A sixth Epstein-Barr virus nuclear protein (EBNA3B) is expressed in latently infected growth-transformed lymphocytes.

In the Epstein-Barr virus BamHI E genomic fragment, there are three distantly homologous long open reading frames, BERF1, BERF2b, and BERF4, each of which is preceded by a short open reading frame. The most leftward and most rightward short and long open reading frame pairs encode 145- and 155-kilodalton proteins in latently infected cells (EBNA3A and EBNA3C, respectively). In this report, we demonstrate that the middle long open reading frame, BERF2b, encodes part of a 165-kilodalton nuclear protein in every latently infected cell. Therefore, this protein is designated EBNA3B.     

A fifth Epstein-Barr virus nuclear protein (EBNA3C) is expressed in latently infected growth-transformed lymphocytes.

Three distantly homologous neighboring long open reading frames in the Epstein-Barr virus (EBV) genome are preceded by short open reading frames. The leftmost short and long open reading frames encode EBNA3, a nuclear protein which is slightly smaller (145 kilodaltons [kDa]) than two other nuclear proteins (150 to 155 kDa) detected in Western blots (immunoblots) of latently infected cell protein (K. Hennessy, F. Wang, E. Woodland-Bushman, and E. Kieff, Proc. Natl. Acad. Sci. USA 83:5693-5697, 1986; I. Joab, D. T. Rowe, M. Bodescot, J.-C. Nicolas, P. J. Farrell, and M. Perricaudet, J. Virol. 61:3340-3344, 1987). We have demonstrated that the most rightward short (BERF3) and long (BERF4) open reading frames are spliced in frame at the 3' end of a 5-kilobase latently infected cell RNA and that this RNA begins within or upstream of the EBV long internal repeat. EBV-immune human antibodies specific for the long open reading frame translation product identified a 155-kDa protein on Western blots of latently infected cell protein and specifically reacted with large nonnucleolar nuclear granules in every latently infected cell. Expression of the cDNA in BALB/c 3T3 cells resulted in translation of full-size EBNA3C but had no effect on cell morphology, contact inhibition, or serum independence.     

A bicistronic Epstein-Barr virus mRNA encodes two nuclear proteins in latently infected, growth-transformed lymphocytes.

EBNA2 is a nuclear protein expressed in all cells latently infected with and growth transformed by Epstein-Barr virus (EBV) infection (K. Hennessy and E. Kieff, Science 227:1230-1240, 1985). The nucleotide sequence of the EBNA2 mRNA (J. Sample, M. Hummel, D. Braun, M. Birkenbach, and E. Kieff, Proc. Natl. Acad. Sci. USA 83:5096-5100, 1986) revealed that it begins with a 924-base open reading frame that has an unusual potential translational initiation site (CAAATGG). This open reading frame is followed by 138 nucleotides with only one highly unlikely translational initiation site (TACATGC), which would translate a pentapeptide before the next stop codon. The last part of the mRNA is the open reading frame which encodes EBNA2. In this paper, we demonstrate that the 924-base open reading frame translates a 40-kilodalton protein in vitro or in murine cells transfected with the EBNA2 cDNA under control of the murine leukemia virus long terminal repeat. A protein of identical size was detected in EBV-transformed, latently infected human lymphocyte nuclei by using antibody specific for the leader open reading frame expressed in bacteria. Therefore, this is a rare example of a mRNA which translates two proteins from nonoverlapping open reading frames. Since the protein encoded by the leader of the EBNA mRNA is expressed in all nuclei of a latently infected cell line, it was designated EBNA-LP. EBNA-LP localizes to small intranuclear particles and differs in this respect from EBNA1, EBNA2, or EBNA3. EBNA-LP is not expressed in an EBV-transformed marmoset lymphocyte cell (B95-8) or in one EBV-infected Burkitt tumor cell line (Raji) but is expressed in three other Burkitt tumor cell lines (Namalwa, P3HR-1, and Daudi).     

Synchronous and sequential activation of latently infected Epstein-Barr virus genomes.

A lymphoid cell system was established that can induce the prompt and synchronous activation of latent Epstein-Barr virus (EBV) genomes and thus allows the identification of viral genes that are activated sequentially depending on their functions. With this system, we proved that disruption of EBV latency is initiated by activation of four EBV genes and that protein synthesis is not required prior to activation of latent EBV. The system should be an in vitro model for studying the mechanism of herpesvirus latency.     

Identification of Marek''s disease virus nuclear antigen in latently infected lymphoblastoid cells.

A new Marek's disease virus (MDV) nuclear antigen (MDNA) was identified in two MDV-transformed T-lymphoblastoid cell lines, MKT-1 and MSB-1, derived from chickens bearing tumors induced by MDV. This MDNA was not detected in MSB-1 cells maintained in iododeoxyuridine, which activates the latent MDV genome. Moreover, it was not found in chicken embryo fibroblasts undergoing productive and cytolytic infection with MDV. Expression of MDNA is not related to strain pathogenicity in chickens, because chicken embryo fibroblasts productively infected with the pathogenic RBIB strain or the nonpathogenic CV-1 strain of MDV did not express this antigen. DNA-protein immunoprecipitation studies revealed that MDNA bound to two sites in the 190,00-base-pair (bp) MDV genome. One of these loci identified by MDNA obtained from MKT-1 and MSB-1 cells corresponded to a 476-bp segment within the short unique region of BamHI-A MDV DNA. A second locus located in a 280-bp segment within the short inverted repeat region of BamHI-A was also identified by MDNA from MSB-1 cells but not by MDNA obtained from MKT-1 cells. Analyses of the nucleotide sequence by DNase digestion showed that MDNA protected a 60-bp segment spanning a 22-bp palindromic sequence of the short unique region and a 103-bp sequence encompassing a 32-bp palindrome in the short inverted repeat region of BamHI-A MDV DNA.     

Effects of adenine arabinoside on lymphocytes infected with Epstein-Barr virus.

Low concentrations of adenine arabinoside inhibited growth of two Epstein-Barr virus producer cell lines in culture, while not significantly affecting a nonproducer cell line and a B-cell-negative line. These observations were extended to include freshly infected cells. Mitogen-stimulated human umbilical cord blood lymphocytes were unaffected by the drug at concentration levels that inhibited [3H]thymidine incorporation into the DNA of Epstein-Barr virus-stimulated cells. DNA synthesis in Epstein-Barr virus-superinfected Raji cells was also adversely affected by adenine arabinoside. However, these same low concentrations of adenine arabinoside in the triphosphate form produced less effect on DNA synthesis in nuclear systems and DNA polymerase assays than on growth or DNA synthesis in whole cells. Therefore the effects reported here of low concentrations of the drug on whole cells may be only in part related to DNA polymerase inhibition. The work reported here suggests that adenine arabinoside has multiple sites of action in infected cells.     

An Epstein-Barr virus transforming protein associates with vimentin in lymphocytes.

The Epstein-Barr virus (EBV) latent infection membrane protein (LMP) is likely to be an important mediator of EBV-induced cell proliferation, since it is one of the few proteins encoded by the virus in latent infection and since production of this protein in Rat-1 cells results in their conversion to a fully transformed phenotype. LMP was previously noted to localize to patches at the cell periphery. In this paper we examine the basis of LMP patching in EBV-infected, transformed lymphocytes. Our data indicate that LMP is associated with the cytoskeletal protein vimentin. Although LMP is fully soluble in isotonic Triton X-100 buffer, only 50% of it is extracted from cells in this solution. The rest remains bound to the cytoskeleton. LMP undergoes phosphorylation, and phosphorylated LMP is preferentially associated with the cytoskeleton. As judged by both immunofluorescence and immunoelectron microscopy, the vimentin network in EBV-transformed lymphocytes or EBV-infected Burkitt tumor lymphocytes is abnormal. Vimentin and LMP often colocalize in a single patch near the plasma membrane. In response to Colcemid treatment of EBV-infected cells, vimentin reorganizes into perinuclear rings, as it does in uninfected cells. LMP is associated with these perinuclear rings. Vimentin (or a vimentin-associated protein) may be a transducer of an LMP transmembrane effect in lymphoproliferation.     

Effect of mild stress in mice latently infected pseudorabies virus.

Stress is one of the important factors that induces reactivation of pseudorabies virus (PrV) in latently infected pigs. We established a murine model of latent PrV infection and examined the effects of mild stress treatment in order to demonstrate that this model simulates natural infection in the pig. Latently infected mice excreted PrV from the nasal cavity under stress treatments consisting of restraint, exposure to cold or transport. Similar reactions have been observed upon treatment with acetylcholine and dexamethasone. The present findings demonstrate that these kinds of mild stress reactivate the virus in murine latent infection models in a manner similar to the induction of latent infection in pigs in the field.     

Pregnancy and parturition of mice latently infected with Pseudorabies virus.

In this study, the influence of pregnancy and parturition on mice in a mouse model of latent infection with Pseudorabies virus (PrV) was analyzed. Latently infected (LI) female mice were paired with mature uninfected males. The mating produced progeny without any clinical signs of Aujeszky's Disease. At weaning, both male and female progeny of LI mice showed significantly lower weight than control mice. PrV was not detected from nasal swab specimens of the female parent mice or the trigeminal ganglia of all mice, except 3 of 50 neonatal mice. These findings demonstrate that pregnancy and parturition induce little reactivation of latent PrV, but do affect the mother's body, as indicated by the decreased weight of progeny at weaning.     

Acute infection with Epstein-Barr virus targets and overwhelms the peripheral memory B-cell compartment with resting, latently infected cells

In this paper we demonstrate that during acute infection with Epstein-Barr virus (EBV), the peripheral blood fills up with latently infected, resting memory B cells to the point where up to 50% of all the memory cells may carry EBV. Despite this massive invasion of the memory compartment, the virus remains tightly restricted to memory cells, such that, in one donor, fewer than 1 in 10(4) infected cells were found in the naive compartment. We conclude that, even during acute infection, EBV persistence is tightly regulated. This result confirms the prediction that during the early phase of infection, before cellular immunity is effective, there is nothing to prevent amplification of the viral cycle of infection, differentiation, and reactivation, causing the peripheral memory compartment to fill up with latently infected cells. Subsequently, there is a rapid decline in infected cells for the first few weeks that approximates the decay in the cytotoxic-T-cell responses to viral replicative antigens. This phase is followed by a slower decline that, even by 1 year, had not reached a steady state. Therefore, EBV may approach but never reach a stable equilibrium.     

A positive autoregulatory loop of LMP1 expression and STAT activation in epithelial cells latently infected with Epstein-Barr virus

STAT3 and STAT5 are constitutively activated and nuclear in nasopharyngeal carcinoma (NPC) cells. In normal signaling, STATs are only transiently activated. To investigate whether Epstein-Barr virus (EBV), and in particular the protein LMP1, contributes to sustained STAT phosphorylation and activation in epithelial cells, we examined STAT activity in two sets of paired cell lines, HeLa, an EBV-converted HeLa cell line, HeLa-Bx1, the NPC-derived cell line CNE2-LNSX, and an LMP1-expressing derivative, CNE2-LMP1. EBV infection was associated with a significant increase in the tyrosine-phosphorylated forms of STAT3 and STAT5 in HeLa-Bx1 cells. This effect correlated with LMP1 expression, since phosphorylated STAT3 and STAT5 levels were also increased in CNE2-LMP1 cells relative to the control CNE2-LNSX cells. No change was observed in STAT1 or STAT6 phosphorylation in these cell lines, nor was there a significant change in the levels of total STAT3, STAT5, STAT1, or STAT6 protein. Tyrosine phosphorylation allows the normally cytoplasmic STAT proteins to enter the nucleus and bind to their recognition sequences in responsive promoters. The ability of LMP1 to activate STAT3 was further established by immunofluorescence assays in which coexpression of LMP1 in transfected cells was sufficient to mediate nuclear relocalization of Flag-STAT3 and by an electrophoretic mobility shift assay which showed that LMP1 expression in CNE2-LNSX cells was associated with increased endogenous STAT3 DNA binding activity. In addition, the activity of a downstream target of STAT3, c-Myc, was upregulated in HeLa-Bx1 and CNE2-LMP1 cells. A linkage was established between interleukin-6 (IL-6)- and LMP1-mediated STAT3 activation. Treatment with IL-6 increased phosphorylated STAT3 levels in CNE2-LNSX cells, and conversely, treatment of CNE2-LMP1 cells with IL-6 neutralizing antibody ablated STAT3 activation and c-Myc upregulation. The previous observation that STAT3 activated the LMP1 terminal repeat promoter in reporter assays was extended to show upregulated expression of endogenous LMP1 mRNA and protein in HeLa-Bx1 cells transfected with a constitutively activated STAT3. A model is proposed in which EBV infection of an epithelial cell containing activated STATs would permit LMP1 expression. This in turn would establish a positive feedback loop of IL-6-induced STAT activation, LMP1 and Qp-EBNA1 expression, and viral genome persistence.