell selection by reovirus implicates the σ1 protein as the primary effector of disease outcome. However, the contribution of σ1s to reovirus disease is unknown. To define the function of σ1s in reovirus pathogenesis, we generated a σ1s-deficient virus by altering a single nucleotide to disrupt the σ1s translational start site. Viruses were recovered that contain nine gene segments from strain type 3 Dearing and either the wild-type or σ1s-null S1 gene segment from strain type 1 Lang. Following peroral inoculation of newborn mice, both viruses replicated in the intestine, although the wild-type virus achieved higher yields than the σ1s-null virus. However, unlike the wild-type virus, the σ1s-deficient virus failed to disseminate to sites of secondary viral replication, including the brain, heart, and liver. Within the small intestine, both viruses were detected in Peyers patches, but only the wild-type virus reached the mesenteric lymph node. Concordantly, wild-type virus, but not σ1s-deficient virus, was detected in the blood of infected animals. Wild-type and σ1s-null viruses produced equivalent titers following intracranial inoculation, indicating that σ1s is dispensable for viral growth in the murine CNS. These results suggest a key role for σ1s in virus spread from intestinal lymphatics to the bloodstream, thereby allowing the establishment of viremia and dissemination to sites of secondary replication within the infected host.

Reverse genetics for mammalian reovirus

Mammalian orthoreoviruses (reoviruses) are highly tractable models for studies of viral replication and pathogenesis. The versatility of reovirus as an experimental model has been enhanced by development of a plasmid-based reverse genetics system. Infectious reovirus can be recovered from cells transfected with plasmids encoding cDNAs of each reovirus gene segment using a strategy that does not require helper virus and is independent of selection. In this system, transcription of each gene segment is driven by bacteriophage T7 RNA polymerase, which can be supplied transiently by recombinant vaccinia virus (rDIs-T7pol) or by cells that constitutively express the enzyme. Reverse genetics systems have been developed for two prototype reovirus strains, type 1 Lang (T1L) and type 3 Dearing (T3D). Each reovirus cDNA was encoded on an independent plasmid for the first-generation rescue system. The efficiency of virus recovery was enhanced in a second-generation system by combining the cDNAs for multiple reovirus gene segments onto single plasmids to reduce the number of plasmids from 10 to 4. The reduction in plasmid number and the use of baby hamster kidney cells that express T7 RNA polymerase increased the efficiency of viral rescue, reduced the incubation time required to recover infectious virus, and eliminated potential biosafety concerns associated with the use of recombinant vaccinia virus. Reovirus reverse genetics has been used to introduce mutations into viral capsid and nonstructural components to study viral protein-structure activity relationships and can be exploited to engineer recombinant reoviruses for vaccine and oncolytic applications.

The Reovirus 1s Protein Is a Determinant of Hematogenous but Not Neural Virus Dissemination in Mice

ABSTRACT Nonstructural protein σ1s is a critical determinant of hematogenous dissemination by type 1 reoviruses, which reach the central nervous system (CNS) by a strictly blood-borne route. However, it is not known whether σ1s contributes to neuropathogenesis of type 3 reoviruses, which disseminate by both vascular and neural pathways. Using isogenic type 3 viruses that vary only in σ1s expression, we observed that mice survived at a higher frequency following hind-limb inoculation with σ1s-null virus than when inoculated with wild-type virus. This finding suggests that σ1s is essential for reovirus virulence when inoculated at a site that requires systemic spread to cause disease. Wild-type and σ1s-null viruses produced comparable titers in the spinal cord, suggesting that σ1s is dispensable for invasion of the CNS. Although the two viruses ultimately achieved similar peak titers in the brain, loads of wild-type virus were substantially greater than those of the σ1s-null mutant at early times after inoculation. In contrast, wild-type virus produced substantially higher titers than the σ1s-null virus in peripheral organs to which reovirus spreads via the blood, including the heart, intestine, liver, and spleen. Concordantly, viral titers in the blood were higher following infection with wild-type virus than following infection with the σ1s-null mutant. These results suggest that differences in viral brain titers at early time points postinfection are due to limited virus delivery to the brain by hematogenous pathways. Transection of the sciatic nerve prior to hind-limb inoculation diminished viral spread to the spinal cord. However, wild-type virus retained the capacity to disseminate to the brain following sciatic nerve transection, indicating that wild-type reovirus can spread to the brain by the blood. Together, these results indicate that σ1s is not required for reovirus spread by neural mechanisms. Instead, σ1s mediates hematogenous dissemination within the infected host, which is required for full reovirus neurovirulence.

Nonstructural protein σ1s mediates reovirus-induced cell cycle arrest and apoptosis.

ABSTRACT Reovirus nonstructural protein σ1s is implicated in cell cycle arrest at the G2/M boundary and induction of apoptosis. However, the contribution of σ1s to these effects in an otherwise isogenic viral background has not been defined. To evaluate the role of σ1s in cell cycle arrest and apoptosis, we used reverse genetics to generate a σ1s-null reovirus. Following infection with wild-type virus, we observed an increase in the percentage of cells in G2/M, whereas the proportion of cells in G2/M following infection with the σ1s-null mutant was unaffected. Similarly, we found that the wild-type virus induced substantially greater levels of apoptosis than the σ1s-null mutant. These data indicate that σ1s is required for both reovirus-induced cell cycle arrest and apoptosis. To define sequences in σ1s that mediate these effects, we engineered viruses encoding C-terminal σ1s truncations by introducing stop codons in the σ1s open reading frame. We also generated viruses in which charged residues near the σ1s amino terminus were replaced individually or as a cluster with nonpolar residues. Analysis of these mutants revealed that amino acids 1 to 59 and the amino-terminal basic cluster are required for induction of both cell cycle arrest and apoptosis. Remarkably, viruses that fail to induce cell cycle arrest and apoptosis also are attenuated in vivo. Thus, identical sequences in σ1s are required for reovirus-induced cell cycle arrest, apoptosis, and pathogenesis. Collectively, these findings provide evidence that the σ1s-mediated properties are genetically linked and suggest that these effects are mechanistically related.