Human cytomegalovirus (HCMV), a ubiquitous human pathogen, is the leading cause of birth defects and morbidity in immunocompromised patients and a potential trigger for vascular disease. HCMV replicates in vascular endothelial cells and drives leukocyte-mediated viral dissemination through close endothelium-leukocyte interaction. However, the genetic basis of HCMV growth in endothelial cells and transfer to leukocytes is unknown. We show here that the UL131-128 gene locus of HCMV is indispensable for both productive infection of endothelial cells and transmission to leukocytes. The experimental evidence for this is based on both the loss-of-function phenotype in knockout mutants and natural variants and the gain-of-function phenotype by trans-complementation with individual UL131, UL130, and UL128 genes. Our findings suggest that a common mechanism of virus transfer may be involved in both endothelial cell tropism and leukocyte transfer and shed light on a crucial step in the pathogenesis of HCMV infection.

A panel of human sera exhibited a ¢128-fold higher neutralizing potency against a human cytomegalovirus (HCMV) clinical isolate propagated and tested in endothelial (or epithelial) cells than against the same virus infecting human fibroblasts. In a group of 18 primary infections, the reverse geometric mean titre was in the range of 10-15 in human fibroblasts within the first 3 months after the onset of infection, whereas the endothelial cell infection-neutralizing activity was already present within the first 10 days, reaching median levels of 122, 320 and 545 at respectively 30, 60 and 90 days after onset, then declining slowly. This difference was also confirmed in the majority of reactivated and remote HCMV infections, as well as in a hyperimmune globulin preparation. The antibody response to HCMV pUL131A, pUL130 and pUL128 locus products, which are required for endothelial/epithelial cell infection, provided a potential molecular basis for such a differential neutralizing activity. In addition, monoclonal/monospecific antibodies raised against the pUL131A, pUL130 and pUL128 proteins were found to display an inhibitory activity on HCMV plaque formation and HCMV leukocyte transfer from HCMV-infected cells. Hence, conventional determination of the neutralizing activity of human sera in fibroblasts is misleading. Antibodies to pUL131A, pUL130 and pUL128 appear to display a major HCMV-neutralizing and dissemination-inhibiting activity.

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We have previously shown that only endotheliotropic strains of human cytomegalovirus (HCMV), such as TB40E, infect monocytes and impair their chemokine-driven migration. The proteins encoded by the UL128-131A region (UL128, UL130, and UL131A) of the HCMV genome, which assemble into a pentameric gH-gL-UL128-UL130-UL131A envelope complex, have been recognized as determinants for HCMV endothelial cell tropism. The genes for these proteins are typically inactivated by mutations in all fibroblast-adapted strains that have lost the diversified tropism of clinical isolates. By using mutant HCMV reconstituted from TB40E-derived bacterial artificial chromosomes (BAC) encoding a wild-type (wt) or mutated form of UL128, we show here that UL128-131A products are essential determinants of infection in monocytes and that pUL128, in particular, can block chemokine-driven motility. The virus BAC4, encoding wt UL128, established infection in monocytes, induced the intracellular retention of several chemokine receptors, and rendered monocytes unresponsive to different chemokines. In contrast, the virus BAC1, encoding a mutated UL128, failed to infect monocytes and to downregulate chemokine receptors. BAC1-exposed monocytes did not express immediateearly (IE) products, retained virions in cytoplasmic vesicles, and exhibited normal chemokine responsiveness. A potential role of second-site mutations in the observed phenotype was excluded by using the revertant viruses BAC1rep and BAC4mut. By incubating noninfected monocytes with soluble recombinant pUL128, we observed both the block of migration and the chemokine receptor internalization. We propose that among the gH-gL-UL128-UL130-UL131A complex subunits, the UL128 protein is the one that triggers monocyte paralysis.

Human cytomegalovirus (HCMV) growth in endothelial cells (EC) requires the expression of the UL131A-128 locus proteins. In this study, the UL130 protein (pUL130), the product of the largest gene of the locus, is shown to be a luminal glycoprotein that is inefficiently secreted from infected cells but is incorporated into the virion envelope as a Golgi-matured form. To investigate the mechanism of the UL130-mediated promotion of viral growth in EC, we performed a complementation analysis of a UL130 mutant strain. To provide UL130 in trans to viral infections, we constructed human embryonic lung fibroblast (HELF) and human umbilical vein endothelial cell (HUVEC) derivative cell lines that express UL130 via a retroviral vector. When the UL130-negative virus was grown in UL130-complementing HELF, the infectivity of progeny virions for HUVEC was restored to the wild-type level. In contrast, the infectivity of the UL130-negative virus for UL130-complementing HUVEC was low and similar to that of the same virus infecting control noncomplementing HUVEC. The UL130-negative virus, regardless of whether or not it had been complemented in the prior cycle, could form plaques only on UL130-complementing HUVEC, not control HUVEC. Because (i) both wild-type and UL130-transcomplemented virions maintained their infectivity for HUVEC after purification, (ii) UL130 failed to complement in trans the UL130-negative virus when it was synthesized in a cell separate from the one that produced the virions, and (iii) pUL130 is a virion protein, models are favored in which pUL130 acquisition in the producer cell renders HCMV virions competent for a subsequent infection of EC.Human cytomegalovirus (HCMV) is a betaherpesvirus that establishes life-long, subclinical infections ubiquitously in human populations (5). HCMV causes serious morbidity in settings of immune system immaturity or depression: it is the leading viral cause of defects at birth and generates a potentially life-threatening disease in immunocompromised patients. In patients with HCMV disease, the virus can be demonstrated in a variety of cells, including hematopoietic cells (monocytes-macrophages, dendritic cells, and neutrophils), endothelial cells (EC), epithelial cells, fibroblasts, neurons, smooth muscle cells, and hepatocytes (5, 38). Much recent work has focused on HCMV infections of EC, for several reasons: (i) arterial endothelia, along with CD34 ϩ myeloid progenitors, have been proposed as sites of HCMV persistence and latency (20); (ii) a bidirectional transmission of HCMV between EC and leukocytes can be demonstrated in vitro and may reflect a mechanism of dissemination in vivo (11,14,34,44); (iii) circulating giant EC may similarly contribute directly to dissemination (32); (iv) HCMV infections of the uterine microvasculature and cytotrophoblasts may underlie motherto-fetus transmission and compromise the placental trophic function in affected pregnancies (25, 47); and (v) HCMV may play a role in atherogenesis, postangioplasty restenosis, posttransplantation endot...

Herpesviruses use gB and gH-gL glycoproteins to execute fusion. Other virus-specific glycoproteins are required for receptor binding and fusion activation. The human cytomegalovirus (HCMV) UL131-128 proteins are essential for the infection of leukocytes, endothelial cells (ECs), and many epithelial cell lines. Here we show that UL131-128 play a role in a chain of events involving gB and gH during HCMV entry into ECs. An HCMV strain bearing the wild-type (wt) UL131-128 locus exhibited a gB transition from a protease-resistant to protease-sensitive form, a conformational change that was suppressed by a thiourea inhibitor of fusion (WY1768); in contrast, gH was susceptible to proteolysis throughout entry. Moreover, gB and gH transiently interacted, and a lipid mixing assay showed that the wt strain had carried out fusion by 60 min postinfection. However, these events were greatly altered when UL131-128-defective strains were used for infection or when there was an excess of soluble pUL128 during wt infection: the gB conformational change became WY1768 resistant, the gB-gH complex was no longer observed, and fusion was prevented. Both gB and gH in this case showed late protease resistance, related to their endocytic uptake. Our data point to the involvement of UL131-128 proteins in driving gB through a WY1768-sensitive fold transition, thus promoting a short-lived gB-gH complex and fusion; they also suggest that HCMV fuses with the EC plasma membrane and that endocytosis ensues only when the virus cannot trigger UL131-128-dependent steps.Human cytomegalovirus (HCMV), the prototypical member of the Betaherpesvirinae subfamily, is the leading infectious cause of congenital defects, a major opportunist in transplant recipients and immunocompromised patients, and a suspected cofactor for cardiovascular diseases, systemic sclerosis, and gastrointestinal cancer (7,27,29,41,42,49). Clinical isolates of HCMV infect various cell types in vitro, including endothelial cells (ECs), thus replicating the broad cell type tropism observed in HCMV infections of immunocompromised subjects (EC-tropic strains). However, laboratory propagation in fibroblasts (FBs) restricts viral tropism (FB-tropic strains), and mutations causing the loss of tropism for ECs, epithelial cells, polymorphonuclear leukocytes, and dendritic cells (DCs) have been mapped to the contiguous UL131, UL130, and UL128 open reading frames (ORFs) (13,25,18,56).There is considerable indirect evidence indicating that UL131-128 proteins act as regulators of virus-cell fusion: (i) FB-tropic strains fail to transfer tegument pp65-UL83 protein to EC nuclei (18), which suggests that infection stops before virus uncoating; (ii) an HCMV deletion mutant lacking the UL128-UL150 ORFs infects retinal pigment epithelial cells after exposure to polyethylene glycol in order to force fusion between viral and cellular membranes (50); and (iii) EC-tropic strains are syncytiogenic at a high multiplicity of infection (18,56).The membrane-spanning glycoproteins gB and gH and the soluble gL...

Human cytomegalovirus (HCMV) encodes a protein related to the large (R1) subunit of ribonucleotide reductase (RR), but does not encode the corresponding small (R2) subunit. The R1 homologue, UL45, lacks many catalytic residues, and its impact on deoxyribonucleotide (dNTP) production remains unknown. Here, UL45 is shown to accumulate at late stages of infection and to be a virion tegument protein. To study UL45 function in its genome context, UL45 was disrupted by transposon insertion. The UL45-knockout (UL45-KO) mutant exhibited a growth defect in fibroblasts at a low m.o.i. and also a cell-to-cell spread defect. This did not result from a reduced dNTP supply because dNTP pools were unchanged in resting cells infected with the mutant virus. Irrespective of UL45 expression, all cellular RR subunits -S-phase RR subunits, and the p53-dependent p53R2 -were induced by infection. p53R2 was targeted to the infected cell nucleus, suggesting that HCMV diverts a mechanism normally activated by DNA damage response. Cells infected with the UL45-KO mutant were moderately sensitized to Fas-induced apoptosis relative to those infected with the parental virus. Together with the report on the UL45-KO endotheliotropic HCMV mutant (Hahn et al., J Virol 76, 9551-9555, 2002), these data suggest that UL45 does not share the prominent antiapototic role attributed to the mouse cytomegalovirus homologue M45 (Brune et al., Science 291, 303-305, 2001). INTRODUCTIONHuman cytomegalovirus (HCMV), a ubiquitous b-herpesvirus that causes severe disease in immunocompromised individuals and in the newborn, replicates in differentiated cells (fibroblasts, microglial, epithelial, endothelial and smooth muscle cells, and monocyte-derived macrophages; Britt & Alford, 1996; Sinzger et al., 1995), and is transported in the bloodstream by abortively infected neutrophils (Gerna et al., 2000). Latent infection is detected in macrophagegranulocyte precursors in the bone marrow and in circulating monocytes (Maciejewski et al., 1992; SoderbergNaucler et al., 1997;Hahn et al., 1998).The class I ribonucleoside diphosphate (ribonucleotide) reductases (RR), which catalyse a limiting step in de novo deoxyribonucleotide (dNTP) synthesis, are essential for DNA replication of both eukaryotic cells and the DNA viruses that infect them (Jordan & Reichard, 1998;Stubbe et al., 2001). RR holoenzymes comprise a catalytic large (R1) subunit, and a small (R2) subunit required for enzyme activation (Jordan & Reichard, 1998). Like many large DNA viruses, the a-and c-herpesviruses encode both subunits of a viral RR, which is important for virus replication in resting or post-mitotic cells (Jacobson et al., 1989;Idowu et al., 1992;Heineman & Cohen, 1994;Aurelian, 1998) because these have minute dNTP pools and virtually no endogenous RR (Engstrom et al., 1985;Jordan & Reichard, 1998;Chabes & Thelander, 2000). In addition, the herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) R1 proteins bear an N-terminal extension (see supplementary data Fig. 1 at JGV Online, http://vir.sg...

IL-27 and IL-35 are heterodimeric cytokines, members of the IL-12 family and considered to have immunomodulatory properties. Their role during neuroinflammation had been investigated using mutant mice devoid of either one of their subunits or lacking components of their receptors, yielding conflicting results. We sought to understand the therapeutic potential of IL-27 and IL-35 delivered by gene therapy in neuroinflammation. We constructed lentiviral vectors expressing IL-27 and IL-35 from a single polypeptide chain, and we validated in vitro their biological activity. We injected IL-27 and IL-35-expressing lentiviral vectors into the cerebrospinal fluid (CSF) of mice affected by experimental neuroinflammation (EAE), and performed clinical, neuropathological and immunological analyses. Both cytokines interfere with neuroinflammation, but only IL-27 significantly modulates disease development, both clinically and neuropathologically. IL-27 protects from autoimmune inflammation by inhibiting granulocyte macrophages colony-stimulating factor (GM-CSF) expression in CD4+ T cells and by inducing program death-ligand 1 (PD-L1) expression in both CNS-resident and CNS-infiltrating myeloid cells. We demonstrate here that IL-27 holds therapeutic potential during neuroinflammation and that IL-27 inhibits GM-CSF and induces pd-l1 mRNA in vivo.