Biomedical subjects

E S Mocarski

Publications and source records attributed to E S Mocarski.

At least 19 recordsLinked to original sources

PubMed2002

Fatal attraction: cytomegalovirus-encoded chemokine homologs.

Members of the cytomegalovirus (CMV) subfamily of betaherpesviruses infecting primates and rodents encode divergent proteins with sequence characteristics and activities of chemokines, a class of small, secreted proteins that control leukocyte migration and trafficking behavior. Human CMV genes UL146 and UL147 encode proteins with sequence characteristics of CXC chemokines, whereas, murine CMV encodes a CC chemokine homolog (MCK-2). Human CMV UL146 encodes a neutrophil-attracting chemokine denoted viral CXC chemokine-1 (vCXCL1) that is as potent as host IL-8 and functions via the CXCR2 receptor, one of two human IL-8 receptors. Murine CMV MCK-2 is composed of a chemokine domain derived from open reading frame (ORF) m131 (and denoted MCK-1) as well as a domain derived from m129 that does not have sequence similarity to any known class of proteins. A synthetic version of murine CMV m131 (MCK-1) protein carries out many of the activities of a positive-acting chemokine, including transient release of intracellular calcium stores and cell adhesion of peritoneal macrophage populations. In the context of the viral genome and infection of the mouse host, the m131-m129 (MCK-2) gene product confers increased inflammation, higher levels of viremia, and higher titers of virus in salivary glands, consistent with a role in promoting dissemination by attracting an important mononuclear leukocyte population. Other characterized primate CMVs, but not other primate betaherpesviruses, encode gene products similar to human UL146 and UL147. Other characterized rodent CMVs encode a gene product similar to the murine CMV chemokine homolog, although not as a spliced gene product. Thus chemokines, like viral proteins that downmodulate MHC class I expression or have sequence homology to host MHC class I proteins, have evolved in primate and rodent CMVs to carry out an analogous set of immunomodulatory functions during infection of the host even though they arise from distinct origins.

Animals↗

PubMed2001

A cytomegalovirus-encoded inhibitor of apoptosis that suppresses caspase-8 activation.

We have identified a human cytomegalovirus cell-death suppressor, denoted vICA, encoded by the viral UL36 gene. vICA inhibits Fas-mediated apoptosis by binding to the pro-domain of caspase-8 and preventing its activation. vICA does not share significant sequence homology with FLIPs or other known suppressors of apoptosis, suggesting that this protein represents a new class of cell-death suppressors. Notably, resistance to Fas-mediated apoptosis is delayed in fibroblasts infected with viruses that encode mutant vICA, suggesting that vICA suppresses death-receptor-induced cell death in the context of viral infection. Although vICA is dispensable for viral replication in vitro, the common targeting of caspase-8 activation by diverse herpesviruses argues for an important role for this antiapoptotic mechanism in the pathogenesis of viral infection in the host, most likely in avoiding immune clearance by cytotoxic lymphocytes and natural killer cells.

Amino Acid Sequence↗

PubMed2001

Distinct and separate roles for herpesvirus-conserved UL97 kinase in cytomegalovirus DNA synthesis and encapsidation.

The human cytomegalovirus UL97 kinase, an important target of antiviral therapy, has an impact on at least two distinct phases of viral replication. Compared with wild-type virus, the UL97 deletion mutant exhibits an early replication defect that reduces DNA accumulation by 4- to 6-fold, as well as a late capsid maturation defect responsible for most of the observed 100- to 1000-fold reduction in replication. Block-release experiments with the antiviral 2-bromo-5,6-dichloro-1-(beta-D-ribofuranosyl)-benzimidazole revealed an important role for UL97 kinase in capsid assembly. Although cleavage of concatemeric DNA intermediates to unit-length genomes remained unaffected, progeny mutant virus maturation was delayed, with accumulation of progeny at significantly reduced levels compared with wild type after release of this block. Transmission electron microscopy confirmed the aberrant accumulation of empty A-like capsids containing neither viral DNA nor an internal scaffold structure, consistent with a failure to stably package DNA in mutant virus-infected cells. The function of UL97 in DNA synthesis as well as capsid assembly suggests that protein phosphorylation mediated by this herpesvirus-conserved kinase increases the efficiency of these two distinct phases of virus replication.

Capsid↗

PubMed2001

Multicenter evaluation of PCR methods for detecting CMV DNA in blood donors.

BACKGROUND: CMV DNA screening may be a useful adjunct to serologic tests in distinguishing potentially infectious blood donations from those that are "CMV-safe." However, there is currently no consensus on the optimal assay method for accurate detection of CMV DNA in donors. STUDY DESIGN AND METHODS: A blinded multicenter evaluation of seven CMV PCR assays was performed by five laboratories by using coded sets of analytical controls and donor blood samples. RESULTS: Five assays displayed sufficient sensitivity for donor screening, as judged by consistent detection of a minimum of 25 CMV genome equivalents (geq) in analytical controls constructed to contain from 1 to 100 CMV geq in background DNA from 250,000 cells, while the other two assays displayed inadequate sensitivity. Three sensitive assays, two based on nested PCR directed at the UL93 and UL32 regions of the CMV genome and another test (Monitor Assay, Roche), did not detect CMV DNA in samples from any of 20 pedigreed CMV-seronegative, Western blot-negative (S-/WB-) donors. Two other assays based on nested PCR occasionally detected CMV DNA in S-WB- samples, and one sensitive nested PCR assay directed at UL123 detected CMV DNA in a large proportion (85%) of S-WB- samples. CONCLUSION: Seven CMV PCR assays currently used for research and/or diagnostic applications displayed marked variations in sensitivity, specificity, and reproducibility when applied to coded analytical and clinical control samples containing cellular DNA from the equivalent of 250,000 WBCs. These results will be useful in the selection of assays with performance characteristics appropriate to donor screening objectives. They may also help explain discrepant findings from previous studies that used PCR to determine CMV DNA prevalence in seronegative and seropositive blood donors.

AIDS-Related Opportunistic Infections↗

PubMed2001

Requirement for uracil-DNA glycosylase during the transition to late-phase cytomegalovirus DNA replication.

Cytomegalovirus gene UL114, a homolog of mammalian uracil-DNA glycosylase (UNG), is required for efficient viral DNA replication. In quiescent fibroblasts, UNG mutant virus replication is delayed for 48 h and follows the virus-induced expression of cellular UNG. In contrast, mutant virus replication proceeds without delay in actively growing fibroblasts that express host cell UNG. In the absence of viral or host cell UNG expression, mutant virus fails to proceed to late-phase DNA replication, characterized by rapid DNA amplification. The data suggest that uracil incorporated early during wild-type viral DNA replication must be removed by virus or host UNG prior to late-phase amplification and encapsidation into progeny virions. The process of uracil incorporation and excision may introduce strand breaks to facilitate the transition from early-phase replication to late-phase amplification.

Amino Acid Sequence↗

PubMed2001

Murine cytomegalovirus CC chemokine homolog MCK-2 (m131-129) is a determinant of dissemination that increases inflammation at initial sites of infection.

The murine cytomegalovirus CC chemokine homolog MCK-2 (m131-129) is an important determinant of dissemination during primary infection. Reduced peak levels of viremia at day 5 were followed by reduced levels of virus in salivary glands starting at day 7 when mck insertion (RM461) and point (RM4511) mutants were compared to mck-expressing viruses. A dramatic MCK-2-enhanced inflammation occurred at the inoculation site over the first few days of infection, preceding viremia. The data further reinforce the role of MCK-2 as a proinflammatory signal that recruits leukocytes to increase the efficiency of viral dissemination in the host.

Animals↗

PubMed2000

Humoral immune response to proteins of human cytomegalovirus latency-associated transcripts.

Latent human cytomegalovirus (CMV) infection of hematopoietic progenitor cells is associated with the presence of latency-associated transcripts that may express 6 proteins larger than 44 amino acids in size (open reading frame [ORF] 55, ORF45, ORF94, ORF59, ORF154, ORF152/UL124). The serologic response to these proteins was evaluated in healthy seropositive individuals as well as in individuals undergoing active CMV infection. Individual recombinant GST-fusion proteins, prepared from bacteria, were found by enzyme-linked immunosorbent assay to be recognized by between 8% and 44% long-term healthy seropositive individuals, with ORF94 and ORF55 being the most broadly and significantly recognized. Although nearly all of serum samples (85%) recognized at least 1 of these proteins, none reacted with all 6. Patterns of antibody prevalence to these proteins in long-term seropositive individuals were similar to many antigens expressed during productive replication (IE1, ppUL57, ppUL83/pp65), but none were broadly detected by a majority of individuals, a characteristic of only a few productive-phase antigens, including ppUL44/ICP36 and ppUL32/pp150. Consistent with prevalence in long-term seropositive individuals, commercial preparations of pooled human gamma globulin were also found to recognize latency-associated proteins. Serologic reactivity to latency-associated proteins was slow to develop following primary infection, in a pattern distinct from any of the characterized replication-phase proteins tested here, and was boosted late after secondary infection or reactivation in solid-organ transplant recipients without showing a correlation with viremia or disease. These results provide evidence that proteins expressed from the latent region during natural infection exhibit immunogenicity comparable with most other characterized viral antigens, although the narrow response to individual latency-associated proteins likely precludes their use in serologic assays to investigate clinical correlates or outcome in transplant recipients.

Antibodies, Viral↗

PubMed2000

Human cytomegalovirus latency-associated protein pORF94 is dispensable for productive and latent infection.

Human cytomegalovirus latency in bone marrow-derived myeloid progenitors is characterized by the presence of latency-associated transcripts encoded in the ie1/ie2 region of the viral genome. To assess the role of ORF94 (UL126a), a conserved open reading frame on these transcripts, a recombinant virus (RC2710) unable to express this gene was constructed. This virus replicated at wild-type levels and expressed productive as well as latency-associated ie1/ie2 region transcripts. During latency in granulocyte-macrophage progenitors, RC2710 DNA was detected at levels indistinguishable from wild-type virus, latent-phase transcription was present, and RC2710 reactivated when latently infected cells were cocultured with permissive fibroblasts. These data suggest pORF94 is not required for either productive or latent infection as assayed in cultured cells despite being the only known nuclear latency-associated protein.

Base Sequence↗

PubMed1999

A cytomegalovirus-encoded mitochondria-localized inhibitor of apoptosis structurally unrelated to Bcl-2.

Human cytomegalovirus (CMV), a herpesvirus that causes congenital disease and opportunistic infections in immunocompromised individuals, encodes functions that facilitate efficient viral propagation by altering host cell behavior. Here we show that CMV blocks apoptosis mediated by death receptors and encodes a mitochondria-localized inhibitor of apoptosis, denoted vMIA, capable of suppressing apoptosis induced by diverse stimuli. vMIA, a product of the viral UL37 gene, inhibits Fas-mediated apoptosis at a point downstream of caspase-8 activation and Bid cleavage but upstream of cytochrome c release, while residing in mitochondria and associating with adenine nucleotide translocator. These functional properties resemble those ascribed to Bcl-2; however, the absence of sequence similarity to Bcl-2 or any other known cell death suppressors suggests that vMIA defines a previously undescribed class of anti-apoptotic proteins.

Apoptosis↗

PubMed1999

Cytomegalovirus-encoded beta chemokine promotes monocyte-associated viremia in the host.

Chemokine homologs are encoded by many large DNA viruses, suggesting that they contribute to control of host leukocyte transmigration and trafficking during viral infection. Murine cytomegalovirus carries a CC (beta) chemokine homolog gene giving rise to two related proteins, murine cytomegalovirus chemokine 1 and 2 (MCK-1 and MCK-2). MCK-1 peptide was found to induce calcium signaling and adherence in murine peritoneal macrophages. Cells bearing human chemokine receptor CCR3 and the human macrophage THP1 cell line were responsive to MCK-1. This pattern suggested that MCK-1 might act as an agonist, promoting leukocyte trafficking during viral infection. Consistent with this prediction, MCK-1/MCK-2 mutant viruses exhibit dramatically reduced peak levels of monocyte-associated viremia in experimentally infected mice. Thus, MCK-1/MCK-2 appears to promote host leukocyte migration to initial sites of infection and may be responsible for attracting monocytes or macrophages that efficiently disseminate virus in the host.

Amino Acid Sequence↗

PubMed1999

Cytomegalovirus encodes a potent alpha chemokine.

Cytomegalovirus is a widespread opportunistic pathogen affecting immunocompromised individuals in whom neutrophils may mediate virus dissemination and contribute to progression of disease. Recent sequence analysis suggests that genes absent or altered in attenuated strains may influence pathogenesis. We have found two genes, UL146 and UL147, whose products have sequence similarity to alpha (CXC) chemokines. UL146 encodes a protein, designated vCXC-1, that is a 117-aa glycoprotein secreted into the culture medium as a late gene product, where its presence correlates with the ability to attract human neutrophils. Recombinant vCXC-1 is a fully functional chemokine, inducing calcium mobilization, chemotaxis, and degranulation of neutrophils. High-affinity vCXC-1 binding is shown to be mediated via CXCR2, but not CXCR1. vCXC-1 exhibits a potency approaching that of human IL-8. As the first example of a virus-encoded alpha chemokine, vCXC-1 may ensure the active recruitment of neutrophils during cytomegalovirus infection, thereby providing for efficient dissemination during acute infection and accounting for the prominence of this leukocyte subset in cytomegalovirus disease.

Amino Acid Sequence↗

PubMed1999

Tetracycline-mediated regulation of gene expression within the human cytomegalovirus genome.

To evaluate the utility of tetracycline gene regulation in the study of human cytomegalovirus gene functions, expression of luciferase under the control of tetracycline-regulatable promoters was studied following transient plasmid transfections and from within recombinant human cytomegalovirus genomes. The tetracycline-regulatable promoter PhCMV*-1 contains sequences from the human cytomegalovirus ie1/ie2 promoter and seven upstream tet operator sites which bind the activator protein tTA only in the absence of tetracycline (Gossen and Bujard (1992). Proc. Natl. Acad. Sci. USA 89, 5547-5551). Two modifications of PhCMV*-1 were also studied: P1129, in which the tet operator sites were reduced from seven to one; and P1125, in which human cytomegalovirus sequences were replaced by adenovirus major late promoter and terminal deoxynucleotidyltransferase initiator sequences. In transient assays, PhCMV*-1 and P1125 exhibited modest differential regulation but were strongly activated by viral infection. P1129 exhibited less viral activation and narrower regulation. In the viral genome, PhCMV*-1 exhibited regulation up to 7-fold during late times of infection, whereas P1125 displayed nearly 100-fold regulation. Regulation of P1125 was fully reversed within 12 to 24 h of adding or removing tetracycline. These results suggest that P1125 may provide sufficient conditional expression to effectively regulate human cytomegalovirus late genes.

Cells, Cultured↗

PubMed1999

Peripheral blood CD14(+) cells from healthy subjects carry a circular conformation of latent cytomegalovirus genome.

The majority of the human population harbors latent cytomegalovirus. Although CD14(+) peripheral blood mononuclear cells have been implicated as sites of latency, the conformation of the latent viral genome in these cells is unknown. In this study, the conformation of viral genomic DNA was assessed in CD14(+) cells from healthy virus seropositive carriers using an electrophoretic separation on native agarose gels in combination with polymerase chain reaction detection. Here we show that the viral genome migrates as a circular plasmid with a mobility equivalent to a circular 230-kb Shigella flexneri megaplasmid marker. Neither linear nor complex or integrated forms of the viral genome were detected. This report provides further evidence that the CD14(+) cell population is an important site of viral latency in the naturally infected human host. Detection of the viral genome as a circular plasmid during latency suggests that this virus maintains its genome in a manner analogous to other herpesviruses where latent viral genome conformation has been studied.

Carrier State↗

PubMed1999

Quantitative analysis of latent human cytomegalovirus.

Cytomegalovirus latency depends on an interaction with hematopoietic cells in bone marrow and peripheral blood. The distribution of viral DNA was investigated by PCR-driven in situ hybridization (PCR-ISH), and the number of viral genomes per cell was estimated by quantitative competitive PCR during both experimental and natural latent infection. During experimental latent infection of cultured granulocyte-macrophage progenitors, the viral genome was detected in >90% of cells at a copy number of 1 to 8 viral genomes per cell. During natural infection, viral genomes were detected in 0.004 to 0.01% of mononuclear cells from granulocyte colony-stimulating factor-mobilized peripheral blood or bone marrow from seropositive donors, at a copy number of 2 to 13 genomes per infected cell. When evaluated by reverse transcription-PCR-ISH, only a small proportion of experimentally infected cells (approximately 2%) had detectable latent transcripts. This investigation identifies the small percentage of bone marrow-derived mononuclear cells that become latently infected during natural infection and suggests that latency may proceed in some cells that fail to encode currently identified latent transcripts.

Cells, Cultured↗

PubMed1998

Modulation of natural killer cell cytotoxicity in human cytomegalovirus infection: the role of endogenous class I major histocompatibility complex and a viral class I homolog.

Natural killer (NK) cells have been implicated in early immune responses against certain viruses, including cytomegalovirus (CMV). CMV causes downregulation of class I major histocompatibility complex (MHC) expression in infected cells; however, it has been proposed that a class I MHC homolog encoded by CMV, UL18, may act as a surrogate ligand to prevent NK cell lysis of CMV-infected cells. In this study, we examined the role of UL18 in NK cell recognition and lysis using fibroblasts infected with either wild-type or UL18 knockout CMV virus, and by using cell lines transfected with the UL18 gene. In both systems, the expression of UL18 resulted in the enhanced killing of target cells. We also show that the enhanced killing is due to both UL18-dependent and -independent mechanisms, and that the killer cell inhibitory receptors (KIRs) and CD94/NKG2A inhibitory receptors for MHC class I do not play a role in affecting susceptibility of CMV-infected fibroblasts to NK cell-mediated cytotoxicity.

Antigens, Viral↗

PubMed1998

Cytomegalovirus remains latent in a common precursor of dendritic and myeloid cells.

Hematopoietic cells and their progenitors play important roles in human cytomegalovirus latency and reactivation. Latent infection has been evaluated in defined populations of myeloid-lineage-committed progenitor cells coexpressing CD33 and CD15 or CD33 and CD14 along with the dendritic cell markers CD1a and CD10. These CD33+ cell populations were found to support latency and expression of viral latency-associated transcripts and to undergo reactivation of productive viral replication when differentiated in the presence of human fibroblasts. Reactivation was also observed when myeloid cells were carried in the presence of fibroblast-conditioned medium or medium supplemented with certain cytokines (interferon gamma, tumor necrosis factor alpha, interleukin 4, or granulocyte-macrophage colony-simulating factor), suggesting that cell differentiation pathways act as determinants of reactivation. More primitive CD34+ hematopoietic cells were also found to be susceptible to viral infection and latency was maintained as these cells differentiated into CD33+-lineage-committed populations. Between 0.01% and 0.001% of CD33+ CD14+ or CD33+ CD15+ bone marrow mononuclear cells isolated from naturally infected individuals were found to express latent transcripts. Thus, cytomegalovirus is carried within a small percentage of myeloid and dendritic cell progenitors in the healthy seropositive host. Virus reactivation may be triggered by factors associated with the inflammatory response.

Antigens, CD↗

PubMed1998

Defective growth correlates with reduced accumulation of a viral DNA replication protein after low-multiplicity infection by a human cytomegalovirus ie1 mutant.

To investigate the importance of the IE1 p72 regulatory protein during human cytomegalovirus replication, a recombinant virus unable to synthesize IE1 p72 was constructed. The Towne strain mutant CR208 lacked exon 4 of the major immediate-early gene and was isolated and complemented in an IE1-expressing immortalized human fibroblast line (ihfie1.3). Replication of CR208 in primary human fibroblasts was completed after an input multiplicity of 10 PFU/cell but was severely-impaired at 0.1 PFU/cell. CR208 formed plaques with lower efficiency on primary fibroblasts than on ihfiel.3 cells, and the relationship between the CR208 inoculum size and the resulting number of undersized plaques was nonlinear, indicating that multiple particles of CR208 were required to initiate lytic replication in a single primary fibroblast. After infection of primary fibroblasts with CR208 at 5 PFU/cell, a normal pattern of viral antigens was detected, although IE1 p72 was absent. During lower-multiplicity infections, IE2 protein was consistently detected at similar levels in a similar proportion of CR208-infected cells relative to the case for a Towne infection, but many fewer CR208-infected cells contained the ppUL44 polymerase accessory protein when evaluated at 24 or 48 h after infection. Furthermore, fibroblasts infected with CR208 at a low multiplicity failed to form viral DNA replication compartments, despite having expressed IE2 p86. These low-multiplicity growth and expression defects were corrected in two rescued derivatives of CR208 able to synthesize IE1 p72. One rescued virus (CR249) carried a deletion removing the large intron between exons 1 and 2 of the ie1-ie2 locus, revealing that this intron was dispensable for growth in cell culture.

Antigens, Viral↗

PubMed1998

Sequences within the herpesvirus-conserved pac1 and pac2 motifs are required for cleavage and packaging of the murine cytomegalovirus genome.

The DNA sequence motifs pac1 [an A-rich region flanked by poly(C) runs] and pac2 (CGCGGCG near an A-rich region) are conserved near herpesvirus genomic termini and are believed to mediate cleavage of genomes from replicative concatemers. To determine their importance in the cleavage process, we constructed a number of recombinant murine cytomegaloviruses with a second cleavage site inserted at an ectopic location within the viral genome. Cleavage at a wild-type ectopic site occurred as frequently as at the natural cleavage site, whereas mutation of this ectopic site revealed that some of the conserved motifs of pac1 and pac2 were essential for cleavage whereas others were not. Within pac1, the left poly(C) region was very important for cleavage and packaging but the A-rich region was not. Within pac2, the A-rich region and adjacent sequences were essential for cleavage and packaging and the CGCGGCG region contributed to, but was not strictly essential for, efficient cleavage and packaging. A second A-rich region was not important at all. Furthermore, mutations that prevented cleavage also blocked duplication and deletion of the murine cytomegalovirus 30-bp terminal repeat at the ectopic site, suggesting that repeat duplication and deletion are consequences of cleavage. Given that the processes of genome cleavage and packaging appear to be highly conserved among herpesviruses, these findings should be relevant to other members of this family.

3T3 Cells↗