Peter A. Barry

Potent Immunosuppressive Activities of Cytomegalovirus-Encoded Interleukin-10

ABSTRACT Cytomegalovirus (CMV) has highly evolved mechanisms for avoiding detection by the host immune system. Recently, in the genomes of human and primate CMV, a novel gene comprising segments of noncontiguous open reading frames was identified and found to have limited predicted homology to endogenous cellular interleukin-10 (IL-10). Here we investigate the biological activities of the CMV IL-10-like gene product and show it to possess potent immunosuppressive properties. Both purified bacterium-derived recombinant CMV IL-10 and CMV IL-10 expressed in supernatants of human cells were found to inhibit proliferation of mitogen-stimulated peripheral blood mononuclear cells (PBMCs), with specific activity comparable to that of recombinant human IL-10. In addition, CMV IL-10 expressed from human cells inhibited cytokine synthesis, as treatment of stimulated PBMCs and monocytes with CMV IL-10 led to a marked decrease in production of proinflammatory cytokines. Finally, CMV IL-10 was observed to decrease cell surface expression of both major histocompatibility complex (MHC) class I and class II molecules, while conversely increasing expression of the nonclassical MHC allele HLA-G. These results demonstrate for the first time that CMV has a biologically active IL-10 homolog that may contribute to immune evasion during virus infection.

Human Cytomegalovirus-Encoded Interleukin-10 Homolog Inhibits Maturation of Dendritic Cells and Alters Their Functionality

ABSTRACT Interleukin-10 (IL-10) suppresses the maturation and cytokine production of dendritic cells (DCs), key regulators of adaptive immunity, and prevents the activation and polarization of naïve T cells towards protective gamma interferon-producing effectors. We hypothesized that human cytomegalovirus (HCMV) utilizes its viral IL-10 homolog (cmvIL-10) to attenuate DC functionality, thereby subverting the efficient induction of antiviral immune responses. RNA and protein analyses demonstrated that the cmvIL-10 gene was expressed with late gene kinetics. Treatment of immature DCs (iDCs) with supernatant from HCMV-infected cultures inhibited both the lipopolysaccharide-induced DC maturation and proinflammatory cytokine production. These inhibitory effects were specifically mediated through the IL-10 receptor and were not observed when DCs were treated with supernatant of cells infected with a cmvIL-10-knockout mutant. Incubation of iDCs with recombinant cmvIL-10 recapitulated the inhibition of maturation. Furthermore, cmvIL-10 had pronounced long-term effects on those DCs that could overcome this inhibition of maturation. It enhanced the migration of mature DCs (mDCs) towards the lymph node homing chemokine but greatly reduced their cytokine production. The inability of mDCs to secrete IL-12 was maintained, even when they were restimulated by the activated T-cell signal CD40 ligand in the absence of cmvIL-10. Importantly, cmvIL-10 potentiates these anti-inflammatory effects, at least partially, by inducing endogenous cellular IL-10 expression in DCs. Collectively, we show that cmvIL-10 causes long-term functional alterations at all stages of DC activation.

B-Virus (Cercopithecine herpesvirus 1) Infection in Humans and Macaques: Potential for Zoonotic Disease

Nonhuman primates are widely used in biomedical research because of their genetic, anatomic, and physiologic similarities to humans. In this setting, human contact directly with macaques or with their tissues and fluids sometimes occurs. Cercopithecine herpesvirus 1 (B virus), an alphaherpesvirus endemic in Asian macaques, is closely related to herpes simplex virus (HSV). Most macaques carry B virus without overt signs of disease. However, zoonotic infection with B virus in humans usually results in fatal encephalomyelitis or severe neurologic impairment. Although the incidence of human infection with B virus is low, a death rate of >70% before the availability of antiviral therapy makes this virus a serious zoonotic threat. Knowledge of the clinical signs and risk factors for human B-virus disease allows early initiation of antiviral therapy and prevents severe disease or death.

Crystal structure of human cytomegalovirus IL-10 bound to soluble human IL-10R1.

Human IL-10 (hIL-10) modulates critical immune and inflammatory responses by way of interactions with its high- (IL-10R1) and low-affinity (IL-10R2) cell surface receptors. Human cytomegalovirus exploits the IL-10 signaling pathway by expressing a functional viral IL-10 homolog (cmvIL-10), which shares only 27% sequence identity with hIL-10 yet signals through IL-10R1 and IL-10R2. To define the molecular basis of this virus–host interaction, we determined the 2.7-Å crystal structure of cmvIL-10 bound to the extracellular fragment of IL-10R1 (sIL-10R1). The structure reveals cmvIL-10 forms a disulfide-linked homodimer that binds two sIL-10R1 molecules. Although cmvIL-10 and hIL-10 share similar intertwined topologies and sIL-10R1 binding sites, their respective interdomain angles differ by ∼40°. This difference results in a striking re-organization of the IL-10R1s in the putative cell surface complex. Solution binding studies show cmvIL-10 and hIL-10 share essentially identical affinities for sIL-10R1 whereas the Epstein–Barr virus IL-10 homolog (ebvIL-10), whose structure is highly similar to hIL-10, exhibits a ∼20-fold reduction in sIL-10R1 affinity. Our results suggest cmvIL-10 and ebvIL-10 have evolved different molecular mechanisms to engage the IL-10 receptors that ultimately enhance the respective ability of their virus to escape immune detection.

Virus-Encoded Homologs of Cellular Interleukin-10 and Their Control of Host Immune Function

The capacity of viruses to successfully infect the immunocompetent host to cause disease argues in favor of virus-encoded functions that specifically target components of the immune system so as to orchestrate an environment that limits the capacity of the host immune response to clear infection. In this respect, many viruses have evolved to coexist with the host immune system by developing an arsenal of strategies to avoid immune surveillance and elimination from the host. These include viruses which have acquired homologs of cellular cytokines or cytokine receptors as a strategy to limit host immune recognition. Cellular interleukin-10 (IL-10) is a pleiotropic immunomodulatory cytokine produced by a wide variety of cells, including monocytes, macrophages, T and B lymphocytes, dendritic cells (DC), keratinocytes, epithelial cells, and mast cells. The properties of IL-10 have been comprehensively reviewed elsewhere (12, 69, 70, 72, 77) and so will not be covered in detail here, but the key features of this cytokine relate mainly to its capacity to exert potent immunosuppressive functions on the expression of a range of cytokines and chemokines (2, 16, 23), as well as the repression of major histocompatibility complex (MHC) molecules and costimulatory molecules (17, 104) and the maturation and function of DC (69). The immunosuppressive properties of IL-10 are primarily restricted to cells of the myeloid lineage (69). In contrast, IL-10 has been shown to exert a stimulatory effect on B lymphocytes (15, 28, 87), mast cells (99), thymocytes (64), and CD8+ T cells (31, 88, 90), highlighting the cell-type-dependent immunomodulatory properties of this cytokine. The immunomodulatory functions manifested by IL-10 require engagement of this cytokine with its cell surface bound receptor. The IL-10 receptor (IL-10R) consists of two different subunits (IL-10R1 and IL-10R2) (52, 60). IL-10 binds first to IL-10R1 with high affinity, and the resulting intermediate IL-10/IL-10R1 complex then binds with lower affinity to IL-10R2. The resulting active signaling complex induces the JAK/Stat signal transduction pathway (69, 72). In the context of viral pathogenesis, infections with a number of different viruses have been documented to upregulate the expression of IL-10, and in some cases, this upregulation has been shown to enhance infection by suppressing the immune function, suggesting that the far-reaching effects of this cytokine have many advantages for invading pathogens (3, 4, 18, 30, 45, 81, 82, 108, 111). IL-10-like open reading frames (ORF) have been identified by sequence homology in multiple members of the Herpesvirales and Poxviridae, all but one of which infect mammalian hosts (Table ​(Table1).1). The one exception, cyprinid herpesvirus 3, is a member of the Alloherpesviridae family of herpesviruses (order, Herpesvirales), which has the common carp (Cyprinus carpio) as its normal host (1). Otherwise, all of the herpesviruses identified to date as encoding IL-10-like ORF, including human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV), are found only in the Beta- and Gammaherpesvirinae subfamilies of the Herpesviridae. No examples of the Alphaherpesvirinae that encode IL-10 homologs have been identified. Of the identified members of the Herpesviridae that encode IL-10 homologs, all but two, equid herpesvirus 2 and ovine herpesvirus 2 (OvHV2) (85, 96, 97), are confined to primate hosts (Table ​(Table1).1). All of the identified Poxviridae that encode IL-10 infect ruminants, including orf virus ([ORFV] sheep and goats), sheeppox virus ([SPPV] sheep), goatpox virus ([GPV] goats), and lumpy skin disease virus ([LSDV] cattle). The genomes of SPPV, GPV, and LSDV are 96 to 97% identical at the nucleic acid level (48, 101). Another member of the Poxviridae that has monkeys as its normal host, yaba-like disease virus, encodes an ORF (Y134R) that exhibits sequence homology to the IL-10-related cytokines IL-19, IL-20, and IL-24 (57). These viral IL-10 (vIL-10) homologs range in size from 139 to 191 amino acids (aa), bracketing the range in sizes for cellular IL-10 proteins (176 to 180 aa). TABLE 1. Viruses with IL-10-like or IL-10-related ORF The role of virus-encoded IL-10 homologs is likely to provide a tool to enable modulation of the local immune response so as to enhance the capacity to replicate, disseminate, and/or persist in an otherwise immunocompetent individual. In fact, there is emerging evidence that virus-encoded IL-10 homologs function in this capacity in a variety of settings. This review will cover those viruses which have thus far been identified as encoding homologs of IL-10. The similarities of each homolog to the IL-10 of the natural host species will be presented together with their biological functions (where known) and the role they may play in viral pathogenesis and evasion of the host immune response.

Temple monkeys and health implications of commensalism, Kathmandu, Nepal

Humans in contact with macaques risk exposure to enzootic primateborne viruses.

Regulation of gene expression directed by the long terminal repeat of the feline immunodeficiency virus

The long terminal repeat (LTR) of a retrovirus contains sequence elements that constitute a promoter for controlling viral gene expression in infected cells. We have examined regulation of LTR-directed gene expression in feline immunodeficiency virus (FIV), a T-lymphocytopathic lentivirus associated with a fatal AIDS-like disease in domestic cats. Two independent virus isolates, designated FIV-Petaluma and FIV-PPR, have been molecularly cloned and show greater than 85% sequence homology. Both clones (termed pF34 and pPPR) produce infectious virus after transfection of permissive feline cells. Basal promoter activity of the LTRs was measured in various cell lines in transient expression assays using plasmids containing the viral LTR linked to the bacterial chloramphenicol acetyltransferase gene. Both LTRs were strong promoters in several cell lines, although in some cell lines the pF34 LTR had four- to fivefold higher basal activity than the pPPR LTR. FIV LTR mutations affecting the first AP4 site, AP1 site, ATF site, or NF-kappa B site resulted in decreased basal activity of the FIV promoter. Mutational analysis also revealed a negative regulatory element. In cotransfection experiments, both pF34 proviral DNA and pPPR proviral DNA appeared to transactivate either the pF34 LTR or the pPPR LTR; however, levels of transactivation were very low. Cotransfection of both LTRs with FIV subgenomic clones containing various viral open reading frames resulted in low level or no transactivation. The LTRs of both FIV clones responded to cell activation signals in human T-lymphoid cells (Jurkat) treated with phytohemagglutinin and phorbol-12-myristate-13-acetate. Promoter function of both FIV LTRs was also enhanced in cells treated with either forskolin, an inducer of intracellular cyclic-AMP (c-AMP), or dibutyryl c-AMP. Analysis of site-specific mutants showed that a potential AP1 site in the U3 domain of the LTR was required for T-cell activation responses mediated by protein kinase C, whereas a putative ATF site was the target for c-AMP-induced responses mediated by protein kinase A. These studies revealed that cellular transcription factors play a significant role in regulation of FIV gene expression.

Cloning of the full-length rhesus cytomegalovirus genome as an infectious and self-excisable bacterial artificial chromosome for analysis of viral pathogenesis.

ABSTRACT Rigorous investigation of many functions encoded by cytomegaloviruses (CMVs) requires analysis in the context of virus-host interactions. To facilitate the construction of rhesus CMV (RhCMV) mutants for in vivo studies, a bacterial artificial chromosome (BAC) containing an enhanced green fluorescent protein (EGFP) cassette was engineered into the intergenic region between unique short 1 (US1) and US2 of the full-length viral genome by Cre/lox-mediated recombination. Infectious virions were recovered from rhesus fibroblasts transfected with pRhCMV/BAC-EGFP. However, peak virus yields of cells infected with reconstituted progeny were 10-fold lower than wild-type RhCMV, suggesting that inclusion of the 9-kb BAC sequence impeded viral replication. Accordingly, pRhCMV/BAC-EGFP was further modified to enable efficient excision of the BAC vector from the viral genome after transfection into mammalian cells. Allelic exchange was performed in bacteria to substitute the cre recombinase gene for egfp. Transfection of rhesus fibroblasts with pRhCMV/BAC-Cre resulted in a pure progeny population lacking the vector backbone without the need of further manipulation. The genomic structure of the BAC-reconstituted virus, RhCMV-loxP(r), was identical to that of wild-type RhCMV except for the residual loxP site. The presence of the loxP sequence did not alter the expression profiles of neighboring open reading frames. In addition, RhCMV-loxP(r) replicated with wild-type kinetics both in tissue culture and seronegative immunocompetent macaques. Restriction analysis of the viral genome present within individual BAC clones and virions revealed that (i) RhCMV exhibits a simple genome structure and that (ii) there is a variable number of a 750-bp iterative sequence present at the S terminus.

Experimental Coinfection of Rhesus Macaques with Rhesus Cytomegalovirus and Simian Immunodeficiency Virus: Pathogenesis

ABSTRACT Human cytomegalovirus (HCMV) possesses low pathogenic potential in an immunocompetent host. In the immunosuppressed host, however, a wide spectrum of infection outcomes, ranging from asymptomatic to life threatening, can follow either primary or nonprimary infection. The variability in the manifestations of HCMV infection in immunosuppressed individuals implies that there is a threshold of host antiviral immunity that can effectively limit disease potential. We used a nonhuman primate model of CMV infection to assess the relationship between CMV disease and the levels of developing anti-CMV immunity. Naive rhesus macaques were inoculated with rhesus cytomegalovirus (RhCMV) followed 2 or 11 weeks later by inoculation with pathogenic simian immunodeficiency virus SIVmac239. Two of four monkeys inoculated with SIV at 2 weeks after inoculation with RhCMV died within 11 weeks with simian AIDS (SAIDS), including activated RhCMV infection. Neither animal had detectable anti-SIV antibodies. The other two animals died 17 and 27 weeks after SIV inoculation with either SAIDS or early lymphoid depletion, although no histological evidence of activated RhCMV was observed. Both had weak anti-SIV antibody titers. RhCMV antibody responses for this group of monkeys were significantly below those of control animals inoculated with only RhCMV. In addition, all animals of this group had persistent RhCMV DNA in plasma and high copy numbers of RhCMV in tissues. In contrast, animals that were inoculated with SIV at 11 weeks after RhCMV infection rarely exhibited RhCMV DNA in plasma, had low copy numbers of RhCMV DNA in most tissues, and did not develop early onset of SAIDS or activated RhCMV. SIV antibody titers were mostly robust and sustained in these monkeys. SIV inoculation blunted further development of RhCMV humoral responses, unlike the normal pattern of development in control monkeys following RhCMV inoculation. Anti-RhCMV immunoglobulin G levels and avidity were slightly below control values, but levels maintained were higher than those observed following SIV infection at 2 weeks after RhCMV inoculation. These findings demonstrate that SIV produces long-lasting insults to the humoral immune system beginning very early after SIV infection. The results also indicate that anti-RhCMV immune development at 11 weeks after infection was sufficient to protect the host from acute RhCMV sequelae following SIV infection, in contrast to the lack of protection afforded by only 2 weeks of immune response to RhCMV. As previously observed, monkeys that were not able to mount a significant immune response to SIV were the most susceptible to SAIDS, including activated RhCMV infection. Rapid development of SAIDS in animals inoculated with SIV 2 weeks after RhCMV inoculation suggests that RhCMV can augment SIV pathogenesis, particularly during primary infection by both viruses.

Neuropathogenesis induced by rhesus cytomegalovirus in fetal rhesus monkeys (Macaca mulatta)

Rhesus cytomegalovirus (RhCMV) infection of rhesus macaques offers opportunities to analyze mechanisms of CMV pathogenesis in a primate species. Four fetal rhesus monkeys were inoculated intraperitoneally with RhCMV early in the second trimester, and pregnancies were terminated by hysterotomy during the third trimester. Three fetuses had evidence of severe CMV disease, including intrauterine growth restriction, ventriculomegaly, microcephaly, lissencephaly, and extensive degenerative changes of the cerebral parenchyma. Histopathologic examination revealed polymicrogyria, gliosis, leptomeningitis, periventricular calcifications, and inclusion-bearing cells. These results demonstrate that the developing macaque brain is susceptible to infection with RhCMV early in the second trimester and that intrauterine infection results in neuropathologic outcomes similar to those observed in humans congenitally infected with CMV.