The post-transcriptional modification of mammalian transcripts by A-to-I RNA editing has been recognized as an important mechanism for the generation of molecular diversity and also regulates protein function through recoding of genomic information. As the molecular players of editing are characterized and an increasing number of genes become identified that are subject to A-to-I modification, the potential impact of editing on the etiology or progression of human diseases is realized. Here we review the recent knowledge on where disturbances in A-to-I RNA editing have been correlated with human disease phenotypes.
Kaposi’s Sarcoma (KS) originates from endothelial cells and it is one of the most overt angiogenic tumors. In Sub-Saharan Africa, where HIV and the Kaposi Sarcoma-associated Herpes Virus (KSHV) are endemic, KS is the most common cancer overall, but model systems for disease study are insufficient. Here we report the development of a novel mouse model of KS where KSHV is retained stably and tumors are elicited rapidly. Tumor growth was sensitive to specific allosteric inhibitors (rapamycin, CCI-779, RAD001) of the pivotal cell growth regulator mTOR. Inhibition of tumor growth was durable up to 130 days and reversible. mTOR blockade reduced VEGF secretion and formation of tumor vasculature. Together, the results demonstrated that mTOR inhibitors exert a direct anti-KS effect by inhibiting angiogenesis and paracrine effectors, suggesting their application as a new treatment modality for KS and other cancers of endothelial origin.
Summary Kaposi’s sarcoma-associated herpesvirus (KSHV) is linked to human malignancies. The majority of tumor cells harbor latent virus and a small percentage undergo spontaneous lytic replication. Both latency and lytic replication are important for viral pathogenesis and spread but the cellular players involved in the switch between the two viral lifecycle phases are not clearly understood. We conducted a siRNA screen targeting the cellular kinome and identified Tousled-like kinases (TLKs) as cellular kinases that control KSHV reactivation from latency. Upon treatment of latent KSHV-infected cells with siRNAs targeting TLKs, we saw robust viral reactivation. Knockdown of TLKs in latent KSHV-infected cells induced expression of viral lytic proteins and production of infectious virus. TLKs were also found to play a role in regulating reactivation from latency of another related oncogenic gammaherpesvirus, Epstein-Barr virus (EBV). Our results establish the TLKs as cellular repressors of gammaherpesviral reactivation.
KSHV inflammatory cytokine syndrome (KICS) is a newly described condition characterized by systemic illness as a result of systemic, lytic KSHV infection. We used Illumina sequencing to establish the DNA vironome of blood from such a patient. It identified concurrent high-level viremia of human herpesvirus (HHV) 8 and 6a. The HHV8 plasma viral load was 5,300,000 copies/ml, which is the highest reported to date; this despite <5 skin lesions and no HHV8 associated lymphoma. This is the first report of systemic HHV6a/KSHV co-infection in a patient. It is the first whole genome KSHV sequence to be determined directly from patient plasma rather than cultured or biopsied tumor material. This case supports KICS as a new clinical entity associated with KSHV.
The sequence of guinea pig cytomegalovirus (GPCMV) was determined by direct sequencing of salivary gland homogenates obtained following sustained, serial in vivo passage of pathogenic virus in guinea pigs. The 233,501-nucleotide salivary gland (SG) genome was noted to have 11 differences compared to the tissue culture-passaged virus, although no variations were noted in putative protein coding sequences.
Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels. One of its many advantages is a lower detection limit compared to other methods of gene expression profiling while using smaller amounts of input for each assay. Automated qPCR setup has improved this field by allowing for greater reproducibility. Its convenient and rapid setup allows for high-throughput experiments, enabling the profiling of many different genes simultaneously in each experiment. This method along with internal plate controls also reduces experimental variables common to other techniques. We recently developed a qPCR assay for profiling of pre-microRNAs (pre-miRNAs) using a set of 186 primer pairs. MicroRNAs have emerged as a novel class of small, non-coding RNAs with the ability to regulate many mRNA targets at the post-transcriptional level. These small RNAs are first transcribed by RNA polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved into the precursor miRNA (pre-miRNA). Pre-miRNAs are exported to the cytoplasm where Dicer cleaves the hairpin loop to yield mature miRNAs. Increases in miRNA levels can be observed at both the precursor and mature miRNA levels and profiling of both of these forms can be useful. There are several commercially available assays for mature miRNAs; however, their high cost may deter researchers from this profiling technique. Here, we discuss a cost-effective, reliable, SYBR-based qPCR method of profiling pre-miRNAs. Changes in pre-miRNA levels often reflect mature miRNA changes and can be a useful indicator of mature miRNA expression. However, simultaneous profiling of both pre-miRNAs and mature miRNAs may be optimal as they can contribute nonredundant information and provide insight into microRNA processing. Furthermore, the technique described here can be expanded to encompass the profiling of other library sets for specific pathways or pathogens. ProtocolThe qPCR pre-miRNA profiling arrays can be set up as fully automated with a Tecan Freedom Evo robot (A) or by hand with the Matrix electronic multichannel pipette (B). 1)Prepare the master mix, primer plates and samples.A. Setup of pre-miRNA assay using the Freedom Tecan Evo robot.
BackgroundIn HIV-infected populations in developed countries, the most recent published cancer incidence trend analyses are only updated through 2008. We assessed changes in the distribution of cancer types and incidence trends among HIV-infected patients in North Carolina up until 2011.MethodsWe linked the University of North Carolina Center for AIDS Research HIV Clinical Cohort, an observational clinical cohort of 3141 HIV-infected patients, with the North Carolina Cancer registry. Cancer incidence rates were estimated across calendar years from 2000 to 2011. The distribution of cancer types was described. Incidence trends were assessed with linear regression.ResultsAcross 15,022 person-years of follow-up, 202 cancers were identified (incidence rate per 100,000 person-years [IR]: 1345; 95% confidence interval [CI]: 1166, 1544). The majority of cancers were virus-related (61%), including Kaposi sarcoma (N = 32) (IR: 213; 95%CI: 146, 301), non-Hodgkin lymphoma (N = 34) (IR: 226; 95%CI: 157, 316), and anal cancer (N = 16) (IR: 107; 95%CI: 61, 173). Non-Hodgkin lymphoma was observed to decrease from 2000 to 2011 (decline of 15 cases per 100,000 person-years per calendar year, 95%CI: -27, -3). No other changes in incidence or changes in incidence trends were observed for other cancers (all P > 0.20).ConclusionsWe observed a substantial burden of a variety of cancers in this population in the last decade. Kaposi sarcoma and non-Hodgkin lymphoma were consistently two of the greatest contributors to cancer burden across calendar time. Cancer rates appeared stable across calendar years, except for non-Hodgkin lymphoma, which appeared to decrease throughout the study period.
Objective Herpesvirus shedding in the oral cavity was analyzed to determine if presence in the oral compartment correlates with systemic changes in HIV-associated immune deficiency as measured by CD4+ counts, plasma HIV viral load (VL) and presence of AIDS-defining events. Design A5254 is a multicenter, cross-sectional, single-visit study to evaluate oral complications of HIV/AIDS and determine the association between clinical appearance, herpesvirus shedding and immune status as ascertained by CD4 count and HIV viral load. 307 HIV infected individuals were evaluated and throat wash collected. Methods Fisher’s exact test and Kruskal-Wallis test were used to assess the association between presence of herpes viruses and the state of immunodeficiency as stratified by a combination of CD4+ count and HIV VL. Relationship between pathogens and HIV VL in plasma was modeled by logistic regression. Results The presence of cytomegalovirus CMV and Herpes Simplex Virus-1 (HSV-1) in throat wash was associated with decreased CD4 counts. By contrast Kaposi Sarcoma-associated herpes virus (KSHV) and EBV were similarly detectable across all levels of CD4 counts. One unit increase in log10(HIV VL) was associated with 1.31 times higher odds of detecting CMV in throat wash when controlling for oral candidiasis, CD4 count, and sites (95% CI 1.04 –1.65, P=0.02). Conclusions Oral CMV shedding was significantly higher in highly immunocompromised HIV+ participants. Our finding supports the recommendations to start antiretroviral therapy (ART) independent of CD4 count as this may have the added benefit to lower the risk of herpes virus transmission among persons infected with HIV and their partners.
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