Natural killer (NK) cells are innate lymphocytes that lack antigen-specific rearranged receptors, a hallmark of adaptive lymphocytes. In some people infected with human cytomegalovirus (HCMV), an NK cell subset expressing the activating receptor NKG2C undergoes clonal-like expansion that partially resembles anti-viral adaptive responses. However, the viral ligand that drives the activation and differentiation of adaptive NKG2C NK cells has remained unclear. Here we found that adaptive NKG2C NK cells differentially recognized distinct HCMV strains encoding variable UL40 peptides that, in combination with pro-inflammatory signals, controlled the population expansion and differentiation of adaptive NKG2C NK cells. Thus, we propose that polymorphic HCMV peptides contribute to shaping of the heterogeneity of adaptive NKG2C NK cell populations among HCMV-seropositive people.

No abstract

To analyze the assembly of herpes simplex virus type 1 (HSV1) by triple-label fluorescence microscopy, we generated a bacterial artificial chromosome (BAC) and inserted eukaryotic Cre recombinase, as well as ␤-galactosidase expression cassettes. When the BAC pHSV1(17 ؉ )blueLox was transfected back into eukaryotic cells, the Cre recombinase excised the BAC sequences, which had been flanked with loxP sites, from the viral genome, leading to HSV1(17 ؉ )blueLox. We then tagged the capsid protein VP26 and the envelope protein glycoprotein D (gD) with fluorescent protein domains to obtain HSV1(17 ؉ )blueLox-GFPVP26-gDRFP and -RFPVP26-gDGFP. All HSV1 BACs had variations in the a-sequences and lost the oriL but were fully infectious. The tagged proteins behaved as their corresponding wild type, and were incorporated into virions. Fluorescent gD first accumulated in cytoplasmic membranes but was later also detected in the endoplasmic reticulum and the plasma membrane. Initially, cytoplasmic capsids did not colocalize with viral glycoproteins, indicating that they were naked, cytosolic capsids. As the infection progressed, they were enveloped and colocalized with the viral membrane proteins. We then analyzed the subcellular distribution of capsids, envelope proteins, and nuclear pores during a synchronous infection. Although the nuclear pore network had changed in ca. 20% of the cells, an HSV1-induced reorganization of the nuclear pore architecture was not required for efficient nuclear egress of capsids. Our data are consistent with an HSV1 assembly model involving primary envelopment of nuclear capsids at the inner nuclear membrane and primary fusion to transfer capsids into the cytosol, followed by their secondary envelopment on cytoplasmic membranes.Herpes simplex virus type 1 (HSV1) causes severe human diseases such as herpes encephalitis or herpes keratoconjunctivitis (18). Its double-stranded DNA genome of 152 kb that codes for more than 80 open reading frames is enclosed in an icosahedral capsid with a diameter of 125 nm. HSV1 is a spherical, enveloped virus with a diameter of about 250 nm. Between the capsid and the viral membrane, there is an amorphous, asymmetric layer, the tegument, which consists of about 20 different proteins (45,74). HSV1 enters cells by fusion at the plasma membrane or after endocytosis by fusion with an endosomal membrane (19,42,66,67,82). After dynein-mediated transport along microtubules (32,56,59,81), capsids reach the nuclear pore where the viral genome is released into the nucleoplasm (68) for viral transcription and DNA replication (74). Progeny viral genomes are packaged into preassembled nuclear capsids, which translocate to the inner nuclear membrane. The subsequent steps of herpesvirus morphogenesis are controversial (12, 13, 65).HSV1 capsids can leave the nucleus by primary envelopment at the inner nuclear membrane (6, 64). According to the luminal or single-envelopment hypothesis, these enveloped virions present in the lumen of the nuclear envelope or the endoplasmic reticulu...

dCleavage of human cytomegalovirus (HCMV) genomes as well as their packaging into capsids is an enzymatic process mediated by viral proteins and therefore a promising target for antiviral therapy. The HCMV proteins pUL56 and pUL89 form the terminase and play a central role in cleavage-packaging, but several additional viral proteins, including pUL51, had been suggested to contribute to this process, although they remain largely uncharacterized. To study the function of pUL51 in infected cells, we constructed HCMV mutants encoding epitope-tagged versions of pUL51 and used a conditionally replicating virus (HCMV-UL51-ddFKBP), in which pUL51 levels could be regulated by a synthetic ligand. In cells infected with HCMV-UL51-ddFKBP, viral DNA replication was not affected when pUL51 was knocked down. However, no unit-length genomes and no DNA-filled C capsids were found, indicating that cleavage of concatemeric HCMV DNA and genome packaging into capsids did not occur in the absence of pUL51. pUL51 was expressed mainly with late kinetics and was targeted to nuclear replication compartments, where it colocalized with pUL56 and pUL89. Upon pUL51 knockdown, pUL56 and pUL89 were no longer detectable in replication compartments, suggesting that pUL51 is needed for their correct subnuclear localization. Moreover, pUL51 was found in a complex with the terminase subunits pUL56 and pUL89. Our data provide evidence that pUL51 is crucial for HCMV genome cleavage-packaging and may represent a third component of the viral terminase complex. Interference with the interactions between the terminase subunits by antiviral drugs could be a strategy to disrupt the HCMV replication cycle.

We have recently introduced a novel procedure for the construction of herpesvirus mutants that is based on the cloning and mutagenesis of herpesvirus genomes as infectious bacterial artificial chromosomes (BACs) in Escherichia coli (M. Messerle, I. Crnković, W. Hammerschmidt, H. Ziegler, and U. H. Koszinowski, Proc. Natl. Acad. Sci. USA 94:14759–14763, 1997). Here we describe the application of this technique to the human cytomegalovirus (HCMV) strain AD169. Since it was not clear whether the terminal and internal repeat sequences of the HCMV genome would give rise to recombination, the stability of the cloned HCMV genome was examined during propagation inE. coli, during mutagenesis, and after transfection in permissive fibroblasts. Interestingly, the HCMV BACs were frozen in defined conformations in E. coli. The transfection of the HCMV BACs into human fibroblasts resulted in the reconstitution of infectious virus and isomerization of the reconstituted genomes. The power of the BAC mutagenesis procedure was exemplarily demonstrated by the disruption of the gpUL37 open reading frame. The transfection of the mutated BAC led to plaque formation, indicating that the gpUL37 gene product is dispensable for growth of HCMV in fibroblasts. The new procedure will considerably speed up the construction of HCMV mutants and facilitate genetic analysis of HCMV functions.

The nucleocytoplasmic export of cytomegaloviral capsids is regulated by formation of a multicomponent nuclear egress complex (NEC), essentially based on viral proteins pUL50 and pUL53. In this study, the generation of recombinant human cytomegaloviruses, expressing tagged versions of pUL50 and pUL53, enabled the investigation of NEC formation in infected primary fibroblasts. For these recombinant viruses, a wild-type-like mode of pUL50-pUL53 interaction and recruitment of both proteins to the nuclear envelope could be demonstrated. Importantly, pUL50 was translocated from an initial cytoplasmic distribution to the nuclear rim, whereas pUL53 accumulated in the nucleus before attaining overall rim colocalization with pUL50. Specified experimental settings illustrated that pUL50 and pUL53 were subject to different pathways of intracellular trafficking. Importantly, a novel nuclear localization signal (NLS) could be identified and functionally verified for pUL53 (amino acids 18-27), whereas no NLS was present in pUL50. Analysis of amino acid replacement mutants further illustrated the differential modes of nuclear import of the two essential viral egress proteins. Taken together, our findings suggest a combination of classical nuclear import (pUL53) and interaction-mediated recruitment (pUL50) as the driving forces for core NEC formation and viral nuclear egress.

Replication of human cytomegalovirus (HCMV) produces large DNA concatemers of head-to-tail-linked viral genomes that upon packaging into capsids are cut into unit-length genomes. The mechanisms underlying cleavage-packaging and the subsequent steps prior to nuclear egress of DNA-filled capsids are incompletely understood. The hitherto uncharacterized product of the essential HCMV UL52 gene was proposed to participate in these processes. To investigate the function of pUL52, we constructed a ⌬UL52 mutant as well as a complementing cell line. We found that replication of viral DNA was not impaired in noncomplementing cells infected with the ⌬UL52 virus, but viral concatemers remained uncleaved. Since the subnuclear localization of the known cleavage-packaging proteins pUL56, pUL89, and pUL104 was unchanged in ⌬UL52-infected fibroblasts, pUL52 does not seem to act via these proteins. Electron microscopy studies revealed only B capsids in the nuclei of ⌬UL52-infected cells, indicating that the mutant virus has a defect in encapsidation of viral DNA. Generation of recombinant HCMV genomes encoding epitope-tagged pUL52 versions showed that only the N-terminally tagged pUL52 supported viral growth, suggesting that the C terminus is crucial for its function. pUL52 was expressed as a 75-kDa protein with true late kinetics. It localized preferentially to the nuclei of infected cells and was found to enclose the replication compartments. Taken together, our results demonstrate an essential role for pUL52 in cleavage-packaging of HCMV DNA. Given its unique subnuclear localization, the function of pUL52 might be distinct from that of other cleavage-packaging proteins.The infection cycle of human cytomegalovirus (HCMV) comprises a phase in the cell nucleus where genome replication and assembly of new capsids take place (24). Replication of the 230-kbp viral DNA genome leads to the formation of concatemers of head-to-tail-linked viral genomes, which are believed to be highly branched. These concatemers are subsequently cleaved into unit-length genomes, which are packaged into preformed capsids. The DNA-filled capsids associate with some tegument proteins at the nuclear membrane and then are transferred into the cytoplasm, where they undergo further coating with tegument proteins. Final envelopment of the capsids most likely occurs in a cytoplasmic virus assembly compartment, which partly overlaps with and is possibly derived from the trans-Golgi network (30).Cleavage-packaging of HCMV genomes and capsid maturation in the nucleus are not completely understood, yet several viral proteins have been implicated as involved in these processes. The HCMV terminase responsible for cleavage of concatemeric DNA was shown to consist of two essential proteins, pUL56 and pUL89 (31, 36). pUL56 binds to viral capsids as well as to the packaging signal located in the a-repeat of the HCMV genome and has been shown to possess ATPase activity. pUL89 directly interacts with pUL56 and seems to be required mainly for DNA cleavage (4,19,31). pUL104 i...

The major immediate-early (MIE) genes of cytomegaloviruses (CMV) are broadly thought to be decisive regulators of lytic replication and reactivation from latency. To directly assess the role of the MIE protein IE1 during the infection of murine CMV (MCMV), we constructed an MCMV with exon 4 of the ie1 gene deleted. We found that, independent of the multiplicity of infection, the resulting recombinant virus, MCMVdie1, which fails to express the IE1 protein, was fully competent for early gene expression and replicated in different cultured cell types with identical kinetics to those of parental or revertant virus. Immunofluorescence microscopy studies revealed that MCMVdie1 was greatly impaired in its capacity to disrupt promyelocytic leukemia bodies in NIH 3T3 cells early after infection, a process that has been proposed to increase viral transcription efficiency. We examined MCMVdie1 in the murine model using both immunocompetent BALB/c and severe combined immunodeficient (SCID) mice. When MCMVdie1 was inoculated into these two types of mice, significantly lower viral titers were detected in infected organs than in those of the wild-type virus-infected animals. Moreover, the ie1-deficient MCMV exhibited a markedly reduced virulence. While all animals infected with 5 ؋ 10 4 PFU of parental virus died by 30 days postinfection, SCID mice infected with a similar dose of MCMVdie1 did not succumb before 60 days postinfection. The in vivo defective growth phenotype of MCMVdie1 was abrogated upon rescue of ie1. These results demonstrate the significance of the ie1 gene for promoting an acute MCMV infection and virulence yet indicate that MCMV is able to grow in vivo, although impaired, in the absence of the ie1 gene.Similar to other herpesviruses, the transcription of the cytomegalovirus (CMV) genome during the lytic infection is temporarily regulated (for a review, see reference 47). The immediate-early (IE or ␣) genes are the first ones to be expressed in the replicative cycle, and their expression does not depend on prior viral protein synthesis. Together with some virion proteins, the IE products activate viral genes and alter the infected cell to generate an appropriate milieu that favors viral replication. Transcription of early (E or ␤) genes requires the expression of at least one of the IE proteins, and only after viral replication has started, the transcription of late (L or ␥) genes proceeds. The majority of the CMV IE transcripts originate from the major IE (MIE) locus. This locus is structurally similar between human CMV (HCMV) and the closely related mouse CMV (MCMV) (14,53,59). The primary transcript from the MIE region is under the control of the strong MIE enhancer-promoter and is differentially spliced to generate two predominant transcripts, the ie1 transcript that consists of exons 1 to 4, and the ie2 transcript that is composed of exons 1 to 3 and 5. In HCMV, the ie1 and ie2 transcripts are translated into the acidic 72-kDa IE1 and the 86-kDa IE2 nuclear phosphoproteins, respectively (for a review, ...