CLIP‐seq to Identify KSHV ORF57‐Binding RNA in Host B Cells

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV), a human gamma-herpesvirus, is etiologically linked to the development of several malignancies, mainly Kaposi's sarcoma. Expressed as an early viral protein, KSHV ORF57 is essential for lytic replication and virion production. ORF57 selectively binds to a subset of viral RNA and affects nearly all aspects of viral RNA processing. To globally identify all viral and host RNA associated with KSHV ORF57 in the infected cells, we have utilized UV cross-linking and immunoprecipitation (CLIP) of KSHV ORF57 combined with high-throughput RNA sequencing (CLIP-seq) to identify ORF57-binding RNA in BCBL-1 cells at genome-wide level. This unit provides step-by-step details on this new method that is applicable for any pathogen or host RNA-binding proteins by slight modification. © 2016 by John Wiley & Sons, Inc.

Kaposi's sarcoma-associated herpesvirus polyadenylated nuclear RNA: a structural scaffold for nuclear, cytoplasmic and viral proteins

“…A CLIP assay utilizing anti-ORF57 antibodies indicated that ORF57–PAN contact sequences clustered within the 5΄ half (nt G42–G48, U60–G80), 3΄ half (nt U888–U966) and in the middle of PAN (nt G353–C402) ( 48 ). These results were later confirmed by two other groups ( 49 , 50 ), and it was proposed that ORF57 binds to the nascent PAN RNA transcript, multimerizes and forms multiple RNP contacts. In support of these observations, our ΔSHAPE analysis identified multiple ORF57–PAN RNA interactions, including sites of weaker binding within the 5΄ MRE core (nt 45–47) and strong binding within nt 120–122 (H5) and nt 338–341 in Domain I, nt 481–483, 589–591 (H18), 602–604 (H17) in Domain II and nt 775–780 and 949–951 ( 21 ) in Domain III (Figure 4 ).…”

Cedratvirus getuliensis replication cycle: an in-depth morphological analysis

“…After isolation, the virus genome was sequenced, and subsequent bioinformatics analyses were developed; we observed high homology and synteny among the genomes of Cedratvirus getuliensis and other Cedratviruses (in preparation). For co-culture and isolation procedures, Acanthamoeba castellanii cells (ATCC 30010) were cultivated in Peptone-yeast extract with glucose (PYG) 13 medium supplemented with 25 mg/ml amphotericin B (Fungizone; Cristalia, São Paulo, Brazil), 500 U/ml penicillin (Schering-Plough, Brazil) and 50 mg/ml gentamicin (Schering-Plough, Brazil). A total of 7 × 10E6 cells was infected with C. getuliensis at a multiplicity of infection (MOI) of 0.01 and incubated at 32 °C.…”

“…After 72–96 h of incubation, the amoebas were analyzed to determine whether infection occurred. Based on these data, the virus titers were determined using the endpoint method 13 , 14 .…”

“…Thirty minutes post-infection, cells and supernatant were collected and centrifuged at 800 g per 10 minutes. The resultant pellet was washed three times with Page’s amoeba saline (PAS) 13 . After, cells were submitted to three rounds of freezing and thawing, to allow the viral particles release, and then subjected to titration using the endpoint method 13 , 14 .…”

“…The resultant pellet was washed three times with Page’s amoeba saline (PAS) 13 . After, cells were submitted to three rounds of freezing and thawing, to allow the viral particles release, and then subjected to titration using the endpoint method 13 , 14 . In parallel, the supernatant of cytochalasin assay was also submitted to titration for comparison.…”