Emma Poole

HIV-1 Gag-RNA interaction occurs at a perinuclear/centrosomal site; analysis by confocal microscopy and FRET.

The Gag polyprotein is the major structural protein of human immunodeficiency virus‐1 (HIV‐1) constituting the viral core. Between translation on cytoplasmic polysomes and assembly into viral particles at the plasma membrane, it specifically captures the RNA genome of the virus through binding RNA structural motifs (packaging signals –Ψ) in the RNA. RNA is believed to be a structural facilitator of Gag assembly. Using a combined approach of immunofluorescence detection of Gag protein and in situ hybridisation detection of viral genomic RNA, we demonstrate that Gag protein colocalises early after expression with Ψ+ RNA in the perinuclear region and also colocalises with centrioles. Colocalised RNA and protein subsequently traffic through the cytoplasm to the plasma membrane of the cell. Gag expressed from Ψ– RNA diffuses throughout the cell. It is not found at centrioles and shows delayed cytoplasmic colocalisation with the RNA genome. RNA capture through Ψ does not influence binding of Gag to microfilaments. Gag does not bind to tubulin during export. The presence of the packaging signal may coordinate capture of Ψ+ RNA by Gag protein at the centrosome followed by their combined transport to the site of budding. HIV‐1 Ψ thus acts as a subcellular localisation signal as well as a high‐affinity‐binding site for Gag.

Role for Bovine Viral Diarrhea Virus Erns Glycoprotein in the Control of Activation of Beta Interferon by Double-Stranded RNA

ABSTRACT Production of alpha/beta interferon in response to viral double-stranded RNA (dsRNA) produced during viral replication is a first line of defense against viral infections. Here we demonstrate that the Erns glycoprotein of the pestivirus bovine viral diarrhea virus can act as an inhibitor of dsRNA-induced responses of cells. This effect is seen whether Erns is constitutively expressed in cells or exogenously added to the culture medium. The Erns effect is specific to dsRNA since activation of NF-κB in cells infected with Semliki Forest virus or treated with tumor necrosis factor alpha was not affected. We also show that Erns contains a dsRNA-binding activity, and its RNase is active against dsRNA at a low pH. Both the dsRNA binding and RNase activities are required for the inhibition of dsRNA signaling, and we discuss here a model to account for these observations.

Latency-Associated Degradation of the MRP1 Drug Transporter During Latent Human Cytomegalovirus Infection

Hide-and-Seek Virus Human cytomegalovirus (HCMV) establishes latent infection in human progenitor dendritic cells, causing significant morbidity and mortality on reactivation, which may occur in transplantation patients who are immunosuppressed. Neither detection nor selective removal of rare latent HCMV-infected cells has been possible. Weekes et al. (p. 199) have found that the multidrug-resistant ABC transporter, multidrug resistance–associated protein-1 (MRP1) is down-regulated during latent HCMV infection. Consequently, cytotoxic MRP1-specific substrates are not exported from HCMV-infected cells and accumulate—leading to cell death, which could potentially provide a mechanism for eliminating infected cells prior to transplantation. A virally encoded protein eliminates a cell surface drug transporter, promoting latent human cytomegalovirus infection. The reactivation of latent human cytomegalovirus (HCMV) infection after transplantation is associated with high morbidity and mortality. In vivo, myeloid cells and their progenitors are an important site of HCMV latency, whose establishment and/or maintenance require expression of the viral transcript UL138. Using stable isotope labeling by amino acids in cell culture–based mass spectrometry, we found a dramatic UL138-mediated loss of cell surface multidrug resistance–associated protein-1 (MRP1) and the reduction of substrate export by this transporter. Latency-associated loss of MRP1 and accumulation of the cytotoxic drug vincristine, an MRP1 substrate, depleted virus from naturally latent CD14+ and CD34+ progenitors, all of which are in vivo sites of latency. The UL138-mediated loss of MRP1 provides a marker for detecting latent HCMV infection and a therapeutic target for eliminating latently infected cells before transplantation.

The UL144 gene product of human cytomegalovirus activates NFκB via a TRAF6-dependent mechanism

Molecular mimicry of cytokines and cytokine receptors is a strategy used by poxviruses and herpesviruses to modulate host immunity. The human cytomegalovirus (HCMV) UL144 gene, situated in the UL/b′ region of the viral genome, has amino‐acid sequence similarity to members of the tumour necrosis factor receptor superfamily. We report that UL144 is a potent activator of NFκB‐induced transcription in a TRAF6‐dependent manner. This NFκB activation enhances expression of the chemokine CCL22 through the NFκB responsive elements found in its promoter. In contrast to the clinical HCMV isolates, extensively passaged laboratory strains lack the UL/b′ region and hence do not encode UL144. Consistent with this, infection with viruses that carry UL/b′ causes NFκB activation and CCL22 expression, a phenotype that is not observed after infections with strains lacking the UL/b′ region. Moreover, knockdown of UL144, TRAF6 or NFκB by specific siRNA in infections with UL144‐encoding HCMV prevents the activation of CCL22 expression normally observed after infection with UL/b′ positive HCMV. Upregulation of CCL22, which attracts Th2 and regulatory T cells, may help HCMV evade immune surveillance.

Virally induced changes in cellular microRNAs maintain latency of human cytomegalovirus in CD34+ progenitors

One site of latency of human cytomegalovirus (HCMV; human herpesvirus 5) is known to be CD34(+) haematopoietic progenitor cells, and it is likely that carriage of latent virus has profound effects on cellular gene expression in order to optimize latency and reactivation. As microRNAs (miRNAs) play important roles in regulating stem-cell gene expression, this study asked whether latent carriage of HCMV led to changes in cellular miRNA expression. A comprehensive miRNA screen showed the differential regulation of a number of cellular miRNAs during HCMV latency in CD34(+) progenitor cells. One of these, hsa-miR-92a, was robustly decreased in three independent miRNA screens. Latency-induced change in hsa-miR-92a results in an increase in expression of GATA-2 and subsequent increased expression of cellular IL-10, which aids the maintenance of latent viral genomes in CD34(+) cells, probably resulting from their increased survival.

Identification of TRIM23 as a Cofactor Involved in the Regulation of NF-κB by Human Cytomegalovirus

ABSTRACT Human cytomegalovirus (HCMV) regulates NF-κB during infection by a variety of mechanisms. For example, the HCMV gene product, UL144, is known to activate NF-κB in a tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6)-dependent manner, causing the upregulation of the chemokine CCL22 (MDC). Viral UL144 is expressed from the UL/b′ region of the HCMV genome at early times postinfection and is a TNFR1-like homologue. Despite this homology to the TNFR1 receptor superfamily, UL144 does not bind to members of the TNF ligand superfamily. We show here that the upregulation of NF-κB by UL144 is dependent upon cellular tripartite motif 23 (TRIM23) protein. We propose a mechanism by which UL144 activates NF-κB through a direct interaction with the cellular protein TRIM23 in a complex containing TRAF6. In contrast, TRIM23 is not involved in conventional double-stranded RNA signaling via NF-κB. Therefore, we present a novel role for TRIM23 that is specific to UL144-mediated activation of NF-κB during the course of virus infection.

Human cytomegalovirus latency alters the cellular secretome, inducing cluster of differentiation (CD)4+ T-cell migration and suppression of effector function

After primary infection, human cytomegalovirus (HCMV) persists as a life-long latent infection, with host immunosuppression often resulting in clinical reactivation. During lytic infection, major changes in the expression of secreted cellular proteins (the secretome) occur that have profound effects on host–cell interactions, particularly at the level of the host immune response. In contrast, little is known about changes in the secretome that accompany latent infection, yet this is likely to be of major importance for the life-long carriage of this persistent human pathogen in the face of constant immunosurveillance. We have analyzed the secretome of cells carrying latent HCMV and have identified changes in several secreted cellular proteins known to be involved in regulation of the immune response and chemoattraction. Here, we show that a latency-associated increase in CC chemokine ligand (CCL)8 results in the recruitment of cluster of differentiation (CD)4+ T cells to supernatants from latently infected CD34+ cells but that these latent supernatants, also rich in immunosuppressive factors, inhibit cytokine secretion and cytotoxicity of HCMV-specific T-helper (Th)1 CD4+ T cells. These results identify a strategy by which sites of latent HCMV can firstly recruit CD4+ T cells and then inhibit their antiviral effector functions, thereby aiding the maintenance of latent infection in the face of the host immune response.

Human cytomegalovirus latency-associated proteins elicit immune-suppressive IL-10 producing CD4⁺ T cells.

Human cytomegalovirus (HCMV) is a widely prevalent human herpesvirus, which, after primary infection, persists in the host for life. In healthy individuals, the virus is well controlled by the HCMV-specific T cell response. A key feature of this persistence, in the face of a normally robust host immune response, is the establishment of viral latency. In contrast to lytic infection, which is characterised by extensive viral gene expression and virus production, long-term latency in cells of the myeloid lineage is characterised by highly restricted expression of viral genes, including UL138 and LUNA. Here we report that both UL138 and LUNA-specific T cells were detectable directly ex vivo in healthy HCMV seropositive subjects and that this response is principally CD4+ T cell mediated. These UL138-specific CD4+ T cells are able to mediate MHC class II restricted cytotoxicity and, importantly, show IFNγ effector function in the context of both lytic and latent infection. Furthermore, in contrast to CD4+ T cells specific to antigens expressed solely during lytic infection, both the UL138 and LUNA-specific CD4+ T cell responses included CD4+ T cells that secreted the immunosuppressive cytokine cIL-10. We also show that cIL-10 expressing CD4+ T-cells are directed against latently expressed US28 and UL111A. Taken together, our data show that latency-associated gene products of HCMV generate CD4+ T cell responses in vivo, which are able to elicit effector function in response to both lytic and latently infected cells. Importantly and in contrast to CD4+ T cell populations, which recognise antigens solely expressed during lytic infection, include a subset of cells that secrete the immunosuppressive cytokine cIL-10. This suggests that HCMV skews the T cell responses to latency-associated antigens to one that is overall suppressive in order to sustain latent carriage in vivo.

The Myeloid Transcription Factor GATA-2 Regulates the Viral UL144 Gene during Human Cytomegalovirus Latency in an Isolate-Specific Manner

ABSTRACT It is generally accepted that, following primary infection, human cytomegalovirus (HCMV) establishes lifelong latency in CD34+ progenitor cells and other derivative cells of the myeloid lineage. In this study, we show that the viral UL144 gene is expressed during latent infection in two cell types of the myeloid lineage, CD34+ and CD14+ monocytes, and that the UL144 protein is functional in latently infected monocytes. However, this latency-associated expression of UL144 occurs only in certain isolates of HCMV and depends on the presence of functional GATA-2 transcription factor binding sites in the UL144 promoter, in contrast to the viral latency-associated gene LUNA, which we also show is regulated by GATA-2 but expressed uniformly during latent infection independent of the virus isolate. Taken together, these data suggest that the HCMV latency-associated transcriptome may be virus isolate specific and dependent on the repertoire of transcription factor binding sites in the promoters of latency-associated genes.

Sleepless latency of human cytomegalovirus

As with all human herpesviruses, human cytomegalovirus (HCMV) persists for the lifetime of the host by establishing a latent infection, which is broken by periodic reactivation events. One site of HCMV latency is in the progenitor cells of the myeloid lineage such as CD34+ cells and their CD14+ derivatives. The development of experimental techniques to isolate and culture these primary cells in vitro is enabling detailed analysis of the events that occur during virus latency and reactivation. Ex vivo differentiation of latently infected primary myeloid cells to dendritic cells and macrophages results in the reactivation of latent virus and provides model systems in which to analyse the viral and cellular functions involved in latent carriage and reactivation. Such analyses have shown that, in contrast to primary lytic infection or reactivation which is characterised by a regulated cascade of expression of all viral genes, latent infection is associated with a much more restricted viral transcription programme with expression of only a small number of viral genes. Additionally, concomitant changes in the expression of cellular miRNAs and cellular proteins occur, and this includes changes in the expression of a number of secreted cellular proteins and intracellular anti-apoptotic proteins, which all have profound effects on the latently infected cells. In this review, we concentrate on the effects of one of the latency-associated viral proteins, LAcmvIL-10, and describe how it causes a decrease in the cellular miRNA, hsa-miR-92a, and a concomitant upregulation of the GATA2 myeloid transcription factor, which, in turn, drives the expression of cellular IL-10. Taken together, we argue that HCMV latency, rather than a period of viral quiescence, is associated with the virally driven manipulation of host cell functions, perhaps every bit as complex as lytic infection. A full understanding of these changes in cellular and viral gene expression during latent infection could have far-reaching implications for therapeutic intervention.