The BHLF1 Locus of Epstein-Barr Virus Contributes to Viral Latency and B-Cell Immortalization.

The Epstein-Barr virus (EBV) BHLF1 gene encodes an abundant linear and several circular RNAs believed to perform noncoding functions during virus replication, although an open reading frame (ORF) is retained among an unknown percentage of EBV isolates. Evidence suggests that BHLF1 is also transcribed during latent infection, which prompted us to investigate the contribution of this locus to latency. Analysis of transcripts transiting BHLF1 revealed that its transcription is widespread among B-cell lines supporting the latency I or III program of EBV protein expression and is more complex than originally presumed. EBV-negative Burkitt lymphoma cell lines infected with either wild-type or two different BHLF1 mutant EBVs were initially indistinguishable in supporting latency III. However, cells infected with BHLF1- virus ultimately transitioned to the more restrictive latency I program, whereas cells infected with wild-type virus either sustained latency III or transitioned more slowly to latency I. Upon infection of primary B cells, which require latency III for growth in vitro, both BHLF1- viruses exhibited variably reduced immortalization potential relative to the wild-type virus. Finally, in transfection experiments, efficient protein expression from an intact BHLF1 ORF required the EBV posttranscriptional regulator protein SM, whose expression is limited to the replicative cycle. Thus, one way in which BHLF1 may contribute to latency is through a mechanism, possibly mediated or regulated by a long noncoding RNA, that supports latency III critical for the establishment of EBV latency and lifelong persistence within its host, whereas any retained protein-dependent function of BHLF1 may be restricted to the replication cycle.IMPORTANCE Epstein-Barr virus (EBV) has significant oncogenic potential that is linked to its latent infection of B lymphocytes, during which virus replication is not supported. The establishment of latent infection, which is lifelong and can precede tumor development by years, requires the concerted actions of nearly a dozen EBV proteins and numerous small non-protein-coding RNAs. Elucidating how these EBV products contribute to latency is crucial for understanding EBV's role in specific malignancies and, ultimately, for clinical intervention. Historically, EBV genes that contribute to virus replication have been excluded from consideration of a role in latency, primarily because of the general incompatibility between virus production and cell survival. However, here, we provide evidence that the genetic locus containing one such gene, BHLF1, indeed contributes to key aspects of EBV latency, including its ability to promote the continuous growth of B lymphocytes, thus providing significant new insight into EBV biology and oncogenic potential.

Triple lysine and nucleosome-binding motifs of the viral IE19 protein are required for human cytomegalovirus S-phase infections.

Herpesvirus genomes are maintained as extrachromosomal plasmids within the nuclei of infected cells. Some herpesviruses persist within dividing cells, putting the viral genome at risk of being lost to the cytoplasm during mitosis because karyokinesis (nuclear division) requires nuclear envelope breakdown. Oncogenic herpesviruses (and papillomaviruses) avoid genome loss during mitosis by tethering their genomes to cellular chromosomes, thereby ensuring viral genome uptake into newly formed nuclei. These viruses use viral proteins with DNA- and chromatin-binding capabilities to physically link viral and cellular genomes together in a process called tethering. The known viral tethering proteins of human papillomavirus (E2), Epstein-Barr virus (EBNA1), and Kaposi's sarcoma-associated herpesvirus (LANA) each contain two independent domains required for genome tethering, one that binds sequence specifically to the viral genome and another that binds to cellular chromatin. This latter domain is called a chromatin tethering domain (CTD). The human cytomegalovirus UL123 gene encodes a CTD that is required for the virus to productively infect dividing fibroblast cells within the S phase of the cell cycle, presumably by tethering the viral genome to cellular chromosomes during mitosis. The CTD-containing UL123 gene product that supports S-phase infections is the IE19 protein. Here, we define two motifs in IE19 required for S-phase infections: an N-terminal triple lysine motif and a C-terminal nucleosome-binding motif within the CTD.IMPORTANCEThe IE19 protein encoded by human cytomegalovirus (HCMV) is required for S-phase infection of dividing cells, likely because it tethers the viral genome to cellular chromosomes, thereby allowing them to survive mitosis. The mechanism through which IE19 tethers viral genomes to cellular chromosomes is not understood. For human papillomavirus, Epstein-Barr virus, and Kaposi's sarcoma-associated herpesvirus, viral genome tethering is required for persistence (latency) and pathogenesis (oncogenesis). Like these viruses, HCMV also achieves latency, and it modulates the properties of glioblastoma multiforme tumors. Therefore, defining the mechanism through which IE19 tethers viral genomes to cellular chromosomes may help us understand, and ultimately combat or control, HCMV latency and oncomodulation.

Murine noroviruses bind glycolipid and glycoprotein attachment receptors in a strain-dependent manner.

Human norovirus infections are the most common cause of acute nonbacterial gastroenteritis in humans worldwide, and glycan binding plays an important role in the susceptibility to these infections. However, due to the lack of an efficient cell culture system or small animal model for human noroviruses, little is known about the biological role of glycan binding during infection. Murine noroviruses (MNV) are also enteric viruses that bind to cell surface glycans, but in contrast to their human counterparts, they can be grown in tissue culture and a small animal host. In this study, we determined glycan-binding specificities of the MNV strains MNV-1 and CR3 in vitro, identified molecular determinants of glycan binding, and analyzed infection in vivo. We showed that unlike MNV-1, CR3 binding to murine macrophages was resistant to neuraminidase treatment and glycosphingolipid depletion. Both strains depended on N-linked glycoproteins for binding, while only MNV-1 attachment to macrophages was sensitive to O-linked glycoprotein depletion. In vivo, CR3 showed differences in tissue tropism compared to MNV-1 by replicating in the large intestine. Mapping of a glycan-binding site in the MNV-1 capsid by reverse genetics identified a region topologically similar to the histo-blood group antigen (HBGA)-binding sites of the human norovirus strain VA387. The recombinant virus showed distinct changes in tissue tropism compared to wild-type virus. Taken together, our data demonstrate that MNV strains evolved multiple strategies to bind different glycan receptors on the surface of murine macrophages and that glycan binding contributes to tissue tropism in vivo.

Contributions of CTCF and DNA methyltransferases DNMT1 and DNMT3B to Epstein-Barr virus restricted latency.

Establishment of persistent Epstein-Barr virus (EBV) infection requires transition from a program of full viral latency gene expression (latency III) to one that is highly restricted (latency I and 0) within memory B lymphocytes. It is well established that DNA methylation plays a critical role in EBV gene silencing, and recently the chromatin boundary protein CTCF has been implicated as a pivotal regulator of latency via its binding to several loci within the EBV genome. One notable site is upstream of the common EBNA gene promoter Cp, at which CTCF may act as an enhancer-blocking factor to initiate and maintain silencing of EBNA gene transcription. It was previously suggested that increased expression of CTCF may underlie its potential to promote restricted latency, and here we also noted elevated levels of DNA methyltransferase 1 (DNMT1) and DNMT3B associated with latency I. Within B-cell lines that maintain latency I, however, stable knockdown of CTCF, DNMT1, or DNMT3B or of DNMT1 and DNMT3B in combination did not result in activation of latency III protein expression or EBNA gene transcription, nor did knockdown of DNMTs significantly alter CpG methylation within Cp. Thus, differential expression of CTCF and DNMT1 and -3B is not critical for maintenance of restricted latency. Finally, mutant EBV lacking the Cp CTCF binding site exhibited sustained Cp activity relative to wild-type EBV in a recently developed B-cell superinfection model but ultimately was able to transition to latency I, suggesting that CTCF contributes to but is not necessarily essential for the establishment of restricted latency.

Ganglioside-linked terminal sialic acid moieties on murine macrophages function as attachment receptors for murine noroviruses.

Noroviruses are the major cause of nonbacterial gastroenteritis in humans. However, little is known regarding the norovirus life cycle, including cell binding and entry. In contrast to human noroviruses, the recently discovered murine norovirus 1 (MNV-1) readily infects murine macrophages and dendritic cells in culture. Many viruses, including the related feline calicivirus, use terminal sialic acids (SA) as receptors for infection. Therefore, we tested whether SA moieties play a role during MNV-1 infection of murine macrophages. Competition with SA-binding lectins and neuraminidase treatment led to a reduction in MNV-1 binding and infection in cultured and primary murine macrophages, suggesting a role for SA during the initial steps of the MNV-1 life cycle. Because SA moieties can be attached to glycolipids (i.e., gangliosides), we next determined whether MNV-1 uses gangliosides during infection. The gangliosides GD1a, GM1, and asialo-GM1 (GA1) are natural components of murine macrophages. MNV-1 bound to ganglioside GD1a, which is characterized by an SA on the terminal galactose, but not to GM1 or asialo-GM1 in an enzyme-linked immunosorbent assay. The depletion of gangliosides using an inhibitor of glycosylceramide synthase (d-threo-P4) led to a reduction of MNV-1 binding and infection in cultured and primary murine macrophages. This defect was specifically rescued by the addition of GD1a. A similar phenotype was observed for MNV field strains WU11 (GV/WU11/2005/USA) and S99 (GV/Berlin/2006/DE). In conclusion, our data indicate that MNV can use terminal SA on gangliosides as attachment receptors during binding to murine macrophages.

Human Cytomegalovirus Genomes Survive Mitosis via the IE19 Chromatin-Tethering Domain.

The genomes of DNA tumor viruses regain nuclear localization after nuclear envelope breakdown during mitosis through the action of a viral protein with a chromatin-tethering domain (CTD). Here, we report that the human cytomegalovirus (HCMV) genome is maintained during mitosis by the CTD of the viral IE19 protein. Deletion of the IE19 CTD or disruption of the IE19 splice acceptor site reduced viral genome maintenance and progeny virion formation during infection of dividing fibroblasts, both of which were rescued by IE19 ectopic expression. The discovery of a viral genome maintenance factor during productive infection provides new insight into the mode of HCMV infection implicated in birth defects, organ transplant failure, and cancer.IMPORTANCE Human cytomegalovirus (HCMV) is the leading infectious cause of birth defects, represents a serious complication for immunocompromised HIV/AIDS and organ transplant patients, and contributes to both immunosenescence and cardiovascular diseases. HCMV is also implicated in cancers such as glioblastoma multiforme (GBM) and infects ex vivo-cultured GBM tumor cells. In dividing tumor cells, the genomes of DNA tumor viruses regain nuclear localization after nuclear envelope breakdown during mitosis. This mitotic survival is mediated by a viral protein with a chromatin-tethering domain (CTD). Here, we report that the HCMV genome is maintained in dividing fibroblasts by the CTD of the viral IE19 protein. The discovery of a viral genome maintenance factor during productive infection could help explain viral genome dynamics within HCMV-positive tumors as well as during latency.

UL84-independent replication of human cytomegalovirus strain TB40/E.

The UL84 gene of human cytomegalovirus is implicated in the initiation of viral DNA replication during lytic infection. UL84 is essential for replication of a cloned viral origin of lytic replication (oriLyt) in vitro and mutants of strains AD169 or Towne with deletions or insertions in UL84 fail to grow in cells permissive for wild type virus. Here we show that UL84 is dispensable for replication of a strain TB40/E clone derived from a bacterial artificial chromosome. The genomes of the fibroblast-adapted strains AD169 and Towne are altered substantially from the consensus for strains that have not been propagated extensively in cell culture. The parental TB40/E genome conforms to the consensus genomic organization. Accordingly, natural HCMV strains may possess replication capability that extends beyond the known oriLyt-dependent replication system of laboratory strains.