Characterization of virus-induced interferon-gamma responses in mice previously infected with murine cytomegalovirus.

These studies demonstrate that in vitro stimulation of spleen cells from murine cytomegalovirus (MCMV) immune mice with MCMV-infected fibroblasts induces production of interferon (IFN)-gamma. This response is specific to MCMV, is not generalized to heterologous viruses, and also is not H-2 restricted. Both early and late CMV antigens induce IFN-gamma. In in vitro cell depletion and direct cell selection experiments, T lymphocytes were responsible for IFN-gamma production. Although both CD4 and CD8 cells appear to be required to induce the response, the cell subset that releases the IFN-gamma is not yet undefined. In vivo, this IFN-gamma response appears early after acute infection and persists > or =1 year. The response is not seen in T cell-deficient mice. Thus, previous MCMV infection results in a virus-specific IFN-gamma response in spleen cells exposed to MCMV antigens. The pathophysiologic significance of these observations is now under study.

The murine cytomegalovirus M25 open reading frame encodes a component of the tegument.

The murine cytomegalovirus (MCMV) monoclonal antibody 5C7:6 was used in Western analysis to probe MCMV infected murine embryo cells (MEC). This antibody recognizes three virus specific polypeptides of 130, 105, and 95 kDa and pulse-chase experiments demonstrated that these three proteins, although antigenically related, are distinct. The 105- and 95-kDa species were expressed with early kinetics, whereas the 130-kDa protein was synthesized as a true late. By screening a lambdagt11 MCMV cDNA library, the gene encoding these proteins was identified as the M25 open reading frame previously reported by Dallas et al. (Dallas, P. B., Lyons, P. A., Hudson, J. B., Scalzo, A. A., and Shellam, G. R., 1994, Virology 200, 643-650). Immunofluorescent studies monitored the location of pM25, present in the nucleus at 15 h after infection, condensing around the periphery of the nucleus at 18 h, before finally accumulating in the cytoplasm. Immunoelectron microscopy detected gold particles associated with the viral tegument of enveloped virions located in the cytoplasm and extracellular space but not with naked nucleocapsids. Western analysis of MCMV purified virions depicted the presence of the 130-kDa protein, the predominant M25 species, in mature virus particles. Together these findings provide compelling evidence that the 130-kDa M25 polypeptide is a component of the viral tegument.

Sequence requirements for the nuclear localization of the murine cytomegalovirus M44 gene product pp50.

The murine cytomegalovirus (MCMV) M44 gene product pp50 is normally present in the nuclei of virus-infected cells. During transient expression of pp50 in COS-1 cells, the phosphoprotein was readily detectable in the nuclei, indicating that it possesses a nuclear localization signal (NLS). Studies on the subcellular locations of N- and C-terminal deletion mutants of pp50 suggested that alterations in both the C terminus and the highly conserved N-terminal domains of pp50 affect nuclear localization. In particular, the C-terminal 11 amino acids of pp50, which includes a "KKQK" motif, were able to mediate the import of a beta-galactosidase fusion protein into the nucleus. The pair of lysine residues in this motif constitutes an essential element of the C-terminal NLS as mutation of this motif to AAQK directly affected the nuclear localization of either pp50 or beta-galactosidase fusion proteins containing the C-terminal portion of pp50. Furthermore our results indicated that the functionality of the C-terminal NLS is dependent on the structural integrity of the highly conserved N-terminal portion of the molecule, as deletion of amino acids 157-201 alone adversely affected nuclear localization. In the absence of a functional C-terminal NLS, the subcellular localization of pp50 is sensitive to potential conformational changes induced by mutations within the N-terminal half of the molecule. Under those circumstances, mutation of the YK residues at position 22-23 or deletion of amino acids 267-283 was sufficient to produce a protein that was impaired in nuclear import or retention.

Chronic infection of human umbilical vein endothelial cells by human herpesvirus-6.

Human herpesvirus-6 (HHV-6) exhibits a predominant tropism for CD4+ T-lymphocytes, but can infect other components of the blood as well as surrounding tissue and organs. To understand the role of the endothelium in the transmission and haematogenous spread of this virus, human umbilical vein endothelial cells (HUVEC) were infected with HHV-6 and monitored for viral gene expression. The presence of both early and late viral antigens was demonstrated by indirect immunofluorescence in 37.6 and 6.5%, respectively, of HUVEC. However, attempts to detect the release of infectious virus were not successful, indicating infection is semipermissive in nature. Upon continued passage of infected HUVEC monolayers, HHV-6 antigen-positive cells persisted up to 27 days post-infection. Furthermore, the virus could be recovered from HUVEC monolayers that contained fewer than 1% antigen-positive cells by co-cultivation with peripheral blood mononuclear cells. Together, these findings suggest that endothelial cells may serve as a reservoir for harbouring HHV-6.

Altered production of GM-CSF and IL-8 in cytomegalovirus-infected, IL-1-primed umbilical cord endothelial cells.

The human cytomegaloviruses (HCMVs) appear to have the potential to disrupt production of hematopoietic cytokines. We examined the production of granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-8 by cultured and CMV-infected human umbilical vein endothelial cells (HUVECs) and compared this production with that of uninfected cells. Endothelial cells are, among other things, an integral component of human bone marrow stroma, and are responsible for production of factors that modulate the proliferation and differentiation of human hematopoietic progenitors. HCMV infection increased the production of GM-CSF in IL-1-primed HUVECs without altering GM-CSF levels in infected but unprimed HUVECs. However, this same virus was capable of causing increased production of the inhibitory cytokine IL-8. Both the viral pellet and the cleared viral supernatant appeared to contribute equally to the increased IL-8 and GM-CSF production, because each of these preparations alone was capable of exerting only half the effect seen with whole virus preparations. That both live virus and soluble protein factors within the viral stock contributed to the enhancement in GM-CSF and IL-8 production was further confirmed by inactivation with either ultraviolet or heat treatment of the viral stocks. Although the identity of the factor within the HCMV stock that contributes to this effect remains unknown, studies conducted in the presence of neutralizing antibodies or polymyxin B ruled out a role for tumor necrosis factor-alpha, IL-6, or endotoxin, all known inducers of GM-CSF. These studies indicate that HCMVs can exert both direct and indirect effects on the production of the hematopoietic factor GM-CSF and the inflammatory/inhibitory cytokine IL-8.

Murine cytomegalovirus replication in the lungs of athymic BALB/c nude mice.

Murine cytomegalovirus (MCMV) infection in the lungs of T cell-deficient athymic BALB/c (Nu/ Nu) mice and their immunocompetent heterozygous (Nu/+) littermates was examined. Following intranasal inoculation, MCMV replicated in the lungs of both Nu/Nu and Nu/+ mice, but virus titers were significantly higher in T cell-deficient mice. After subcutaneous inoculation, virus disseminated to lung tissue of athymic mice, leading to progressive MCMV replication in lungs that was not seen in the immunocompetent mice. Athymic mice failed to develop an antibody response to MCMV. Histologically, athymic mice uniformly developed focal interstitial cellular aggregates adjacent to blood vessels or airways, which progressively enlarged and coalesced. Pneumonitis was not seen in the lungs of any Nu/+ mice. Thus, MCMV can replicate in the lungs without pneumonitis in immunocompetent mice, but MCMV produces a progressive focal pneumonitis during deficiency of T cell-mediated immunity.

Intracellular localization of the herpes simplex virus type-1 origin binding protein, UL9.

UL9 is the origin binding protein of herpes simplex virus type-1 (HSV-1). A UL9-specific monoclonal antibody (17B) whose epitope maps to the N-terminal 33 amino acids was used to study the localization of UL9 in infected and transfected cells. We demonstrate the colocalization of UL9 and the HSV-1 single-strand DNA binding protein (ICP8 or UL29) in replication compartments, sites of viral DNA synthesis. On the other hand, UL9 does not completely colocalize with ICP8 in prereplicative sites, structures observed under conditions that inhibit viral DNA polymerase. Cells transfected with various deletion or pyruvate kinase fusion constructs were analyzed by indirect immunofluorescence assay to define the nuclear localization signal (NLS) of UL9. Deletion analysis showed that the region required for nuclear localization lies within the C-terminal DNA binding domian (amino acids 535-851). Various regions of UL9 were tested in fusion constructs for their ability to direct the normally cytoplasmic chicken pyruvate kinase protein to the nucleus. A fusion construct containing the carboxy-terminal 107 residues (amino acids 745-851) localized efficiently to the nucleus, whereas a fusion construct containing the N-terminal 660 amino acids of UL9 was unable to do so. Mutations designed to alter a potential NLS sequence (793-KREFAGARFKLR-804) within the C-terminal 107 residues result in a mutant UL9 protein which falls to localize efficiently to the nucleus. These results suggest that the major NLS of UL9 maps within the C-terminal 107 amino acids.

Evaluation of PCR and nested PCR for laboratory diagnosis of hepatitis C virus infection.

The detection of hepatitis C virus (HCV) RNA by nested polymerase chain reaction (PCR) is believed to be the most reliable method to diagnose HCV infections. A pitfall of nested PCR is that it is prone to contamination. Single step reverse transcription-PCR (RT-PCR) was performed, prospectively, on 80 sera from 59 patients with a set of primers that amplified a 273 bp sequence unique to the 5' noncoding (NC) region of the HCV genome. Nested PCR, was performed on all PCR negative specimens with a set of primers that amplified a 255 bp internal to the original primers. Single step RT-PCR was positive on 45 sera from 35 patients following gel electrophoresis and on two additional sera from two patients following Southern blot hybridization. Nested PCR was positive on two more sera following gel electrophoresis of the nested PCR products. These two patients were seropositive and subsequent serum from one patient was positive by single step PCR. Three additional sera were positive following Southern blot analysis of the nested PCR products. Two patients were seropositive and had elevated serum alanine aminotransferase (ALT) levels. The third patient was seronegative with normal ALT level and was considered a false positive. The remaining seronegative control specimens were PCR negative by both methods. The majority of PCR positive patients (82%) had elevated ALT levels, while the majority of PCR negative seropositive patients had normal ALT levels. We conclude that single step PCR is a sensitive test for the laboratory diagnoses of the majority of the HCV infections.

A rapid and sensitive radioimmunoassay for the detection of human cytomegalovirus binding and infection of human fibroblasts.

A rapid and sensitive radioimmunoassay for the quantitation of HCMV binding and infection of human fibroblasts (HFF) was developed. The protocol involves the use of a monoclonal antibody (27-156) reactive with HCMV gB (alpha-gB), followed by an 125I-labeled second antibody to mouse IgG. Antibody to gB bound specifically to HFF inoculated with HCMV when compared to sham inoculated cells or cells inoculated with HSV (strain KOS). Antibody to gB also bound to HFF infected with HCMV 48 h prior to assay. The binding of antibody to HFF inoculated with HCMV was found to be dependent on antibody concentration and to demonstrate saturable kinetics. Moreover, antibody binding was directly dependent on the concentration of the virus inoculum, using either conventional viral preparations or gradient purified HCMV. The binding of antibody to HFF inoculated with HCMV at 4 degrees C was found to be dependent on antibody concentration and to demonstrate saturable kinetics. Displacement of HCMV binding to HFF with the proteoglycan heparin sulfate could be detected, thus allowing for competitive binding studies. This binding assay allows for the relative quantitation of HCMV binding to cells and will be useful for examining the early events of cell-viral interactions.

Replication of human cytomegalovirus in cells deficient in beta 2-microglobulin gene expression.

To study the roles of beta 2-microglobulin (beta 2-m) and major histocompatibility complex (MHC) class I expression in human cytomegalovirus (HCMV) infection, the ability of HCMV strain AD-169 to infect and replicate in a human melanoma cell line (FO-1), which is beta 2-m-deficient and cannot express MHC class I on its cell surface, was examined. Susceptibility of FO-1 cells was compared with human foreskin fibroblasts (HFF) and FO-1H cells (FO-1 cells that have been transfected with the human beta 2-m gene, restoring MHC I expression on the cell surface). As judged by the HCMV immediate early 1 (IE-1) antigen expression, HCMV was able to infect FO-1 cells, although somewhat less efficiently than HFF. However, the expression of HCMV late (L) antigen and the production of virus was significantly less for FO-1 cells than for HFF. Analysis of the FO-1H transfectants revealed that expression of IE-1 and L HCMV antigens was comparable to FO-1 cells, which lack MHC I. Treatment of FO-1 and FO-1H cells with sodium butyrate prior to inoculation did not alter the expression of MHC I in either cell type, but did increase susceptibility of both cell types to HCMV infection, as well as the expression of L antigens and production of virus. These studies indicate that HCMV infection of FO-1 cells is independent of beta 2-m and MHC class I expression.

The role of tumor necrosis factor-alpha in acute murine cytomegalovirus infection in BALB/c mice.

The role of tumor necrosis factor-alpha (TNF alpha) in acute lethal and sublethal murine cytomegalovirus (MCMV) infection in BALB/c mice was examined. During the course of acute infection, TNF alpha was not detectable in the serum or bronchoalveolar lavage (BAL) fluids, while TNF alpha was uniformly detected in both serum and BAL following intravenous administration of lipopolysaccharide (LPS). Administration of recombinant murine (rMu) TNF alpha did not consistently alter the virus content of tissues during acute infection. Passive transfer of purified polyclonal immunoglobulin containing neutralizing antibody to TNF alpha did not alter mortality or MCMV replication in tissues during acute infection but did block the TNF alpha response when LPS was administered to BALB/c mice. Thus, TNF alpha appears to play little role in the course and outcome of acute MCMV infection.

The effect of short-chain fatty acids on the susceptibility of human umbilical vein endothelial cells to human cytomegalovirus infection.

We have compared the replication of three strains of human cytomegalovirus (HCMV), HCMV AD-169, HCMV Towne, or HCMV RC-256, an insertional mutant of Towne containing the LacZ gene of E. coli, in human umbilical vein endothelial cells (HUVEC) and human forskin fibroblasts (HFF). We also examine the effects of salts of short-chain fatty acids on the susceptibility of HUVEC to infection by HCMV. All three virus strains replicated in both cell types, but 10-to 100-fold less virus was produced in HUVEC cells than HFF. For all virus strains, expression of HCMV IE-1 antigen in HFF was > 70% 24 h after inoculation. In contrast, the number of HUVEC exhibiting IE-1 antigen at 24 h was < 15%. Treatment of HUVEC with sodium butyrate, sodium hexanoate, or sodium propionate prior to virus inoculation increased the IE-1 and late HCMV antigen expression in a dose- and time-dependent manner. Virus yield was also increased. This increased susceptibility was inhibited by cycloheximide and tunicamycin, indicating a requirement for new cellular protein synthesis. Treatment with both sodium hexanoate and propionate after virus inoculation increased HUVEC susceptibility to HCMV infection. Treatment of HUVEC with sodium butyrate after virus inoculation also increased HCMV IE-1 antigen expression, but only after removal of the drug. These studies demonstrate that the susceptibility of HUVEC to HCMV infection can be increased by the treatment of the host cell with salts of short-chain fatty acids, such as sodium butyrate, before or after virus inoculation.

Acute murine cytomegalovirus infection induces lethal hepatitis.

Events were examined that might contribute to mortality in acute murine cytomegalovirus (MCMV) infection after intraperitoneal inoculation. Specifically, viral replication in the liver, spleen, and pancreas and the concomitant biochemical abnormalities induced by MCMV during lethal and nonlethal acute viral infection were compared. Mortality was limited to susceptible strains of mice infected by the intraperitoneal (ip) route. In addition, the virus content of the lung, liver, spleen, and pancreas was 100- to 1000-fold greater with lethal infection in the ip-infected group than in those with nonlethal infection. Serum transaminase and lipase levels were markedly elevated in susceptible mice inoculated with MCMV ip. Histopathologic and immunocytochemical changes in the liver, coupled with elevated serum transaminase levels indicating severe hepatitis, appear sufficient to explain the early mortality seen with the ip route of infection.

Characterization of a 52K protein of murine cytomegalovirus and its immunological cross-reactivity with the DNA-binding protein ICP36 of human cytomegalovirus.

We have developed a hybridoma, designated 25G11, which produced a monoclonal antibody (MAb) reactive with a 52K protein of murine cytomegalovirus (MCMV). This MAb, 25G11, was reactive with a protein band of 52K in MCMV-infected cell lysates and with a protein of 49K in human CMV (HCMV)-infected cell lysates as detected by immunoblot analysis. With purified MCMV virions, 25G11 gave a faintly immunoreactive band of 52K. However, no immunoreactive protein band was detected with purified HCMV virions, nor with purified HCMV or MCMV envelope preparations. By immunocytochemistry, 25G11 detected viral antigen primarily in the nucleus of HCMV- or MCMV-infected cells. The antibody 25G11 was used to screen a lambda gt11 library of HCMV DNA fragments. One of the isolated clones (lambda 32323B) was employed for gene mapping on the HCMV genome, which suggested that the immunoreactive HCMV protein was the DNA-binding protein (ICP36). Analysis of the recombinant fusion protein with antibody 25G11 and with an MAb (CH16) specific for an HCMV DNA-binding protein confirmed the identity of the cross-reacting protein as ICP36. Furthermore, we found that whereas the epitope recognized by 25G11 was conserved between HCMV and MCMV proteins, the epitope recognized by CH16 was unique to HCMV and thus represents a variable region in the protein.

Murine models of cytomegalovirus-associated pneumonitis.

Data derived from the murine models described in this report now serve as the cornerstone for our understanding of at least some of the mechanisms by which MCMV in combination with other cofactors can trigger the series of events that result in the development of interstitial pneumonia in the MCMV-infected mouse. What is clear from the studies completed to date is that MCMV by itself is not particularly pathogenic when it replicates in the lung, but in the presence of a perturbation in the immune system of the host, can produce serious pulmonary disease. It is hoped that the knowledge gained from these studies in mice will someday be shown to be relevant to humans.

Synergistic effect of murine cytomegalovirus on the induction of acute graft-vs-host disease involving MHC class I differences only. Analysis of in vitro T cell function.

The development of acute graft-vs-host disease (GVHD) is a common outcome after the injection of fully MHC disparate parental T cells into unirradiated F1 mice. Murine cytomegalovirus (MCMV) infection has been previously shown to augment the development of acute GVHD in the parent-into-F1 (P----F1) model, such that 10-fold fewer parental cells are required. In the present study, we have investigated the effect of MCMV infection on the induction of non-lethal GVHD that occurs in P----F1 combinations involving MHC class I only or class II only differences. Using P----F1 combinations involving either an H-2K only difference or an H-2D only difference, MCMV infection of F1 mice 3 days before the injection of parental spleen cells led to a profound T cell immunodeficiency that strongly resembled that observed in acute GVHD. Further studies examining the H-2K disparate P----F1 combination, C57Bl/6---- (C57Bl/6xB6.C-H-2bm1) F1 and combined MCMV infection showed that the immunodeficiency is characterized by a profound loss of in vitro Th cell production of IL-2 and an intrinsic defect in T effector function as shown by an inability of rIL-2 to restore defective CTL responses. Additional experiments in these mice revealed the presence of suppressor cells as well as significant parent-anti-F1 CTL activity possibly accounting for the suppressor effect. This pattern of immunodeficiency was not seen after the administration of either MCMV or MHC class I disparate parental cells alone. MCMV infection did not detectably alter the immunodeficiency observed in a P----F1 combination involving a MHC class II difference only. These results indicate that MCMV infection can alter the pattern of GVHD in the setting of an MHC class I disparity, but not in the setting of class II disparity, such that it resembles acute GVHD. These results may have relevance to the human transplant setting where intercurrent CMV infection has been associated with an adverse clinical outcome.

Reovirus type 3 binds to antagonist domains of the beta-adrenergic receptor.

Reovirus type 3 interfered with the binding of beta-adrenergic antagonist ligands to receptors on Y1 adrenal, C6 glioma, and mouse L cells. This inhibition of beta-adrenergic binding was dose related. Reovirus did not interfere with dopaminergic binding or isoproterenol-induced activation of adenylate cyclase. In addition, reovirus infected Y1 cells, which bind beta-adrenergic antagonist ligands but lack agonist-induced activity. These results suggest that reovirus infection is initiated by binding to antagonist (nonfunctional) domains of the adrenergic receptor complex.

In vivo administration of monoclonal antibody to the NK 1.1 antigen of natural killer cells: effect on acute murine cytomegalovirus infection.

Monoclonal antibody to the NK 1.1 antigen, found on the natural killer cells of a number of strains of mice, specifically suppresses NK cell function when given in vivo. Using this monoclonal antibody, we have examined the effects of specific suppression of natural killer (NK) cells in vivo on acute murine cytomegalovirus (MCMV) infection in C57BL/10ScN mice. Administration of antibody to NK 1.1 substantially lowered the resistance of C57BL/10ScN mice to lethal virus challenge. In addition, antibody administration prior to intraperitoneal infection significantly increased MCMV replication in salivary glands, lungs, and spleens. In C3H/HeN mice, a strain that lacks the NK 1.1 antigen, antibody to NK 1.1 had no effect on virus replication or lethal infection. Thus, in vivo administration of monoclonal antibody to NK 1.1 alters the course of acute MCMV infection. These findings further substantiate the role of NK cells in defense against acute MCMV infection.