Jpn. J. Infect. Dis., 52, 1999

Laboratory and Epidemiology Communications

lntegrase of Human Endogenous Retrovirus K‑ 10 Supports
the Replication of Replication‑Incompetent tnt‑ HumanImmunodeficiency Virus Type 1 Mutant
Toshihiko Ogata, Nobuo Okui, Ryuta Sakuma, Noriko Kobayashi
and Yoshihiro Kitamura*Division of Molecular Genetics, National Institute of lnfectious Diseases,
Gakuen 41 711 , Musashimurayama‑shi, 7Tokyo 208‑0011
Communicated by Hiroshi Yoshikura(Accepted January 6, 2000)

The integration of proviral DNA is an essential step forretroviral replication and is mediated by a viral integrase (IN).

presence of HERV IN in HIVHQHY Virions released from thetransfected 293T cells, suggesting that HERV IN was actually

In general, IN utilizes, as a donor substrate, long teminal

incorporated into HIV‑1 virions. Similarly, We prepared

repeats (IJR) of homologous origin but not of heterologous
origin (I)･ Howev.er, in vitro studies suggested that humanendogenous retrovlruS (HERV) K‑ 10 IN could utilize not only
the homologous LTR but also human immunodeficiency

VprPCHERVIN
VprPαH E RVINvirus type 1 (HIV‑1) IJR as a substrate (4). This reportdemonstrates that tnt‑negative HIV‑ 1 mutant replicated incells simultaneously expresslng HERV IN･ This suggests thatVpr

PC川VLAllN

the HERV IN could utilize IJR of HIV‑I, which is only
VprPChl rVIN

distantly related to HERV (2).

Vpr‑PC (protease deavage sequence)‑IN fusion protein has
been shown to be incorporated into virions and cleaved into

Vpr PC HIVLAIIN(HHCCHERV)VprPC and IN during virion maturation (5,9). Therefore, We

VprPCn lVIN(HHCCH ERV)

constructed a vector which expressed VprPC fused to IN ofHERV (pC‑VprPC/HERVIN) (Fig. 1). Tb detemine whether
HERV IN protein could be incorporated into HIV‑ 1 virions,Fig. 1 ･ Schematic representation of VprPC fusion proteins･

we transfected 293T cells (3) with pC‑VprPC/tN together withThe left open boxes, Vpr of HIVLA.; dashed boxes, a linker peptide

PHQHY, an HIV‑ I clone encoding non‑functional mutatedcontaimingthe innuenza virus hemagglutinin (HA) and a junctionalIN, which had base substitutions in the 12仇and 16th histidinepeptide ofRT and IN (from refs･ 5, 9); closed boxes, integrase of HERV

residues (6)･ Immunoblot analysis (Fig. 2) revealed theK‑ 10; open boxes, integrase of HIVLAトkDa12U ncleaved

VprPC/HERVIN

45 ･一･･

C 一eavedHERVIN

31 ‑‑Fig. 2. Immunoblot analysis ofviriOn proteins.

Virions were collected by centrifugation, and lysed in RIFA buffer･ Virion proteins were separated in a 15% polyacrylamide
geIand blotted onto a nitrocellulose membrane. The bound proteins were probed withanti‑HERVIN rabbit serum which was
not cross‑reactive to HIVll IN and then withanti‑rabbit HRP‑conjugated goat serum, and visualized using an ECL plus kit(AmershamPharmacia Biotech, AB･, Uppsala, Sweden)･ Analyzed are NIA‑3HQrTY Virions released from 293T cells inthe
presence of VprPC/HIVLAIIN (lane 1) or VprPC/HERVIN (lane 2)･ The size markers (3 I and 45 kDa) are positionedalongside
the left of the gel. HIV protease partially cleaved the incorporated VprPC/HERVIN (50 kDa) to generate VprPC ( I 8 kDa)andHERV IN (32 kDa) (Fig, 1).
*Corresponding author: Fax: +8ト42‑567‑5632, E‑mail:yochan @ nih･ go･Jp251

Jpn. J. Infect. Dis., 52, 1999

Kanda and K. Yoshiike for their encouragement. The work
was supported by a grant from the Ministry of Education,
Science, Sports and Culture of Japan.

Table･ Infectivities of the virions incorporatlng Various INs
Controla HIVLAI INb HERV INb HHCCHERVb
Nunberorblue cells̀ 302士2.94

60±1.632.2±0.81

0±0.00REFERENCESa WLld‑typeHIV‑ 1 (strain LAI) released from the 293T cells transfectedwith the infectious clone DNA (pLAl).

I. Brown, P.0. (1990): Integration of retroviral DNA･ Curr･Vlrions released from the 293T cells transfected withDNA of anTop. Microbiol. Immunol., 157, 19‑48･

integration‑defective HIV‑ 1 mutant (HQHY)alongwith pC‑VprPC/

2. Doolittle, 良. F, Feng, D. F, Johnson, M. S･ and McClure,

HIVIN, pC‑VprPC/HERVIN, or pC‑V prPC/HHCCHERVIN･c MAGIC cells (4x IO4 cells/well in a 24‑well plate) were infected with

M. A. (1989): Origins and evolutionary relationships ofretroviruses. Q. Rev. Bio1., 64, 1‑30.

each sample (equivalent to I ng of p24)･ The cells were stainedwith

3. DuBridge, R. B., Tang, P., Hsia, H･ C･,Leong, P･ M･,

X‑gal for cellular β ‑galactosidase expression two days post‑infecdon.

The blue‑stained cells were counted. Results obtained from five

Miller, J. H. and Calos, M. P. (1987): Analysis of muta‑

independent experiments were presented as ̀average ± standard

tion in human cells by uslng an Epstein‑Ba汀Vims shuttle

deviation. 'system. Mol. Cell. Bio1., 7, 379‑387.
4. Kitamura, Y., Ayukawa, T., Ishikawa, T., Kanda, T･ andYoshiike, K. (1996): Human endogenous retrovirus KIO

HIVHQHY Virions that incorporated the intact IN of HIV‑ lLAI

as a positive controland those which incorporated non‑functional HERV IN, HIVIN(HHCCHERV) (ami.no acidresidues 17‑39 0f HIV IN were replaced with 23 amlnO acid

5. Liu, H., Wu, X., Xiao, H., Conway, ∫.A. and Kappes, ∫.

residues, V NAAGLKNKFDVTWK(〕AKDⅣqH) as a nega‑

deficiency viruStypel integrase into virions independentencodes a functional integrase. ∫. Viro1., 70, 3302‑3306･C. (1997): Incorporation of functional human immuno‑

ofthe Gag‑Pol precursor protein･ J･ ViroI･, 71 , 7704‑77 10･

tive control.

We compared infectivities of the virions incorporating

6. Masuda, T., Planelles, V., Krogstad, P. and Chen, I･ S･various INs in MAGIC cells (7,8). The inocula contained the( 1995): Genetic analysis of human immunodeficiency

same amount of p24･ The virions that incorporatedthe intact

virus typel integrase and the U3 att site: unusual pheno‑

HERV IN actually produced blue colonies, and their estimatedtype of mutants in the zinc finger‑like domain･ J･ Virol･,

infectivity was about 3.7% of the infectivity of the virions

69, 6687‑6696.

7. Nakayama, E. E., Shioda, T., Tatsumi, M･, Xin, X･, Yu,

享ncorporatingthe intact HIV‑1 IN (恥ble)･Asthe in vitro
Integration activity of HER∀ IN was about 10‑30% of HIV‑ 1D., Ohgimoto, S., Kato, A., Sakai, Y･, Ohnishi, Y･ andIN when HIV‑I IJR was used as a donor substrate (4), thevalue of 3.7% may be reasonable. Virions incorporating
HIVINHHCCHERV Showed no detectable infectivity. Theseexperiments suggestthat HERV IN mediatedthe integration
reaction of HIV‑I IJR in vivo.

Nagai, Y. (1998): Importance of the N‑glycan in the V3
100p of HIVl envelope protein for CXCR一斗but not

CCR‑5‑dependent fusion. FEBSLett･, 426, 367‑372･
8. Okui, N., Kobayashi, N. and Kitamura, Y･ (1998):
Production of uninfectious human immunodeficiency

The above expenments may raise the possibility of anvirus type I containing viral protein R fused to a slngle‑
chainantibody against viral integrase. J. Virol･, 72, 69601

interaction between human endogenous retrovirus and HIV‑l during the replication process. In light of this,the develop‑

6964.

ment of integrase negative HIV mutant should be performed

9. Wu, X., Liu, H., Xiao, H., Conway, ∫. A., Hunter, E･ andKappeS, J. C. (1997). Functional RT and IN incorporated
into HIV‑I particles independently of the Gag/Potwith caution.

We thank Dr. M. Tatsumi for MAGIC cells, and Drs. T.

precursor protein. EMBO ∫., 16, 5113‑5122･252