Determining the role of the Epstein-Barr virus Cp EBNA2-dependent enhancer during the establishment of latency by using mutant and wild-type viruses recovered from cottontop marmoset lymphoblastoid cell lines.

Epstein-Barr virus (EBV) nuclear antigen (EBNA) 2 (EBNA2) is involved in upregulating the expression of both EBNAs and latency-associated membrane proteins. Transcription of the six EBNA genes, which are expressed in EBV-immortalized primary B cells, arises from one of two promoters, Cp and Wp, located near the left end of the viral genome. Wp is exclusively used to drive EBNA gene transcription during the initial stages of infection in primary B cells; induction of transcription from Cp follows. We previously have mapped an EBNA2-dependent enhancer upstream of Cp (M. Woisetschlaeger et al., Proc. Natl. Acad. Sci. USA 88:3942-3946, 1991) and, more recently, have demonstrated that deletion of this enhancer results in EBV-immortalized lymphoblastoid cell lines (LCLs) that are heavily biased toward the use of Wp to drive transcription of the EBNA genes (L. Yoo et al., J. Virol. 71:9134-9142, 1997). To assess the immortalizing capacity of this mutant EBV and to monitor the early events after infection of primary B cells, B cells isolated from cottontop marmosets were used to generate LCLs immortalized with the Cp EBNA2 enhancer deletion mutant virus. As previously reported, all EBV-infected marmoset LCLs examined could be triggered to produce significant levels of virus. Infection of human B cells with wild-type or Cp EBNA2 enhancer mutant viruses recovered from marmoset B-cell lines demonstrated that (i) the Cp EBNA2 enhancer mutant virus immortalizes primary human B cells nearly as efficiently as wild-type virus and (ii) the Cp EBNA2-dependent enhancer plays an important role in the induction of Cp activity during the early stages of infection. The latter is consistent with the phenotype of LCLs immortalized with the Cp EBNA2 enhancer mutant EBV. Finally, using an established LCL in which EBNA2 function is regulated by beta-estradiol, we showed that the loss of EBNA2 function results in an approximately 4-fold decrease in the steady-state levels of Cp-initiated transcripts and a concomitant increase in the steady-state levels of Wp-initiated transcripts. Taken together, these results provide strong evidence that EBNA2 plays an important role in regulating Cp activity. These results also demonstrate that diminished induction of Cp activity does not appear to affect the ability of EBV to immortalize primary B cells in cultures. Finally, as shown here, infection of marmoset B cells with immortalization-competent mutants of EBV provides a convenient reservoir for the production of mutant viruses.

Mutations in the tyrosine phosphatase CD45 gene in a child with severe combined immunodeficiency disease.

The hematopoietic-specific transmembrane protein tyrosine phosphatase CD45 functions to regulate Src kinases required for T- and B-cell antigen receptor signal transduction. So far, there have been no reports to our knowledge of a human deficiency in a tyrosine-specific phosphatase. Here, we identified a male patient with a deficiency in CD45 due to a large deletion at one allele and a point mutation at the other. The point mutation resulted in the alteration of intervening sequence 13 donor splice site. The patient presented at 2 months of age with severe combined immunodeficiency disease. The population of peripheral blood T lymphocytes was greatly diminished and unresponsive to mitogen stimulation. Despite normal B-lymphocyte numbers, serum immunoglobulin levels decreased with age. Thus, CD45 deficiency in humans results in T- and B-lymphocyte dysfunction.

Partial glycosylation at asparagine-2181 of the second C-type domain of human factor V modulates assembly of the prothrombinase complex.

Thrombin-activated factor Va exists as two isoforms, factor Va(1) and factor Va(2), which differ in the size of their light chains and their affinity for biological membranes. The heterogeneity of the light chain remained following incubation of factor Va with N-glycanase. However, we found that the factor V C2 domain, which contains a single potential glycosylation site at Asn-2181, was partially glycosylated when expressed in COS cells. To confirm the structural basis for factor Va(1) and factor Va(2), we mutated Asn-2181 to glutamine (N2181Q) and expressed this mutant using a B domain deletion construct (rHFV des B) in COS cells. Thrombin activation of N2181Q released a light chain with mobility identical to that of factor Va(2) on SDS-PAGE. The functional properties of purified N2181Q were similar to those of factor Va(2) in prothrombinase assays carried out in the presence of limiting concentrations of phosphatidylserine. The binding of human factor Va(1) and factor Va(2) to 75:25 POPC/POPS vesicles was also investigated in equilibrium binding assays using proteins containing a fluorescein-labeled heavy chain. The affinity of human factor Va(2) binding to POPC/POPS vesicles was approximately 3-fold higher than that of factor Va(1). These results indicate that partial glycosylation of factor V at asparagine-2181 is the structural basis of the light chain doublet and that the presence of this oligosaccharide reduces the affinity of factor Va for biological membranes.

B-cell lines immortalized with an Epstein-Barr virus mutant lacking the Cp EBNA2 enhancer are biased toward utilization of the oriP-proximal EBNA gene promoter Wp1.

During Epstein-Barr virus (EBV) latent infection of B lymphocytes in vitro, six viral nuclear antigens (EBNAs) are expressed from one of two promoters, Cp or Wp, whose activities have previously been shown to be mutually exclusive in established lymphoblastoid cell lines. Initially after infection, the EBNA genes are transcribed from Wp, which is present in multiples copies within the major internal repeat of EBV. Approximately 48 to 72 h postinfection, Wp is downregulated, with a corresponding increase in transcription from Cp. An EBNA2-responsive enhancer exists upstream of Cp, and a role for EBNA2 in the induction of Cp activity during the establishment of viral latency has previously been proposed (Woisetschlaeger et al., Proc. Natl. Acad. Sci. USA 87:1725-1729, 1991). To critically assess the potential role for this enhancer region in determining relative usage of Cp and Wp, an EBNA2 enhancer deletion mutant virus was generated. Lymphoblastoid cell lines were screened by PCR and Southern blotting for the presence of mutant virus harboring the EBNA2 enhancer deletion. A quantitative S1 nuclease protection assay was developed to allow comparison of relative Cp and Wp activities for the cell lines containing mutant virus and those of the wild-type recombinants which lacked the enhancer deletion. In general, the wild-type recombinants had higher levels of Cp-initiated transcripts than Wp-initiated transcripts. In contrast, the Cp EBNA2 enhancer deletion mutants exhibited a strong bias toward Wp activity. Notably, only the first Wp (oriP-proximal Wp; Wp1) appears active in these mutants. S1 nuclease protection assays using a probe which hybridizes to the W2 exon, contained in both Cp- and Wp-initiated transcripts, indicated that the total level of transcription from Cp and Wp remained the same in wild-type and EBNA2 enhancer mutant cell lines. The presence of both Cp and Wp activity in the wild-type recombinants, as well as in newly derived lymphoblastoid cell lines established with the prototype B95.8 virus, demonstrated that Cp and Wp activities are not always mutually exclusive.

Mature B cells are required for acute splenic infection, but not for establishment of latency, by murine gammaherpesvirus 68.

Murine gammaherpesvirus 68 (gamma HV-68; also referred to as MHV-68) is a gammaherpesvirus which infects murid rodents. Previous studies showed that CD8 T cells are important for controlling gamma HV-68 replication during the first 2 weeks of infection and suggested a role for B cells in latent or persistent gamma HV-68 infection. To further define the importance of B cells and CD8 T cells during acute and chronic gamma HV-68 infection, we examined splenic infection in mice with null mutations in the transmembrane domain of the mu-heavy-chain constant region (MuMT; B-cell and antibody deficient) or in the beta2-microglobulin gene (beta2 -/-; CD8 deficient). Immunocompetent mice infected intraperitoneally with gamma HV-68 demonstrated peak splenic titers 9 to 10 days postinfection, cleared infectious virus 15 to 20 days postinfection, and harbored low levels of latent virus at 6 weeks postinfection. Beta2-/- mice showed peak splenic gamma HV-68 titers similar to those of normal mice but were unable to clear infectious virus completely from the spleen, demonstrating persistent infectious virus 6 weeks postinfection. These data indicate that CD8 T cells are important for clearing infectious gamma HV-68 from the spleen. Infected MuMT mice did not demonstrate detectable infectious gamma HV-68 in the spleen at any time after infection, indicating that mature B lymphocytes are necessary for acute splenic infection by gamma HV-68. Despite the lack of measurable acute infection, MuMT spleen cells harbored latent virus 6 weeks postinfection at a level about 100-fold higher than that in normal mice. These data demonstrate establishment of latency by a herpesvirus in an organ in the absence of acute viral replication in that organ. In addition, they demonstrate that gamma HV-68 can establish latency in a cell type other than mature B lymphocytes.

Sleep patterning and behaviour in cats with pontine lesions creating REM without atonia.

Lesions of the dorsal pontine tegmentum release muscle tone and motor behaviour, much of it similar to orienting during wakefulness, into rapid eye movement sleep (REM), a state normally characterized by paralysis. Sleep after pontine lesions may be altered, with more REM-A episodes of shorter duration compared to normal REM. We examined behaviour, ponto-geniculo-occipital (PGO) waves (which may be central markers of orienting) and sleep in lesioned cats: (i) to characterize the relationship of PGO waves to behaviour in REM-A; (ii) to determine whether post-lesion changes in the timing and duration of REM-A episodes were due to activity-related awakenings: and (iii) to determine whether alterations in sleep changed the circadian sleep/wake cycle in cats. Behavioural release in REM-A was generally related to episode length, but episode length was not necessarily shorter than normal REM in cats capable of full locomotion in REM-A. PGO wave frequency was reduced overall during REM-A, but was higher during REM-A with behaviour than during quiet REM-A without overt behaviour. Pontine lesions did not significantly alter the circadian sleep/wake cycle: REM-A had approximately the same Light/Dark distribution as normal REM. Differences in the patterning of normal REM and REM-A within sleep involve more than mere movement-induced awakenings. Brainstem lesions that eliminate the atonia of REM may damage neural circuitry involved in REM initiation and maintenance; this circuitry is separate from circadian control mechanisms.