MicroRNAs and Unusual Small RNAs Discovered in Kaposi's Sarcoma-Associated Herpesvirus Virions

Abstract

It is widely held that any given virus uses only one type of nucleic acid for genetic information storage. However, this consensus has been challenged slightly by several recent studies showing that many RNA species are present within a range of DNA viruses that include Kaposi's sarcoma-associated herpesvirus (KSHV). RNAs extracted from purified DNA virus particles exhibit great diversity in terms of length, abundance, temporal expression, cellular localization, and coding capacity during viral infection. In addition to known RNA species, the current study showed that small regulatory RNAs were present in KSHV virions. A large number of viral and cellular microRNAs (miRNAs), as well as unusual small RNAs (usRNAs), were detected in KSHV virions by using deep sequencing. Both viral and host miRNAs detected in small RNAs extracted from KSHV virions were further shown to colocalize with KSHV virions directly by in situ hybridization (ISH)-electron microscopy (EM) (ISH-EM). Some of these miRNAs were differentially present in the host cells and KSHV virions, suggesting that they are not randomly present in KSHV virions. The virional miRNAs could be transported into host cells, and they are biologically functional during de novo viral infection. Our study revealed miRNAs and usRNAs as a novel group of components in KSHV virions.

Transcriptome Analysis of Kaposi's Sarcoma-Associated Herpesvirus during De Novo Primary Infection of Human B and Endothelial Cells

“…The three white bands observed on the sucrose gradient (Fig. 1A) were consistent with previous reports (26,36), representing the A-type (empty virus particles), B-type (intermediate virus particles), and C-type (mature virions) KSHV particles. The lower band representing the mature C-type KSHV virion particles was collected with a syringe and diluted before pelleting the virions by ultracentrifugation.…”

“…After quantifying, 10 pM concentrations of the mature library samples were used for the RNA-seq analysis. In comparison with previous reports (26,27), the transcriptome analysis with the CLC Genomic Workbench 7 software using KSHV as the reference genome detected a larger number of KSHV mRNAs in the virions. A previous DNA microarray study identified 11 KSHV mRNAs in the virions (27), but in this study, we identified more than 60 KSHV-specific transcripts, including mRNAs for latent genes ( Table 1).…”

“…KSHV virions were purified as previously described (26,27). Approximately 100 million TRExBCBL1-RTA cells were induced with doxycycline (1 g/ml) for 5 days, after which the culture supernatant was collected, centrifuged at 4,000 rpm for 10 min, and filtered through a 0.45-m filter to remove cellular debris before concentrating the virus by centrifugation at 25,000 rpm for 2 h at 4°C.…”

“…Indeed, several recent studies have shown that herpesviruses, including KSHV particles, contain a variety of viral proteins (24,25) and diverse RNA species such as viral mRNA, noncoding RNA, viral and cellular microRNAs (miRNAs), and unusual small RNA (usRNA) (26,27). These RNAs are selectively packaged during virion budding and released into the target cells during de novo infection, and they have been shown to be biologically functional (27).…”

“…The KSHV ORF59 transcript is also present in the virions and translated very early during primary infection (27). Recent studies have similarly shown that several of the viral and cellular miRNAs are selectively packaged into the virions and released into the target cells during infection (26). However, the exact composition of the virion transcripts in the KSHV particles and their functions remain unclear (27).…”