Transcription of hepatitis delta virus RNA by RNA polymerase II.

Previous studies have indicated that the replication of the RNA genome of hepatitis delta virus (HDV) involves redirection of RNA polymerase II (Pol II), a host enzyme that normally uses DNA as a template. However, there has been some controversy about whether in one part of this HDV RNA transcription, a polymerase other than Pol II is involved. The present study applied a recently described cell system (293-HDV) of tetracycline-inducible HDV RNA replication to provide new data regarding the involvement of host polymerases in HDV transcription. The data generated with a nuclear run-on assay demonstrated that synthesis not only of genomic RNA but also of its complement, the antigenome, could be inhibited by low concentrations of amanitin specific for Pol II transcription. Subsequent studies used immunoprecipitation and rate-zonal sedimentation of nuclear extracts together with double immunostaining of 293-HDV cells, in order to examine the associations between Pol II and HDV RNAs, as well as the small delta antigen, an HDV-encoded protein known to be essential for replication. Findings include evidence that HDV replication is somehow able to direct the available delta antigen to sites in the nucleoplasm, almost exclusively colocalized with Pol II in what others have described as transcription factories.

Experimental study on the action of allitridin against human cytomegalovirus in vitro: Inhibitory effects on immediate-early genes.

Garlic (Allium sativum) extraction has been reported having anti-HCMV efficacy. This study was aimed to investigate the effect of allitridin (diallyl trisulfide, a compound from A. sativum extraction) on the replication of HCMV and the expression of viral immediate-early genes. In HCMV plaque-reduction assay, allitridin appeared a dose-dependent inhibitory ability with EC(50) value of 4.2 microg/ml (selective index, SI=16.7). Time-of-addition and time-of-removal studies showed that allitridin inhibited HCMV replication in earlier period of viral cycle before viral DNA synthesis. Both immediate early gene (ie1) transcription and IEA (IE(1)72 and IE(2)86) expression was suppressed by allitridin, but not by GCV in a single HCMV cycle format. In addition, allitridin appeared stronger inhibition on IE(2)86 than on IE(1)72. Decrease of viral DNA load in infected cells was also detected under allitridin treatment, probably due to an indirect consequence of the reduction in ie gene transcription. In summary, this study indicated that allitridin has anti-HCMV activity and the mechanism is associated with suppression of ie gene transcription.

Effects of human cytomegalovirus infection on apoptosis and expression of apoptosis-regulating factors.

The present study aimed to find out dynamic changes of apoptosis in human cytomegalovirus (HCMV) infected cells and the influence of HCMV infection on activation of caspase-3 and the expression of apoptosis-regulating genes, bcl-2 and fas mRNA. The sequential changes of apoptotic cell rate in high and low MOI (MOI = 2.5 and 0.25 respectively) of HCMV infected human embryonic lung fibroblasts (HELFs) at 1 h, 12 h, 24 h, 36 h, 48 h, 72 h and 96 h post-infection were measured by flow cytometry. The expression levels of caspase-3 protein and bcl-2 and fas mRNA in HCMV infected cells (MOI = 0.25) at 72 h post-infection were detected by Western blot and in situ hybridization methods, respectively. It was found that the ratio of apoptotic cells in normal controls was consistently lower, but the rates in low and high MOI infected cells were gradually increased with time prolonged, reached peak at 96 h (8.85%) and 72 h (25.63%), respectively. By Western blot analysis, only a narrow band of 32 kD (1 kD = 0.992 1 ku) procaspase-3 was found in normal cells, but a wider procaspase-3 band and a much wider band of 17 kD proteins (p17) appeared in the infected cells. Meanwhile, the expression of bcl-2 mRNA was higher and that of fas mRNA was lower in the normal HELF cells, whereas there were significantly lower bcl-2 mRNA and higher fas mRNA expression levels in HCMV infected cells. It was concluded that HCMV was a stronger inducer of apoptosis in HELF cells. Caspase-3, as the marker of undergoing apoptosis, was expressed increasingly and activated in the infected cells, indicating its action in HCMV-inducing apoptosis. Down-regulating bcl-2 mRNA expression and up-regulating fas mRNA expression were also involved in the mechanism of HCMV-induced apoptosis.

[Effects of allitridin on the expression of human cytomegalovirus immediate early antigens-IE72 and IE86 in human embryonic lung cells].

OBJECTIVE: To investigate the effect of allitridin injection on the expression of human cytomegalovirus (HCMV) immediate-early antigens (IEAs including IE72 and IE86) in human embryonic lung cells. METHOD: HCMV AD 169 Virus strain infected cell model (MOI = 2.5 and 0.25, respectively) were established, and then treated with ICm5 and MTC doses of allitridin. Western blot was used to analyze the of IE72 and IE86 expression after the treatment, ganciclovir(GCV, IC50 and 2.3 x IC50) treatment as control. RESULT: No matter what kind of MOI was used, both IE86 and IE72 antigens' expression was effectively suppressed by allitridin treatment, and the inhibitory rate of IE86 was almost twice of IE72's. Compared with GCV, allitridin had stronger inhibitory effect on IE86 expressing, although its efficacy on IE72 was weaker than GCV. CONCLUSION: Allitridin could suppress the expression of IE72 and IE86, especially for IE86 expressing, maybe it is ore of key role in the mechanism of allitridin against HCMV.

Thymidine phosphorylase expression in B-cell lymphomas and its significance: a new prognostic marker?

OBJECTIVE: To explore thymidine phosphorylase (TP) expression in B-cell lymphomas (BCLs). TP is expressed by tumor and stromal cells in a variety of cancers. STUDY DESIGN: Paraffin-embedded tissues from follicular lymphomas, diffuse large BCLs (DLBCLs), and benign lymph nodes were studied using immunohistochemical staining with antibodies for TP and CD68. Prognostic markers were used to stain DLBCLs. We correlated TP expression in DLBCL indirectly with prognostic immunomarkers and directly with survival data. RESULTS: TP expression in BCLs was noted in a subset of malignant B cells. TP expression in higher-grade lymphoma was identified in 66% of cases and 11% of lower-grade lymphomas. Macrophages/stromal cells demonstrated an intense cytoplasmic and/or nuclear staining pattern in both lymphoma and benign lymph nodes, confirmed by CD68 coexpression. Increased macrophage/ stromal cells in higher-grade lymphomas are associated with enhanced TP expression in neoplastic B cells (observation only). Sixty-eight percent of TP-positive DLBCLs were of nongerminal center origin, indicating poorer prognosis. CONCLUSION: TP is more likely expressed by malignant B cells in higher-grade lymphomas, and expression of TP possibly results from changes intrinsic to the tumor cells or interactions between microenvironment and tumor. TP positivity in DLBCL correlates with nongerminal center origin and worse outcome.

Expression of thymidine phosphorylase in lymph nodes involved with mycosis fungoides and sézary syndrome.

Thymidine phosphorylase may be overexpressed in both neoplastic cells and tumor stromal cells in a variety of malignancies. Our study explores thymidine phosphorylase expression in lymph nodes (LNs) from patients with mycosis fungoides (MF) or Sézary syndrome (SS). In MF/SS, the LNs may have a pathologic diagnosis of either dermatopathic lymphadenopathy (LN-DL) or involvement by MF/SS (LN-MF). We performed immunohistochemical staining on MF/SS lymph nodes using antibodies to thymidine phosphorylase, CD68, CD21, CD3, and CD4. In both LN-DL and benign nodes, thymidine phosphorylase staining was noted only in macrophages, dendritic cells, and endothelial cells. In LN-MF, thymidine phosphorylase expression was also noted in subsets of intermediate to large neoplastic T cells. Concurrent CD68, CD21, CD3, and CD4 staining supported the above observations. Similar results were noted in the skin and in LN-MF with large cell transformation. Other T-cell lymphomas were also examined (total 7 cases); only enteropathy-type T-cell lymphoma (1 case) showed TP positivity in neoplastic T lymphocytes. We demonstrated that thymidine phosphorylase staining is present in neoplastic T cells in mycosis fungoides. The exact mechanism needs further investigation.

Action of inhibitors on accumulation of processed hepatitis delta virus RNAs.

Hepatitis delta virus (HDV) replication involves processing and accumulation of three RNA species: the genome, its exact complement (the antigenome), and a polyadenylated mRNA that acts as a template for the small delta antigen (deltaAg), the only protein of HDV and essential for genome replication. In a recently reported experimental system, addition of tetracycline induced synthesis of a DNA-directed source of deltaAg, producing within 24 h a significant increase in accumulation of newly transcribed and processed HDV RNAs. This induction was used here to study the action of various inhibitors on accumulation. For example, potent and HDV-specific inhibition, in the absence of detected host toxicity, could be obtained with ribavirin, mycophenolic acid, and viramidine. An interpretation is that these inhibitors reduced the available GTP pool, leading to a specific inhibition of the synthesis and accumulation of HDV RNA-directed RNA species. In contrast, no inhibition was observed with L-FMAU (2'-fluoro-5-methyl-beta-L-arabinofuranosyl-uridine), alpha interferon, or pegylated alpha interferon. After modifications to the experimental system, it was also possible to examine the effects of three known host RNA polymerase inhibitors on HDV genome replication: amanitin, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), and actinomycin. Of most interest, amanitin at low doses blocked accumulation of HDV RNA-directed mRNA but had less effect on HDV genomic and antigenomic RNAs. Additional experiments indicated that this apparent resistance to amanitin inhibition of genomic and antigenomic RNA relative to mRNA may not reflect a difference in the transcribing polymerase but rather relative differences in the processing and stabilization of nascent RNA transcripts.

Development of a novel system to study hepatitis delta virus genome replication.

Hepatitis delta virus (HDV) genome replication requires the virus-encoded small delta protein (deltaAg). During replication, nucleotide sequence changes accumulate on the HDV RNA, leading to the translation of deltaAg species that are nonfunctional or even inhibitory. A replication system was devised where all deltaAg was conditionally provided from a separate and unchanging source. A line of human embryonic kidney cells was stably transfected with a single copy of cDNA encoding small deltaAg, with expression under tetracycline (TET) control. Next, HDV genome replication was initiated in these cells by transfection with a mutated RNA unable to express deltaAg. Thus, replication of this RNA was under control of the TET-inducible deltaAg. In the absence of TET, there was sufficient deltaAg to allow a low level of HDV replication that could be maintained for at least 1 year. When TET was added, both deltaAg and genomic RNA increased dramatically within 2 days. With clones of such cells, designated 293-HDV, the burst of HDV RNA replication interfered with cell cycling. Within 2 days, there was a fivefold enhancement of G1/G0 cells relative to both S and G2/M cells, and by 6 days, there was extensive cell detachment and death. These findings and those of other studies that are under way demonstrate the potential applications of this experimental system.

Alternative processing of hepatitis delta virus antigenomic RNA transcripts.

Intrinsic to the life cycle of hepatitis delta virus (HDV) is the fact that its RNAs undergo different forms of posttranscriptional RNA processing. Transcripts of both the genomic RNA and its exact complement, the antigenomic RNA, undergo ribozyme cleavage and RNA ligation. In addition, antigenomic RNA transcripts can undergo 5' capping, 3' polyadenylation, and even RNA editing by an adenosine deaminase. This study focused on the processing of antigenomic RNA transcripts. Two approaches were used to study the relationship between the events of polyadenylation, ribozyme cleavage, and RNA ligation. The first represented an examination under more controlled conditions of mutations in the poly(A) signal, AAUAAA, which is essential for this processing. We found that when a separate stable source of deltaAg-S, the small delta protein, was provided, the replication ability of the mutated RNA was restored. The second approach involved an examination of the processing in transfected cells of specific Pol II DNA-directed transcripts of HDV antigenomic sequences. The DNA constructs used were such that the RNA transcripts were antigenomic and began at the same 5' site as the mRNA produced during RNA-directed HDV genome replication. A series of such constructs was assembled in order to test the relative abilities of the transcripts to undergo processing by polyadenylation or ribozyme cleavage at sites further 3' on a multimer of HDV sequences. The findings from the two experimental approaches led to significant modifications in the rolling-circle model of HDV genome replication.