Construction and identification of the recombinant expression lentivirus vector carrying KSHV Rta gene

PDF(2616 KB)

Journal of Jiangsu University(Medicine Edition) ›› 2014, Vol. 24 ›› Issue (02) : 93-98,104.

Article

Construction and identification of the recombinant expression lentivirus vector carrying KSHV Rta gene

CHEN Fei, YAN Qin, LU Chun

Author information +

Abstract

Objective: To construct the recombinant lentivirus carrying Kaposi′s sarcoma-associated herpesvirus (KSHV) replication and transcriptional activator（Rta） gene. Methods: The fragment of Rta gene from expression vector pcDNA3.1-Rta was cloned into the lentivirus vector pHAGE-CMV-MCS-IzsGreen, then the recombinant vector pHAGE-Rta, packaging vector psPAX2 and envelope vector pMD2.G were cotransfected into the 293 T cells. Filtered culture media were harvested and the viral titer was checked by observing the expression of green fluorescent protein (GFP). BCBL-1 cells were infected with the LentivirusRta of which multiplicity of infection (MOI) was 1.0, 5.0 and 10.0, respectively, and Rta protein was detected by western blotting; meanwhile, the expression of KSHV lytic protein viral interleukin-6 (vIL-6) and the production of KSHV virion were examined by western blotting and virion release assay, respectively. Results: Recombinant lentivirus with high titer and efficient infection were obtained by the lentivirus vectors system. BCBL-1 cells could be efficiently infected, and the expression of Rta protein in BCBL-1 cells infected with Lentivirus-Rta was detectable. The expression of Rta could promote KSHV lytic replication,upregulate the expression of vIL-6 protein and the production of KSHV virion. Conclusion: The recombinant lentivirus vector carrying Rta gene was constructed successfully; and the expression of Rta in BCBL-1 cells infected with Lentivirus-Rta could reactivate KSHV from latency successfully.

Cite this article

EndNote

Ris (Procite)

Bibtex

Download Citations

CHEN Fei, YAN Qin, LU Chun. Construction and identification of the recombinant expression lentivirus vector carrying KSHV Rta gene[J]. Journal of Jiangsu University(Medicine Edition), 2014, 24(02): 93-98,104

References

[1\]Katano H, Sata T. Human herpesvirus 8 virology, epidemiology and related diseases

J

. Jpn J Infect Dis, 2000, 53(4):137-155.

2

Chang Y, Cesarman E, Pessin MS,et al. Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi′s sarcoma

J

. Science, 1994, 266(5192):1865-1869.

3

Davis M, Sturzl M, Blasig C. Expression of human herpesvirus 8-encoded cyclin D in Kaposi′s sarcoma spindle cells

J

. J Natl Cancer Inst, 1997, 89(24):1868-1874.
[4]Sadler R, Wu L, Forghani B. A complex translational program generates multiple novel proteins from the latently expressed kaposin (K12) locus of Kaposi′s sarcomaassociated herpesvirus

J

. J Virol, 1999, 73(7): 5722-5730.
[5]Lu C, Gordon GM, Chandran B, et al. Human herpesvirus 8 reactivation and human immunodeficiency virus type 1 gp120

J

. Arch Pathol Lab Med, 2002,126(8):941-946.

6

Naranatt PP, Krishnan HH, Svojanovsky SR, et al. Host gene induction and transcriptional reprogramming in Kaposi′s sarcomaassociated herpesvirus (KSHV/HHV-8)infected endothelial, fibroblast, and B cells: insights into modulation events early during infection

J

. Cancer Res， 2004, 64(1): 72-84.
[7]Zhu X, Guo Y, Yao S, et al. Synergy between Kaposi′s sarcomaassociated herpesvirus (KSHV) vIL6 and HIV1 Nef protein in promotion of angiogenesis and oncogenesis: role of the AKT signaling pathway

J

. Oncogene, 2013.

E p u b a h e a d o f p r i n t

[8]王平，黄敏，卢春. 含HIV-1Vif基因重组慢病毒表达载体的构建及其对KSHV裂解性周期复制影响的初探

J

. 南京医科大学学报：自然科学版，2010，30(3)：286-290，355.

9] B o w s e r B S, D e W i r e S M, D a m a n i a B . T r a n s c r i p t i o n a l r e g u l a t i o n o f t h e K 1 g e n e p r o d u c t o f K a p o s i' s s a r c o m a - a s s o c i a t e d h e r p e s v i r u s \[J

. J Virol, 2002, 76(24):12574-12583.

10] B o w s e r B S, M o r r i s S, S o n g M J, e t a l . C h a r a c t e r i z a t i o n o f K a p o s i' s s a r c o m a  a s s o c i a t e d h e r p e s v i r u s (K S H V) K 1 p r o m o t e r a c t i v a t i o n b y R t a \[J

. Virology, 2006, 348(2):309-327.
[11]Deng H, Chu JT, Rettig MB, et al. Rta of the human herpesvirus 8/Kaposi sarcomaassociated herpesvirus upregulates human interleukin6 gene expression

J

. Blood, 2002, 100(5): 1919-1921.
[12]Gonzlez CM, Wong EL, Bowser BS, et al. Identification and characterization of the Orf49 protein of Kaposi′s sarcomaassociated herpesvirus

J

. J Virol, 2006, 80(6):3062-3070.
[13]Lukac DM, Kirshner JR, Ganem D. Transcriptional activation by the product of open reading frame 50 of Kaposi′s sarcomaassociated herpesvirus is required for lytic viral reactivation in B cells[J]. J Virol, 1999, 73(11): 9348-9361.
[14]Sakakibara S, Ueda K, Chen J, et al. Octamerbinding sequence is a key element for the autoregulation of Kaposi′s sarcomaassociated herpesvirus ORF50/Lyta gene expression [J]. J Virol, 2001, 75(15): 6894-6900.
[15]Song MJ, Li X, Brown HJ, et al. Characterization of interactions between RTA and the promoter of polyadenylated nuclear RNA in Kaposi′s sarcoma-associated herpesvirus/human herpesvirus 8 [J]. J Virol, 2002, 76(10): 5000-5013.
[16]Ueda K, Ishikawa K, Nishimura K, et al. Kaposi′s sarcomaassociated herpesvirus (human herpesvirus 8) replication and transcription factor activates the K9 (vIRF) gene through two distinct cis elements by a nonDNAbinding mechanism [J]. J Virol, 2002, 76(23): 12044-12054.
[17]Wong EL, Damania B. Transcriptional regulation of the Kaposi′s sarcomaassociated herpesvirus K15 gene [J]. J Virol, 2006, 80(3): 1385-1392.
[18]Lan K, Kuppers DA, Verma SC, et al. Kaposi′s sarcoma-associated herpesvirusencoded latencyassociated nuclear antigen inhibits lytic replication by targeting Rta: a potential mechanism for virusmediated control of latency [J]. J Virol, 2004, 78(12):6585-6594.
[19]Chen J, Ueda K, Sakakibara S, et al. Activation of latent Kaposi′s sarcomaassociated herpesvirus by demethylation of the promoter of the lytic transactivator [J]. Proc Natl Acad Sci U S A, 2001, 98(7):4119-4124.
[20]Lu F, Zhou J, Wiedmer A, et al. Chromatin remodeling of the Kaposi′s sarcomaassociated herpesvirus ORF50 promoter correlates with reactivation from latency [J]. J Virol, 2003, 77 (21):11425-11435.
[21]West JT, Wood C. The role of Kaposi′s sarcomaassociated herpesvirus/human herpesvirus-8 regulator of transcription activation (RTA) in control of gene expression [J]. Oncogene, 2003, 22(33):5150-5163.
[22]Myoung J, Ganem D. Generation of a doxycyclineinducible KSHV producer cell line of endothelial origin: maintenance of tight latency with efficient reactivation upon induction [J]. J Virol Methods, 2011, 174(1/2):12-21.
[23\]Gradoville L, Gerlach J, Grogan E, et al. Kaposi′s sarcomaassociated herpesvirus open reading frame 50/Rta protein activates the entire viral lytic cycle in the HHB2 primary effusion lymphoma cell line

J

. J Virol, 2000, 74(13): 6207-6212.

24

Deng H, Young A, Sun R. Autoactivation of the rta gene of human herpesvirus8/Kaposi′s sarcomaassociated herpesvirus

J

. J Gen Virol, 2000, 81(Pt 12):3043-3048.

25

Nakamura H, Lu M, Gwack Y, et al. Global changes in Kaposi′s sarcomaassociated virus gene expression patterns following expression of a tetracyclineinducible Rta transactivator

J

. J Virol, 2003, 77(7): 4205-4220.

26

Lukac DM, Renne R, Kirshner JR, et al. Reactivation of Kaposi′s sarcomaassociated herpesvirus infection from latency by expression of the ORF 50 transactivator, a homolog of the EBV R protein

J

. Virology, 1998, 252(2):304-312.

27

Yang Z, Yan Z, Wood C. Kaposi′s sarcomaassociated herpesvirus transactivator RTA promotes degradation of the repressors to regulate viral lytic replication