The Molecular Tweezer CLR01 Inhibits Antibody-Resistant Cell-to-Cell Spread of Human Cytomegalovirus

Human cytomegalovirus (HCMV) uses two major ways for virus dissemination: infection by cell-free virus and direct cell-to-cell spread. Neutralizing antibodies can efficiently inhibit infection by cell-free virus but mostly fail to prevent cell-to-cell transmission. Here, we show that the ‘molecular tweezer’ CLR01, a broad-spectrum antiviral agent, is not only highly active against infection with cell-free virus but most remarkably inhibits antibody-resistant direct cell-to-cell spread of HCMV. The inhibition of cell-to-cell spread by CLR01 was not limited to HCMV but was also shown for the alphaherpesviruses herpes simplex viruses 1 and 2 (HSV-1, -2). CLR01 is a rapid acting small molecule that inhibits HCMV entry at the attachment and penetration steps. Electron microscopy of extracellular virus particles indicated damage of the viral envelope by CLR01, which likely impairs the infectivity of virus particles. The rapid inactivation of viral particles by CLR01, the viral envelope as the main target, and the inhibition of virus entry at different stages are presumably the key to inhibition of cell-free virus infection and cell-to-cell spread by CLR01. Importance: While cell-free spread enables the human cytomegalovirus (HCMV) and other herpesviruses to transmit between hosts, direct cell-to-cell spread is thought to be more relevant for in vivo dissemination within infected tissues. Cell-to-cell spread is resistant to neutralizing antibodies, thus contributing to the maintenance of virus infection and virus dissemination in the presence of an intact immune system. Therefore, it would be therapeutically interesting to target this mode of spread in order to treat severe HCMV infections and to prevent dissemination of virus within the infected host. The molecular tweezer CLR01 exhibits broad-spectrum antiviral activity against a number of enveloped viruses and efficiently blocks antibody-resistant cell-to-cell spread of HCMV, thus representing a novel class of small molecules with promising antiviral activity.     

Design of a US28 ORF Deletion Virus in a Temperature-Sensitive Cytomegalovirus Strain Fails to Promote Lytic Replication in Hematopoietic Cells

Human cytomegalovirus (CMV) is a ubiquitous pathogen that latently resides in hematopoietic cells. Latently infected individuals with dysfunctional immune systems often experience CMV reactivation, which can cause devastating disease and mortality. While factors dictating the balance between latency and reactivation are not completely understood, CMV US28 is required for maintaining latent infection, and viral mutants that alter US28 function result in a lytic-like, rather than latent, infection in hematopoietic cells. In turn, viral lytic factors alter the host cell, making it challenging to characterize the US28-specific changes in the cellular milieu. To circumvent this, we generated a temperature-sensitive TB40/E recombinant virus, TB40/EgfpC510G (tsC510G), into which we engineered an amino acid change at position 510 (C510G) of IE2, as previously described in the CMV Towne strain. Using tsC510G, we then deleted the US28 ORF, termed tsC510G-US28Δ. Consistent with previous findings, tsC510G-US28Δ fails to undergo latency in Kasumi-3 cells at the permissive temperature. However, parallel cultures maintained at the non-permissive temperature showed a significant reduction in infectious center frequency, as measured by limiting dilution assay. Thus, we generated a new US28 mutant virus for use as a tool to study US28-specific changes in latently infected hematopoietic cells in the absence of induced lytic replication.     

HCMV gH/gL/UL128-131 interferes with virus entry into epithelial cells: evidence for cell type-specific receptors

Human cytomegalovirus (HCMV) forms two different membrane protein complexes, gH/gL/gO and gH/gL/UL128/UL130/UL131, that function in different cell types. gH/gL/gO appears to be important for HCMV entry into or spread between fibroblasts, processes that occur at neutral pH. We demonstrated that HCMV entry into epithelial and endothelial cells requires gH/gL/UL128–131 and involves endocytosis and low pH. A complex of all five HCMV proteins, gH, gL, UL128, UL130, and UL131, is the functionally important mediator of this entry pathway into epithelial/endothelial cells. Here, we report that expression of gH/gL/UL128–131 in ARPE-19 epithelial cells causes the cells to be resistant to HCMV infection. Another HCMV glycoprotein, gB, did not interfere, and expression of all five gH/gL/UL128–131 proteins was required for this interference. gH/gL/UL128–131 interference was at the stage of virus entry into cells rather than the initial adsorption onto cell surfaces or after-entry defects. By contrast, expression of gH/gL/UL128–131 in primary human fibroblasts did not block HCMV infection. Previously, interference by retrovirus and herpes-simplex-virus entry mediators resulted from sequestration or obstruction of receptors. We concluded that epithelial cells express gH/gL/UL128–131 receptors that mediate HCMV entry. Fibroblasts either lack the gH/gL/UL128–131 receptors, the receptors are more numerous, or fibroblasts express other functional receptors.     

Expansion of CD94/NKG2C+ NK cells in response to human cytomegalovirus-infected fibroblasts

CD94/NKG2C(+) natural killer (NK) cells are increased in healthy individuals infected with human cytomegalovirus (HCMV), suggesting that HCMV infection may shape the NK cell receptor repertoire. To address this question, we analyzed the distribution of NK cell subsets in peripheral blood lymphocytes (PBLs) cocultured with HCMV-infected fibroblasts. A substantial increase of NK cells was detected by day 10 in samples from a group of HCMV(+) donors, and CD94/NKG2C(+) cells outnumbered the CD94/NKG2A(+) subset. Fibroblast infection was required to induce the preferential expansion of CD94/NKG2C(+) NK cells that was comparable with allogeneic or autologous fibroblasts, and different virus strains. A CD94-specific monoclonal antibody (mAb) abrogated the effect, supporting an involvement of the lectinlike receptor. Purified CD56(+) populations stimulated with HCMV-infected cells did not proliferate, but the expansion of the CD94/NKG2C(+) subset was detected in the presence of interleukin-15 (IL-15). Experiments with HCMV deletion mutants indicated that the response of CD94/NKG2C(+) NK cells was independent of the UL16, UL18, and UL40 HCMV genes, but was impaired when cells were infected with a mutant lacking the US2-11 gene region. Taken together the data support that the interaction of CD94/NKG2C with HCMV-infected fibroblasts, concomitant to the inhibition of human leukocyte antigen (HLA) class I expression, promotes an outgrowth of CD94/NKG2C(+) NK cells.     

A Viral Pilot for HCMV Navigation?

gH/gL virion envelope glycoprotein complexes of herpesviruses serve as entry complexes and mediate viral cell tropism. By binding additional viral proteins, gH/gL forms multimeric complexes which bind to specific host cell receptors. Both Epstein–Barr virus (EBV) and human cytomegalovirus (HCMV) express alternative multimeric gH/gL complexes. Relative amounts of these alternative complexes in the viral envelope determine which host cells are preferentially infected. Host cells of EBV can modulate the gH/gL complex complement of progeny viruses by cell type-dependent degradation of one of the associating proteins. Host cells of HCMV modulate the tropism of their virus progenies by releasing or not releasing virus populations with a specific gH/gL complex complement out of a heterogeneous pool of virions. The group of Jeremy Kamil has recently shown that the HCMV ER-resident protein UL148 controls integration of one of the HCMV gH/gL complexes into virions and thus creates a pool of virions which can be routed by different host cells. This first mechanistic insight into regulation of the gH/gL complex complement of HCMV progenies presents UL148 as a pilot candidate for HCMV navigation in its infected host.     

Constitutive Signaling by the Human Cytomegalovirus G Protein Coupled Receptor Homologs US28 and UL33 Enables Trophoblast Migration In Vitro

Human cytomegalovirus (HCMV) encodes four homologs of G protein coupled receptors (vGPCRs), of which two, designated UL33 and US28, signal constitutively. UL33 and US28 are also conserved with chemokine receptors: US28 binds numerous chemokine classes, including the membrane bound chemokine, fractalkine; whereas UL33 remains an orphan receptor. There is emerging data that UL33 and US28 each contribute to HCMV associated disease, although no studies to date have reported their potential contribution to aberrant placental physiology that has been detected with HCMV congenital infection. We investigated the signaling repertoire of UL33 and US28 and their potential to enable trophoblast mobilization in vitro. Results demonstrate the constitutive activation of CREB by each vGPCR in ACIM-88 and HTR-8SVneo trophoblasts; constitutive NF-kB activation was detected for US28 only. Constitutive signaling by each vGPCR enabled trophoblast migration. For US28, fractalkine exhibited inverse agonist activity and dampened trophoblast migration. UL33 stimulated expression of both p38 mitogen activated (MAP) and Jun N-terminal (JNK) kinases; while p38 MAP kinase stimulated CREB, JNK was inhibitory, suggesting that UL33 dependent CREB activation was regulated by p38/JNK crosstalk. Given that chemokines and their receptors are important for placental development, these data point to the potential of HCMV UL33 and US28 to interfere with trophoblast responses which are important for normal placental development.     

Structural and biochemical studies of HCMV gH/gL/gO and Pentamer reveal mutually exclusive cell entry complexes

Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and the leading viral cause of birth defects after congenital infection. The glycoprotein complexes gH/gL/gO and gH/gL/UL128/UL130/UL131A (Pentamer) are key targets of the human humoral response against HCMV and are required for HCMV entry into fibroblasts and endothelial/epithelial cells, respectively. We expressed and characterized soluble forms of gH/gL, gH/gL/gO, and Pentamer. Mass spectrometry and mutagenesis analysis revealed that gL-Cys144 forms disulfide bonds with gO-Cys351 in gH/gL/gO and with UL128-Cys162 in the Pentamer. Notably, Pentamer harboring the UL128-Cys162Ser/gL-Cys144Ser mutations had impaired syncytia formation and reduced interference of HCMV entry into epithelial cells. Electron microscopy analysis showed that HCMV gH/gL resembles HSV gH/gL and that gO and UL128/UL130/UL131A bind to the same site at the gH/gL N terminus. These data are consistent with gH/gL/gO and Pentamer forming mutually exclusive cell entry complexes and reveal the overall location of gH/gL-, gH/gL/gO-, and Pentamer-specific neutralizing antibody binding sites. Our results provide, to our knowledge, the first structural view of gH/gL/gO and Pentamer supporting the development of vaccines and antibody therapeutics against HCMV.Human cytomegalovirus (HCMV) is a member of the β-herpesvirus subfamily with >60% seropositivity in adults worldwide (1). HCMV infection is typically asymptomatic, but can cause severe disease or death in immunocompromised solid organ and hematopoietic stem cell transplant recipients. In addition, HCMV can infect the placenta and cross this barrier to infect developing fetuses, causing severe birth defects (2). Given the severity and importance of this disease, obtaining an effective vaccine is considered a public health priority (3).The ability of HCMV to cause disease in a wide range of organs and tissue types is reflected at the cellular level by the virus infecting epithelial cells, endothelial cells, fibroblasts, dendritic cells, hepatocytes, neurons, macrophages, and leukocytes (4). Similar to other herpesviruses, the envelope glycoproteins gB and gH/gL form the conserved fusion machinery required for viral entry (5, 6). Recent structural and mutagenesis analysis suggested that gB is responsible for mediating virus and host membrane fusion during viral entry (7, 8). The role of gH/gL in fusion is less clear because crystal structures of herpes simplex virus 2 (HSV-2), pseudo-rabies virus (PrV), and Epstein–Barr virus (EBV) gH/gL did not reveal any similarity to known viral fusion proteins (9–11). It has been proposed that gH/gL is involved in the entry process through activation of gB (12). In addition to gB and gH/gL, most herpesviruses encode additional glycoproteins that are able to interact with gH/gL and are capable of either mediating binding to specific cellular receptors or regulating the activity of the gH/gL–gB complex (5, 6).HCMV entry into both epithelial and endothelial cells requires a pentameric glycoprotein complex (Pentamer) formed between gH/gL and the UL128, UL130, and UL131A proteins (13, 14). Mutations in the UL131A–UL128 gene locus are sufficient to eliminate epithelial/endothelial tropism and occur spontaneously within only a few passages of wild-type (WT) HCMV in fibroblasts (15, 16). In addition, Pentamer cell surface overexpression interferes with HCMV entry into epithelial cells, but not into fibroblasts, suggesting the presence of a cell-type-specific Pentamer receptor (17).HCMV entry into fibroblasts is mediated by the gH/gL/gO complex at the cell surface at neutral pH (18–21). gO is a highly glycosylated protein and has been shown to covalently interact with gH/gL (22, 23). It has been proposed that gO might function as a molecular chaperone to promote gH/gL incorporation, but not gH/gL/gO, into the virion (21). However, it has been recently demonstrated that gH/gL/gO and Pentamer are much more abundant on the HCMV envelope than gH/gL alone (24).Highly potent HCMV-neutralizing monoclonal antibodies were isolated from the memory B-cell repertoire of HCMV-immune donors and shown to bind the Pentamer. These antibodies were capable of neutralizing HCMV infection of epithelial/endothelial cells, but not fibroblasts (25, 26). In addition, several studies have demonstrated that the Pentamer is the main target of the neutralizing humoral response to HCMV infection in epithelial/endothelial cells (27–29). Consistent with these observations, immunization with the Pentamer has been shown to elicit a strong neutralizing antibody response in mouse, rabbit, and rhesus macaque models (30–32). Together these data indicate that the Pentamer represents a key antigenic target for vaccine development against HCMV infection.Here we report the purification and biochemical characterization of HCMV gH/gL, gH/gL/gO, and Pentamer. In addition, we describe the architecture of these complexes by electron microscopy (EM) and characterize their interaction with MSL-109, a previously described HCMV-neutralizing antibody isolated from the spleen of a HCMV-seropositive individual (33, 34). Our data provide new insights into the structure and function of the HCMV gH/gL/gO and Pentamer complexes.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Viperin (cig5), an IFN-inducible antiviral protein directly induced by human cytomegalovirus

Little is known about the mechanism by which IFNs inhibit human cytomegalovirus (HCMV) replication. Indeed, infection of fibroblasts with HCMV initiates the expression of a subset of type I IFN-inducible genes whose role in the infectious process is unclear. We describe here the identification of a cytoplasmic antiviral protein that is induced by IFNs, by HCMV infection, and by the HCMV envelope protein, glycoprotein B (gB). Stable expression of the protein in fibroblasts inhibits productive HCMV infection, down-regulating several HCMV structural proteins (gB, pp28, and pp65) known to be indispensable for viral assembly and maturation. We have named the protein viperin (for virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible). HCMV infection causes the redistribution of the induced viperin from its normal endoplasmic reticulum association, first to the Golgi apparatus and then to cytoplasmic vacuoles containing gB and pp28. Expression before HCMV infection reduces viperin redistribution from the endoplasmic reticulum to the Golgi apparatus and prevents vacuolar localization, perhaps reflecting the mechanism used by HCMV to evade the antiviral function.     

Neutralizing Antibodies Limit Cell-Associated Spread of Human Cytomegalovirus in Epithelial Cells and Fibroblasts

Human cytomegalovirus (HCMV) can cause severe clinical disease in immunocompromised individuals, such as allograft recipients and infants infected in utero. Neutralizing activity of antibodies, measured as the ability to prevent the entry of cell-free virus, has been correlated with the reduction in HCMV transmission and the severity of HCMV-associated disease. However, in vivo HCMV amplification may occur mainly via cell-to-cell spread. Thus, quantifying the inhibition of cell-to-cell transmission could be important in the evaluation of therapeutic antibodies and/or humoral responses to infection or immunization. Here, we established a quantitative plaque reduction assay, which allowed for the measurement of the capacity of antibodies to limit HCMV spread in vitro. Using an automated fluorescence spot reader, infection progression was assayed by the expansion of viral plaques during the course of infection with various GFP-expressing viruses. We found that in contrast to non-neutralizing monoclonal antibodies (mAbs), neutralizing mAbs against both glycoprotein B and H (gB and gH) could significantly inhibit viral plaque expansion of different HCMV strains and was equally efficient in fibroblasts as in epithelial cells. In contrast, an anti-pentamer mAb was active only in epithelial cells. Taken together, our data demonstrate that specific anti-HCMV mAbs can significantly limit cell-associated virus spread in vitro.     

Human cytomegalovirus cell-to-cell spread in the absence of an essential assembly protein

The human cytomegalovirus UL99-coded pp28 is a myristoylated phosphoprotein located in the virion tegument domain, which resides between the capsid and envelope. A previous study has demonstrated that BADsubUL99, a pp28-deficient mutant virus, fails to assemble enveloped virus particles. Capsids, coated with tegument proteins, accumulate in the cytoplasm of mutant virus-infected cells. This phenotype indicates that pp28 is required for the acquisition of an envelope; it presumably acts by directing tegument-associated capsids to bud through an intracellular membrane derived from the cell's secretory apparatus that has been modified to contain viral transmembrane glycoproteins. Here we demonstrate that BADsubUL99 can spread from cell to cell, even though highly sensitive assays fail to detect infectious virus progeny in cultures of infected fibroblasts. We propose that, in the absence of pp28, tegument-coated capsids might nevertheless bud through cellular membranes, including the plasma membrane. If this suggestion is correct, the enveloped particle could potentially infect an adjacent cell to mediate the cell-to-cell spread that is observed. This mode of spread might also occur after infection with wild-type virus, and it could facilitate immune evasion, assuming that the resulting particles do not have a normal complement of virus-coded envelope glycoproteins.     

UL40-mediated NK evasion during productive infection with human cytomegalovirus

Human cytomegalovirus (HCMV) exploits a range of strategies to evade and modulate the immune response. Its capacity to down-regulate MHC I expression was anticipated to render infected cells vulnerable to natural killer (NK) attack. Kinetic analysis revealed that during productive infection, HCMV strain AD169 first enhanced and then inhibited lysis of primary skin fibroblasts by a CD94/NKG2A(+)NKG2D(+)ILT2(+) NK line. The inhibition of cytotoxicity against strain AD169-infected fibroblasts was abolished by prior treatment of targets or effectors with anti-MHC I and anti-CD94 monoclonal antibodies, respectively, implying a CD94/HLA-E-dependent mechanism. An HCMV strain AD169, UL40 deletion mutant could not inhibit CD94/NKG2A(+) NK killing against skin fibroblasts. The contribution of UL40 to evasion of primary NK cells then was tested in a system where targets and effectors were MHC-matched. Primary NK cells activated with IFNalpha as well as cultured primary NK cell lines showed increased killing against DeltaUL40-infected fibroblasts compared with AD169-infected targets. This effect was abrogated by depletion of CD94(+) cells. These findings demonstrate that HCMV encodes a mechanism of evasion specifically targeted against a proportion of CD94(+) NK cells and show that this system functions during a productive infection.     

Human cytomegalovirus sequences expressed in latently infected individuals promote a latent infection in vitro

Latency enables human cytomegalovirus (HCMV) to persist in the hematopoietic cells of infected individuals indefinitely and prevents clearance of the pathogen. Despite its critical importance to the viral infectious cycle, viral mechanisms that contribute to latency have not been identified. We compared the ability of low-passage clinical and laboratory-adapted strains of HCMV to establish a latent infection in primary human CD34(+) cells. The low-passage strains, Toledo and FIX, established an infection with the hallmarks of latency, whereas the laboratory strains, AD169 and Towne, replicated producing progeny virus. We hypothesized that ULb' region of the genome, which is unique to low-passage strains, may encode a latency-promoting activity. We created and analyzed recombinant viruses lacking segments or individual open reading frames (ORFs) in the ULb' region. One 5-kb segment, and more specifically the UL138 ORF, was required for HCMV to establish and/or maintain a latent infection in hematopoietic progenitor cells infected in vitro. This is the first functional demonstration of a virus-coded sequence required for HCMV latency. Importantly, UL138 RNA was expressed in CD34(+) cells and monocytes from HCMV-seropositive, healthy individuals. UL138 might be a target for antivirals against latent virus.     

Antibody-driven design of a human cytomegalovirus gHgLpUL128L subunit vaccine that selectively elicits potent neutralizing antibodies

The use of neutralizing antibodies to identify the most effective antigen has been proposed as a strategy to design vaccines capable of eliciting protective B-cell immunity. In this study, we analyzed the human antibody response to cytomegalovirus (human cytomegalovirus, HCMV) infection and found that antibodies to glycoprotein (g)B, a surface glycoprotein that has been developed as a HCMV vaccine, were primarily nonneutralizing. In contrast, most of the antibodies to the complex formed by gH, gL, protein (p)UL128, pUL130, and pUL131 (the gHgLpUL128L pentamer) neutralized HCMV infection with high potency. Based on this analysis, we developed a single polycistronic vector encoding the five pentamer genes separated by “self-cleaving” 2A peptides to generate a stably transfected CHO cell line constitutively secreting high levels of recombinant pentamer that displayed the functional antigenic sites targeted by human neutralizing antibodies. Immunization of mice with the pentamer formulated with different adjuvants elicited HCMV neutralizing antibody titers that persisted to high levels over time and that were a hundred- to thousand-fold higher than those found in individuals that recovered from primary HCMV infection. Sera from mice immunized with the pentamer vaccine neutralized infection of both epithelial cells and fibroblasts and prevented cell-to-cell spread and viral dissemination from endothelial cells to leukocytes. Neutralizing monoclonal antibodies from immunized mice showed the same potency as human antibodies and targeted the same as well as additional sites on the pentamer. These results illustrate with a relevant example a general and practical approach of analytic vaccinology for the development of subunit vaccines against complex pathogens.Human cytomegalovirus (HCMV) is a ubiquitously distributed member of the Herpesviridae family that establishes a lifelong infection and represents a major threat for human health. Primary infection during pregnancy is the most frequent cause of congenital birth defects, with an overall 0.6% incidence, whereas severe infections develop in immunocompromised patients (1, 2). In addition, HCMV has been proposed as an agent associated with immune senescence (3) and atherosclerosis (4).HCMV has a broad cell tropism and exploits multiple glycoprotein complexes present on the virion envelope for binding and fusion with host cells. Some glycoproteins (g), such as gM/gN and gB, are used to infect several cell types, whereas glycoprotein complexes containing gH and gL mediate cell type-specific virus entry (5, 6). A pentameric complex comprising gH, gL, protein (p)UL128, pUL130, and pUL131 [gHgLpUL128locus (L)] was shown to be required by clinical HCMV isolates to infect endothelial, epithelial, and myeloid cells (7–10). In vitro cultured HCMV viruses with mutations in the UL128–131 locus lose tropism for endothelial and epithelial cells but retain the expression of the gHgL-containing complex, which is sufficient to infect fibroblasts (11).Because of the high incidence rate of HCMV infections and its impact on public health, considerable efforts have been made in the last decade to develop treatments or vaccines capable of preventing HCMV infection (12). The major target populations for a HCMV vaccine are seronegative women of childbearing age, whereas infants represent another potential population contributing to viral dissemination (13). In addition, patients on a list for organ transplantation (especially those with HCMV-seronegative who are at risk for life-threatening HCMV disease) would benefit from a HCMV vaccine. The administration of the HCMV-attenuated Towne vaccine prevented the development of disease in kidney transplant recipients, although it did not prevent infection (14).The abundant virion protein gB was shown to elicit vigorous T-cell and antibody responses and represents the basis of most vaccines developed so far (15). However, in recent phase II trials, a MF59-adjuvanted gB vaccine showed modest efficacy in preventing infection (16) and reducing duration of viremia in transplant recipients (17). These findings may be explained by the finding that most antibodies induced by the vaccines lack virus-neutralizing activity (18), whereas those that neutralized did not block efficiently infection of epithelial cells (19). Therefore, a HCMV vaccine capable of eliciting neutralizing antibodies that prevent the infection of multiple cellular targets and block viral dissemination is considered a high priority (20).Passively administered polyclonal antibodies isolated from seropositive donors were suggested to be effective in preventing infection of the fetus (21). These findings were not confirmed in a recent randomized study where the same antibody preparation showed a modest, not significant, effect on the rate of congenital HCMV infection, possibly due to the low level of neutralizing antibodies contained in Ig preparation (22).We previously isolated from HCMV immune donors antibodies that bound to conformational epitopes on the gHgLpUL128L pentameric complex and were extraordinarily potent in neutralizing HCMV infection of epithelial, endothelial, and myeloid cells (23). The pentamer-specific antibodies neutralized viral infection at picomolar concentrations and were a thousand-fold more potent than antibodies to gB, gH, or gMgN complex (23). More recently, we showed that an early antibody response to the pentamer was associated with lack of viral transmission to the fetus from HCMV-infected pregnant mothers, suggesting that pentamer-specific antibodies are responsible for the inhibition of viral spread in vivo (24).In this study, we report a systematic analysis of the human antibody response to HCMV infection, which indicates that the gHgLpUL128L pentamer is the target of the most effective neutralizing antibodies. Based on this information, we developed a novel process to produce in a secreted form a recombinant pentamer vaccine from a mammalian CHO cell line stably transfected by a single polycistronic vector encoding the five different HCMV pentamer genes separated by autonomous “self-cleaving” 2A peptides. We found that this vaccine can elicit in mice titers of neutralizing antibodies 100–1,000-fold higher than those induced by natural infection. These antibodies neutralized infection of both epithelial cells and fibroblasts and prevented viral dissemination from endothelial cells to leukocytes.     

Human cytomegalovirus gene expression during infection of primary hematopoietic progenitor cells: a model for latency

Human cytomegalovirus (HCMV) resides latently in hematopoietic cells of the bone marrow. Although viral genomes can be found in CD14+ monocytes and CD34+ progenitor cells, the primary reservoir for latent cytomegalovirus is unknown. We analyzed human hematopoietic subpopulations infected in vitro with a recombinant virus that expresses a green fluorescent protein marker gene. Although many hematopoietic cell subsets were infected in vitro, CD14+ monocytes and various CD34+ subpopulations were infected with the greatest efficiency. We have developed an in vitro system in which to study HCMV infection and latency in CD34+ cells cultured with irradiated stromal cells. Marker gene expression was substantially reduced by 4 days postinfection, and infectious virus was not made during the culture period. However, viral DNA sequences were maintained in infected CD34+ cells for >20 days in culture, and, importantly, virus replication could be reactivated by coculture with human fibroblasts. Using an HCMV gene array, we examined HCMV gene expression in CD34+ cells. The pattern of viral gene expression was distinct from that observed during productive or nonproductive infections. Some of these expressed viral genes may function in latency and are targets for further analysis. Altered gene expression in hematopoietic progenitors may be indicative of the nature and outcome of HCMV infection.     

Intracellular Trafficking of the Human Cytomegalovirus-Encoded 7-trans-Membrane Protein Homologs pUS27 and pUL78 during Viral Infection: A Comparative Analysis

Human cytomegalovirus (HCMV) encodes four G protein-coupled receptor (GPCR) homologs, termed pUS27, pUS28, pUL33, and pUL78. In contrast to the extensively characterized vGPCRs pUS28 and pUL33, knowledge concerning pUS27 and pUL78 is limited. Previous studies already demonstrated constitutive internalization of pUS27 and pUL78, as well as an association with the endosomal machinery, however, these results were mainly obtained using transiently transfected cells. To explore the subcellular localization of both receptors during viral infection, we constructed recombinant HCMVs expressing tagged vGPCRs. Colocalization analyses revealed a predominant association of pUS27 or pUL78 with the trans-Golgi network or the endoplasmic reticulum, respectively. Intriguingly, our data emphasize that protein sorting is highly regulated by viral functions as we detected dramatic changes in the colocalization of pUS27 and pUL78 with endosomal markers during progression of HCMV replication. Furthermore, we observed cell type-dependent differences in trafficking of both vGPCRs between fibroblasts and epithelial cells. Most importantly, infection experiments with a recombinant HCMV carrying tagged versions of pUS27 and pUL78 simultaneously, revealed that these two proteins do not colocalize during viral infection. This contrasts to results of transient expression experiments. In conclusion, our results highlight the importance to investigate vGPCR trafficking in a viral context.     

Role of PARP-1 in Human Cytomegalovirus Infection and Functional Partners Encoded by This Virus

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that threats the majority of the world’s population. Poly (ADP-ribose) polymerase 1 (PARP-1) and protein poly (ADP-ribosyl)ation (PARylation) regulates manifold cellular functions. The role of PARP-1 and protein PARylation in HCMV infection is still unknown. In the present study, we found that the pharmacological and genetic inhibition of PARP-1 attenuated HCMV replication, and PARG inhibition favors HCMV replication. PARP-1 and its enzymatic activity were required for efficient HCMV replication. HCMV infection triggered the activation of PARP-1 and induced the translocation of PARP-1 from nucleus to cytoplasm. PARG was upregulated in HCMV-infected cells and this upregulation was independent of viral DNA replication. Moreover, we found that HCMV UL76, a true late protein of HCMV, inhibited the overactivation of PARP-1 through direct binding to the BRCT domain of PARP-1. In addition, UL76 also physically interacted with poly (ADP-ribose) (PAR) polymers through the RG/RGG motifs of UL76 which mediates its recruitment to DNA damage sites. Finally, PARP-1 inhibition or depletion potentiated HCMV-triggered induction of type I interferons. Our results uncovered the critical role of PARP-1 and PARP-1-mediated protein PARylation in HCMV replication.     

Multi-Modal Imaging with a Toolbox of Influenza A Reporter Viruses

Reporter viruses are useful probes for studying multiple stages of the viral life cycle. Here we describe an expanded toolbox of fluorescent and bioluminescent influenza A reporter viruses. The enhanced utility of these tools enabled kinetic studies of viral attachment, infection, and co-infection. Multi-modal bioluminescence and positron emission tomography–computed tomography (PET/CT) imaging of infected animals revealed that antiviral treatment reduced viral load, dissemination, and inflammation. These new technologies and applications will dramatically accelerate in vitro and in vivo influenza virus studies.