Jacquelyn S. Slater

The salivary glands as a privileged site of cytomegalovirus immune evasion and persistence

The salivary glands (SG) provide a haven for persistent cytomegalovirus replication, and in this regard are a privileged site of virus immune evasion. The murine cytomegalovirus (MCMV) model has provided insight into the immunological environment of the SG and the unqiue virus–host relationship of this organ. In response to MCMV infection, a robust T cell-mediated immune response is elicited, comprised predominantly of CD8+ T cells that phenotypically and functionally appear activated. However, they fail to clear virus by an unknown evasion mechanism that is independent of inhibitory NKG2A- or Programmed Death 1-mediated signaling. Virus is eventually eliminated from the SG by effector CD4+ T cells expressing antiviral cytokines. However, this mechanism is severely dampened by high levels of the immunosuppressive cytokine IL-10, selectively expressed by SG CD4+ T cells.

Vigorous Innate and Virus-Specific Cytotoxic T-Lymphocyte Responses to Murine Cytomegalovirus in the Submaxillary Salivary Gland

ABSTRACT To better understand the immunological mechanisms that permit prolonged shedding of murine cytomegalovirus (MCMV) from the salivary gland, the phenotypic and functional characteristics of leukocytes infiltrating the submaxillary gland (SMG) were analyzed in infected BALB/c mice. A robust innate immune response, comprised of CD11c+ major histocompatibility complex class II+ CD11b− CD8α+ dendritic cells and γ/δ T-cell receptor-bearing CD3+ T cells was prominent through at least 28 days postinfection. Concurrently, a dramatic increase in pan-NK (DX5+) CD3+ and CD8+ T cells was observed, while CD4+ T cells, known to be essential for viral clearance from this tissue, increased slightly. The expression particularly of gamma interferon but also of interleukin-10 and CC chemokines was extraordinarily high in the SMG in response to MCMV infection. The gamma interferon was produced primarily by CD4+ and CD8+ T lymphocytes and DX5+ CD3+ T cells. The SMG CD8+ T cells were highly cytolytic ex vivo, and a significant proportion of these cells were specific to an immunodominant MCMV peptide. These peptide-specific clones were not exhausted by the presence of high virus titers, which persisted in the SMG despite the strength of the cell-mediated responses. In contrast, MCMV replication was efficiently cleared from the draining cervical and periglandular lymph nodes, a tissue displaying a substantially weaker antiviral response. Our data indicated that vigorous innate and acquired immune responses are elicited, activated, and retained in response to mucosal inflammation from persistent MCMV infection of the submaxillary gland.

Products of US22 Genes M140 and M141 Confer Efficient Replication of Murine Cytomegalovirus in Macrophages and Spleen

ABSTRACT Efficient replication of murine cytomegalovirus (MCMV) in macrophages is a prerequisite for optimal growth and spread of the virus in its natural host. Simultaneous deletion of US22 gene family members M139, M140, and M141 results in impaired replication of MCMV in macrophages and mice. In this study, we characterized the proteins derived from these three genes and examined the impact of individual gene deletions on viral pathogenesis. The M139, M140, and M141 gene products were identified as early proteins that localize to both the nucleus and cytoplasm in infected cells. Gene M139 encodes two proteins, of 72 and 61 kDa, while M140 and M141 each encode a single protein of 56 (pM140) and 52 (pM141) kDa, respectively. No role for the M139 proteins in MCMV replication in macrophages or mice was determined in these studies. In contrast, deletion of either M140 or M141 resulted in impaired MCMV replication in macrophages and spleen tissue. Replication of the M140 deletion mutant was significantly more impaired than that of the virus lacking M141. Further analyses revealed that the absence of the pM140 adversely affected pM141 levels by rendering the latter protein unstable. Since the replication defect due to deletion of M140 was more profound than could be explained by the reduced half-life of pM141, pM140 must exert an additional, independent function in mediating efficient replication of MCMV in macrophages and spleen tissue. These data indicate that the US22 genes M140 and M141 function both cooperatively and independently to regulate MCMV replication in a cell type-specific manner and, thus, to influence viral pathogenesis.

Murine cytomegalovirus-induced suppression of antigen-specific cytotoxic T lymphocyte maturation

Antigen-specific cytotoxic T lymphocyte (CTL) maturation was inhibited in mice acutely infected with murine cytomegalovirus (MCMV). When immunization with Simian virus 40 (SV40) either preceded or followed infection with MCMV by 1 day, the frequency of SV40-specific CTL precursors among lymph node cells (LNC) was significantly reduced compared to noninfected mice. Replication of the herpesvirus in LNC could not be detected; however, MCMV rendered noninfectious by heat treatment was not suppressive to CTL development. Lymph node cells from nonimmunized, MCMV-infected mice contained a cell(s) present in low frequency which suppressed in vitro maturation of SV40 CTL in immune lymph nodes from mice not infected with MCMV. These suppressor cells did not affect the antigen- and interleukin-2-dependent growth nor cytotoxic activity of a mature, SV40-specific CTL clone. These results indicate that MCMV interferes with immunoregulatory functions required for development of antigen-specific CTL precursors to mature effector CTL. The immunosuppression is mediated at least in part by the MCMV-induced suppressor cell(s).

Complex Formation among Murine Cytomegalovirus US22 Proteins Encoded by Genes M139, M140, and M141

ABSTRACT The murine cytomegalovirus (MCMV) proteins encoded by US22 genes M139, M140, and M141 function, at least in part, to regulate replication of this virus in macrophages. Mutant MCMV having one or more of these genes deleted replicates poorly in macrophages in culture and in the macrophage-dense environment of the spleen. In this report, we demonstrate the existence of stable complexes formed by the products of all three of these US22 genes, as well as a complex composed of the products of M140 and M141. These complexes form in the absence of other viral proteins; however, the pM140/pM141 complex serves as a requisite binding partner for the M139 gene products. Products from all three genes colocalize to a perinuclear region of the cell juxtaposed to or within the cis-Golgi region but excluded from the trans-Golgi region. Interestingly, expression of pM141 redirects pM140 from its predominantly nuclear residence to the perinuclear, cytoplasmic locale where these US22 proteins apparently exist in complex. Thus, complexing of these nonessential, early MCMV proteins likely confers a function(s) independent of each individual protein and important for optimal replication of MCMV in its natural host.

Murine cytomegalovirus independently inhibits priming of helper and cytotoxic T lymphocytes

Murine cytomegalovirus (MCMV) inhibits antigen-specific cytotoxic T lymphocyte (CTL) priming in vivo (Campbell et al., 1989). To address the mechanism of this immune suppression, two possibilities were considered: (1) MCMV directly interferes with in vivo priming of CTL precursors (CTLp), or (2) MCMV suppresses T helper cell functions necessary for CTL priming. We therefore quantitated T helper cell function in MCMV-infected, SV40-immune mice and assessed dependency of SV40-specific CTLp priming on T helper cell activity. MCMV infection of H-2b mice significantly suppressed the frequency of IL-2 producing T helper cells generated in SV40-immune mice. This suppression was not due to alterations in the number or percentage of CD4 lymphocytes. The helper cell deficiency correlated with suppressed SV40-specific CTL activity. However, CTLp priming in vivo was found to be independent of CD4 T helper cells and IL-2. Therefore, the suppressive effects of MCMV on helper and cytotoxic T cell functions are independent, implying that MCMV directly inhibits an event in lymphocyte priming common to both helper and cytotoxic T cells.

Enhancement of interleukin-1 activity by murine cytomegalovirus infection of a macrophage cell line

The effect of murine cytomegalovirus (MCMV) infection on interleukin 1 (IL-1) secretion was assessed using the macrophage cell lines P388D1 and J774A.1. The former proved to be nonpermissive for MCMV in that infectious virus and viral immediate early protein (pp89) were not expressed in these cells. MCMV infection of the P388D1 cells had no effect on release of biologically active IL-1. In contrast, J774A.1 cells, which were semipermissive for virus replication and pp89 expression, secreted enhanced levels of IL-1 activity following infection. The enhancement was evident when infection either preceded or followed lipopolysaccharide stimulation of the macrophages. The relative proportion of IL-1 alpha and beta secreted from MCMV-infected cells was similar to noninfected controls. In addition, the bioactivity of intracellular IL-1 alpha escaping membranes of fixed cells was unaffected by virus infection. From these findings, we conclude that limited MCMV expression in the J774A.1 macrophage cell line enhances secretion of IL-1 alpha and beta bioactivity.

Murine Cytomegalovirus US22 Protein pM140 Protects Its Binding Partner, pM141, from Proteasome-Dependent but Ubiquitin-Independent Degradation

ABSTRACT Stable assembly of murine cytomegalovirus (MCMV) virions in differentiated macrophages is dependent upon the expression of US22 family gene M140. The M140 protein (pM140) exists in complex with products of neighboring US22 genes. Here we report that pM140 protects its binding partner, pM141, from ubiquitin-independent proteasomal degradation. Protection is conferred by a stabilization domain mapping to amino acids 306 to 380 within pM140, and this domain is functionally independent from the region that confers binding of pM140 to pM141. The M140 protein thus contains multiple domains that collectively confer a structure necessary to function in virion assembly in macrophages.