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Abstract
Kaposi's sarcoma associated herpesvirus (KSHV) is subdivided into gamma-herpesvirus and causes Kaposi's sarcoma in human immunodeficiency virus (HIV)-infected patients. A defining feature of herpesviral biology is the presence of two alternative genetic lifestyles - a latent infection and a lytic replicative cycle. Almost all herpesviruses examined so far have been shown to express viral miRNAs in latently and/or productively infected cells. KSHV encodes an array of 15 distinct miRNAs, all of which are expressed at readily detectable levels in latently KSHV infected cells. The expression of an array of these viral miRNAs in KSHV-infected cells suggests that down-regulation of host cell mRNAs by miRNA-mediated RNA interference may represent a critical step in the establishment and/or maintenance of latent infections by KSHV. To investigate KSHV miRNAs that are expressed in KSHV-infected cells, KSHV-infected human umbilical cord vein endothelial cells (HUVECs) and BCBL-1 cells were used and their miRNAs were analyzed by a modified real-time PCR method. Some KSHV miRNAs were detected in KSHV-infected HUVECs and their expression was affected by genetic life cycles. In addition, KSHV miRNAs were also detected in BCBL-1 and their expression was not related to treatment of sodium butyrate. These results indicate that KSHV infection in cells inducing KSHV miRNAs expression would be increased upon entry into latent replication.
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Figure 1.
miRNA detection by SYBRpolyA-based techniques. Step 1: polyadenylation of total RNA giving rise to polyadenylated miRNA. Step 2: reverse transcription of poly(A) miRNA using a poly(T) primer that contains a universal adaptor (RACE). Step 3: qPCR amplification using a miRNA-specific forward primer, reverse primer complementary to the universal adaptor.
Figure 2.
KSHV-BAC36 infection of HUVECs. KSHV-BAC36 infected to each cell by low speed centrifugation with 5 μg/ml of polybrene. After incubation, GFP expression under a fluorescence inverted microscope was evaluated to monitor infection. (A) GFP expression of KSHV-BAC36 infected HUVECs. (B) Cell numbers of HUVECs after KSHV-BAC36 infection were counted. (C) Percentage of GFP expressing cell of remaining cells.
Figure 3.
Expression kinetics of KSHV miRNAs after infection of HUVECs with KSHV. The expression levels of KSHV miRNAs at each time points were measured by quantitative real-time RT-PCR method. The graphs show relative amounts of KSHV miRNAs expression at each time points compared with their maximum expression amounts. KSHV miRNAs were detected in KSHV-infected HUVECs and their expression was affected by genetic life cycle.
Figure 4.
Expression kinetics of KSHV miRNAs after treatment of sodium butylate on BCBL-1. The expression levels of KSHV miRNAs at each time points were tested by quantitative real-time RT-PCR method. The graphs show relative amounts of KSHV miRNAs expression at each time points compared with their maximum expression amounts. KSHV miRNAs were detected in BCBL-1 and their expression has no relationship to the treatment of sodium butylate.
Table 1.
Primers used for reverse transcription and qRT-PCR reactions.
| Primer | Sequence |
|---|---|
| RT1 | 5′-GCGAGCACAGAATTAATACGACTCCTGGGCAATTTTTTTTTTTTVN*-3′ |
| RACE1 | 5′-GCGAGCACAGAATTAATACGAC-3′ |
| RT2 | 5′-TGTCAGGCAACCGTATTCACCTCCTGCGCAATTTTTTTTTTTTVN*-3′ |
| RACE2 | 5′-TGTCAGGCAACCGTATTCACC-3′ |
| GAPDHF | 5′-GAAGGTGAAGGTCGGAGTC-3′ |
| GAPDHR | 5′-GAAGATGGTGATGGGATTTC-3′ |
Table 2.
KSHV miRNA forward primers.
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