Anti-apoptotic function of herpes simplex virus -2 latency-associated transcript RL1 sequence and screening of its encoded microRNAs.
BACKGROUND
The latency-associated transcript (LAT) gene of herpes simplex virus (HSV)-2 is the only detectable viral gene expressed during latent infection in neurons. LAT inhibits apoptosis and maintains latency by promoting the survival of infected neurons. However, whether LAT functions during HSV-2 infection via its encoded RNAs or via its encoded proteins remain unknown. Increasing evidence has indicated that LAT is likely to functionally promote the establishment of latent infection via LAT-encoded microRNAs (miRNAs).


OBJECTIVE
To explore whether the RL1 fragment of the five adjacent miRNAs has an effect on cell apoptosis, then provide supporting evidence to elucidate the potential role of these miRNAs and to aid screening of their cellular targets.


METHODS
A number of techniques, including MTT assay, flow cytometry and DNA ladder analysis, were used to verify the role of the RL1 fragment and the contribution of the individual miRNAs to the anti-apoptotic effect.


RESULTS
Five miRNAs (miR-H3, miR-H4-3p, miR-H4-5p, miR-H24 and miR-H19) were detected by quantitative PCR in PC12 cells stably expressing RL1 after pEGFP-RL1 plasmid transfection in vitro. The data indicated that expression of HSV-2 LAT RL1 seems to provide protection against apoptosis of PC12 cells induced by ActD. Antisense miRNAs specifically inhibiting these five miRNAs could efficiently reduce their expression. Transfection of antisense-miR-H3, antisense-miR-H4-5p and antisense-miR-H19 into PC12 cells stably expressing RL1 were able to partly reverse the anti-apoptotic effect of these miRNAs.


CONCLUSIONS
These findings indicate that the apoptotic role of the RL1 fragment is likely to be related to overexpression of miR-H3, miR-H4-5p and miR-H19 in PC12 cells.
School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, Guangdong, China.
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Y
Liu
Department of Dermatology, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou, Guangdong, China. huilany88@hotmail.com.
H L
HL
Yang
School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, Guangdong, China.|Department of Dermatology, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou, Guangdong, China.
F F
FF
Zhong
Department of Dermatology, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou, Guangdong, China.
J Y
JY
Fan
D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D018929:Cell Culture Techniques; D003609:Dactinomycin; D015967:Gene Expression Regulation, Viral / Q000502:physiology*; D018258:Herpesvirus 2, Human / Q000235:genetics*; D035683:MicroRNAs; D016716:PC12 Cells; D051381:Rats; D014764:Viral Proteins / Q000502:physiology*; D017735:Virus Latency / Q000235:genetics*
C477254:LAT protein, Human herpesvirus 2; D035683:MicroRNAs; D014764:Viral Proteins; D003609:Dactinomycin