Previously, we identified the glycoprotein gO gene, UL74, as a hypervariable locus in the human cytomegalovirus (HCMV) genome [Virology 293 (2002) 281]. Here, we analyze gO from 50 isolates from congenitally infected newborns, transplant recipients, and HIV/AIDS patients from Italy, Australia, and UK. These are compared to four gO groups described from USA transplantation patients [J. Virol. 76 (2002) 10841]. Phylogenetic analyses identified seven genotypes. Divergence between genotypes was up to 55% and within 3%. Discrete linkage was shown between seven hypervariable gO and gN genotypes, but not with gB. This suggests interactions, while gN and gO are known to form complexes with distinct conserved glycoproteins gM, gH/gL, respectively, both are involved in fusogenic entry and exit. Codon-based maximum likelihood models showed evidence for sites of positive selection. Further analyses of disease relationships should take into account these newly defined gO/gN groups.

The human herpesvirus 6 (HHV-6) U51 gene defines a new family of betaherpesvirus-specific genes encoding multiple transmembrane glycoproteins with similarity to G protein-coupled receptors, in particular, human chemokine receptors. These are distinct from the HHV-6 U12 and HCMV US28 family. In vitro transcription and translation as well as transient cellular expression of U51 showed properties of a multiple transmembrane protein with a 30-kDa monomer as well as high m.w. aggregates or oligomers. Transient cellularly expressed U51 also appeared to form dimeric intermediates. Despite having only limited sequence similarity to chemokine receptors, U51 stably expressed in cell lines showed specific binding of the CC chemokine RANTES and competitive binding with other β chemokines, such as eotaxin; monocyte chemoattractant protein 1, 3, and 4; as well as the HHV-8 chemokine vMIPII. In epithelial cells already secreting RANTES, U51 expression resulted in specific transcriptional down-regulation. This correlated with reduced secretion of RANTES protein into the culture supernatants. Regulation of RANTES levels may alter selective recruitment of circulating inflammatory cells that the virus can infect and thus could mediate the systemic spread of the virus from initial sites of infection in epithelia. Alternatively, chemokine regulation could modulate a protective inflammatory response to aid the spread of virus by immune evasion. Such mimicry, by viral proteins, of host receptors leading to down-regulation of chemokine expression is a novel immunomodulatory mechanism.

Persistent carriage was associated with high levels of specific IgG antibodies and serum bactericidal activity while recent strain acquisition correlated with a significant induction of antibodies. We conclude that phase-variable genes are driven into lower expression states during long-term persistent meningococcal carriage, in part due to continuous exposure to antibody-mediated selection, suggesting localized hypermutation has evolved to facilitate host persistence.

Human herpesvirus-6A (HHV-6A) betachemokine-receptor U51A binds inflammatory modulators CCL2, CCL5, CCL11, CCL7, and CCL13. This unique specificity overlaps that of human chemokine receptors CCR1, CCR2, CCR3, and CCR5. In model cell lines, expression leads to CCL5 down-regulation with both constitutive and inducible signaling. Here, immunomodulation pathways are investigated in human leukocytes permissive for infection. Constitutive signaling was shown using inositol phosphate assays and inducible calcium signaling by response to CCL2, CCL5 and CCL11. Constitutive signaling targets were examined using an immune response-related microarray and RT-PCR, showing down-regulation of CCL5 and FOG-2, a hematopoietic transcriptional repressor. By RT-PCR and siRNA reversion, CCL5 and FOG-2 were shown down-regulated, during peak U51A expression post infection. Two further active ligands, XCL1 and CCL19, were identified, making U51A competitor to their human receptors, XCR1 and CCR7, on T lymphocytes, NK and dendritic cells. Finally, U51A-expressing cell lines and infected ex vivo leukocytes, showed migration towards chemokine-gradients, and chemokine internalization. Consequently, U51A may affect virus dissemination or host transmission by chemotaxis of infected cells to sites of chemokine secretion specific for U51A (for example the lymph node or lung, by CCL19 or CCL11, respectively) and evade immune-effector cells by chemokine diversion and down-regulation, affecting virus spread and inflammatory pathology.

The human herpes virus 6 (HHV-6)-encoded chemokine receptor U51 constitutively activates phospholipase C (PLC) and inhibits cAMP-responsive element (CRE)-mediated gene transcription via the activation of G q/11 proteins. Yet, chemokines known to bind U51 differentially regulate U51 coupling to G proteins. CCL5/RANTES induced pertussis toxin (PTX)-insensitive increases in PLC activity and changes in intracellular free calcium concentration ([Ca 2ϩ ] i ), whereas both CCL2/MCP-1 and CCL11/eotaxin failed to stimulate PLC activity or increase [Ca 2ϩ ] i . In contrast, all three chemokines counteracted the effects of U51 on CRE activity via the activation of PTX-sensitive G i/o proteins. For each of the tested chemokines, coexpression of U51 with a variety of G␣ subunits, however, revealed a distinct profile for preferred G-protein coupling, which could be shifted by modulation of the relative expression of G proteins. These findings are consistent with a chemokine-selective trafficking of receptor stimulus to distinct G proteins and suggest that the constitutive activity of U51 and the chemokine-induced signaling involve different active states of the receptor. By virtue of its ability to constitutively activate signaling pathways, its G-protein promiscuity, and the chemokinedirected trafficking of receptor stimulus, U51 can be considered a sensitive and versatile virally encoded signaling device, potentially of importance in HHV-6-related pathologies.