Autocrine CCL3 and CCL4 induced by the oncoprotein LMP1 promote Epstein-Barr virus-triggered B cell proliferation.

Epstein-Barr virus (EBV) alters the regulation and expression of a variety of cytokines in its host cells to modulate host immune surveillance and facilitate viral persistence. Using cytokine antibody arrays, we found that, in addition to the cytokines reported previously, two chemotactic cytokines, CCL3 and CCL4, were induced in EBV-infected B cells and were expressed at high levels in all EBV-immortalized lymphoblastoid cell lines (LCLs). Furthermore, EBV latent membrane protein 1 (LMP1)-mediated Jun N-terminal protein kinase activation was responsible for upregulation of CCL3 and CCL4. Inhibition of CCL3 and CCL4 in LCLs using a short hairpin RNA approach or by neutralizing antibodies suppressed cell proliferation and caused apoptosis, indicating that autocrine CCL3 and CCL4 are required for LCL survival and growth. Importantly, significant amounts of CCL3 were detected in EBV-positive plasma from immunocompromised patients, suggesting that EBV modulates this chemokine in vivo. This study reveals the regulatory mechanism and a novel function of CCL3 and CCL4 in EBV-infected B cells. CCL3 might be useful as a therapeutic target in EBV-associated lymphoproliferative diseases and malignancies.

Lack of resistance-associated mutations in UL54 and UL97 genes of circulating cytomegalovirus strains isolated in a medical center in Taiwan.

Human cytomegalovirus (HCMV) is a large DNA virus and a member of the betaherpesvirus family. HCMV infection is extremely common in human populations and can cause severe diseases in immunocompromised hosts. Ganciclovir is the most widely used antiviral drug for cytomegalovirus infection and works by blocking the amplification of HCMV. HCMV strains resistant to ganciclovir have been detected in recent decades and mainly result from mutations in UL97 (protein kinase) and UL54 (DNA polymerase) genes. In order to understand the prevalence of resistance of HCMV in Taiwan, we studied 40 clinical isolates to detect the mutations of UL97 and UL54 that might be related to resistance. The results showed that no mutation known to cause ganciclovir resistance was detected in any strain, but some polymorphisms (N685S, A688V, A885T, N898D in UL54; D605E in UL97) were frequently observed. Our results suggest that resistant HCMV strains are not prevalent in Taiwan.

Argonaute-2 enhances suppression of human cytomegalovirus replication by polycistronic short hairpin RNAs targeting UL46, UL70 and UL122.

BACKGROUND: Human cytomegalovirus (HCMV) is a common human pathogen that causes significant morbidity and mortality. The efficacy of anti-HCMV drugs such as ganciclovir, foscarnet and cidofovir is limited because of drug toxicities and frequent development of resistance. Here, we report an alternative anti-HCMV method using RNA silencing. METHODS: Combinatorial use of second-generation short hairpin RNAs (shRNA-miRs) targeting various transcripts of HCMV and an RNA-silencing endonuclease Argonaute-2 (Ago2) expression vector were applied to inhibit replication of HCMV AD169. Normal human fetal lung MRC-5 fibroblasts were transfected with pSM30-shRNA-miRs harbouring single or multiple shRNA-miR cassettes with or without Ago2 and then infected with HCMV AD169. Production of small interfering RNA (siRNA) was quantified by reverse transcription PCR. Virus secretion was evaluated by plaque reduction assays. RESULTS: The use of shRNA-miRs targeting a single HCMV gene suppressed HCMV AD169 viral titres by 50-70%. Polycistronic shRNA-miRs targeting UL46+UL122 and UL70+UL46+UL122 reached nearly 80% of inhibition. Coexpression of Ago2 with shRNA-miRs targeting UL46+UL122 and UL70+UL46+UL122 achieved a 95% reduction in viral maturation. CONCLUSIONS: Coexpression of Ago2 with shRNA-miRs enhanced the production of mature siRNAs and increased the efficiency of RNA silencing in the suppression of HCMV replication. This strategy may be universally applied to RNA interference-based therapies.