Khalid Saeed    - research student -

Abstract: It is well documented that RNAi is an antiviral mechanism in plants and insects, whereas it is still unclear whether RNAi naturally limit viral infections in vertebrates. In our previous work we have shown that human adenovirus inhibits RNAi by blocking the activity of Dicer and the RNA-induced silencing complex (RISC). The virus-associated RNAs, VA RNAI and VA RNAII bind Dicer through their terminal stems and are cleaved by Dicer into functional siRNA, which are incorporated into RISC. By cloning and sequencing small RNAs we have shown that approximately 80% of Ago2-containing RISC immunopurified from late infected cells is associated with VA RNA-derived small RNAs (mivaRNAs). VA RNAII appears to be the preferred substrate for Dicer and accounts for approximately 60% of all small RNAs in RISC. Collectively, our results suggest that the mivaRNAs are efficiently used for RISC assembly in late-infected cells. Potentially they function as miRNAs regulating translation of cellular mRNAs. It has been reported that human adenovirus type 5 VA RNAI has two transcription initiation sites, which produces two clusters of VA RNAI with 3nt difference at their 5’ end. We have observed that this heterogeneity contributes to the strand bias of VA RNAI incorporating into RISC. We are currently characterizing the mechanism(s) controlling 3’ or 5’ strand incorporation. Our previous results have shown that the efficiency of RNAi is enhanced in a cell line stably over expressing the Ago2 protein, a result that suggests that Ago2 is a limiting factor for RNAi. Adenovirus is an important viral vector system that has been designed for short hairpin RNA delivery to target cells. We speculate that by increasing the amount of the Ago2 protein in target cells, the efficacy of RNAi should be improved. At present, we are testing this hypothesis by constructing a recombinant adenovirus, which expresses the Ago2 protein from an inducible promoter. This virus may be beneficial for the therapeutic application of RNAi.

Abstract: Human Adenovirus type 5 encodes two short RNA polymerase III transcripts, the virus-associated (VA) RNAI and VA RNAII, which can adopt stable hairpin structures that resemble micro-RNA precursors. The terminal stems of the VA RNAs are processed into small RNAs (mivaRNAs) that are incorporated into RISC. It has been reported that VA RNAI has two transcription initiation sites, which produce two VA RNAI species; a major species, VA RNAI(G), which accounts for 75% of the VA RNAI pool, and a minor species, VA RNAI(A), which initiates transcription three nucleotides upstream compared to VA RNAI(G). We show that this 5'-heterogeneity results in a dramatic difference in RISC assembly. Thus, both VA RNAI(G) and VA RNAI(A) are processed by Dicer at the same position in the terminal stem generating the same 3'-strand mivaRNA. This mivaRNA is incorporated into RISC with 200-fold higher efficiency compared to the 5'-strand of mivaRNAI. Of the small number of 5'-strands used in RISC assembly only VA RNAI(A) generated active RISC complexes. We also show that the 3'-strand of mivaRNAI, although being the preferred substrate for RISC assembly, generates unstable RISC complexes with a low in vitro cleavage activity, only around 2% compared to RISC assembled on the VA RNAI(A) 5'-strand.

Abstract: PURPOSE: Pulmonary Tuberculosis presents in many ways; some patients develop a slowly progressive, insidious disease whilst others develop a rapidly progressive, fulminating respiratory failure. no information is available as to whether strain types affect the severity of disease. We hypothesized that patients who develop ARDS due to Mycobacterium tuberculosis are infected with a more virulent strain of mycobacterium tuberculosis that is distinct from the strain causing less aggressive pulmonary disease. METHODS: Patients admitted to AKUH with ARDS due to microbiologically-confirmed pulmonary tuberculosis were reviewed. Preserved specimens were prospectively recultured and isolates spoligotyped for strain identification. The control group was patients diagnosed with pulmonary TB in the outpatient setting on respiratory secretion culture. Clinical data demographics, treatment (steroids, ATT) and outcomes (mortality rate, sensitivity patterns, strain typing of isolates) are reported as means and proportions with standard deviations. RESULTS: 12 cases met criteria for enrollment. 12 consecutively sampled, control patients were included for comparison. 91.6% [11 patients] were admitted from the emergency room. 33% [4 patients] had Acute Respiratory Distress Syndrome [ARDS] at admission while in 66% [8 patients] ARDS developed an average of 1.14 days [range 1–7 days] after admission. 41.6% [5 patients] were treated with intravenous corticosteroids for ARDS caused by mycobacterium TB. Inpatient mortality rate was 61% [11 patients]. The mean length of hospitalization was 11.8 days ± 12.2 [range 1–50]. The most common strains identified in the ARDS group were CAS I (58.3%) and UNQUE (25%).BIEJING and CAS subfamily were identified in 8.3% patients each. In the control group, UNQUE (33.3%) was most common, followed by CAS subfamily (25%) and CAS I (25%). BEIJING strain was the least common (16.6%). CONCLUSION: Patients who develop ARDS due to pulmonary TB, have a different preponderance of mycobacterial strains compared to those who do not develop ARDS. CLINICAL IMPLICATIONS: These results have implications for vaccine development and early steroid use in TB-induced ARDS. DISCLOSURE: Nawal Salahuddin, No Financial Disclosure Information; No Product/Research Disclosure Information