Parental LTRs Are Important in a Construct of a Stable and Efficient Replication-Competent Infectious Molecular Clone of HIV-1 CRF08_BC

ABSTRACT Dicistronic, selectable subgenomic replicons derived from the Con1 strain of hepatitis C virus (HCV) are capable of autonomous replication in cultured Huh7 cells (Lohmann et al., Science 285:110-113, 1999). However, adaptive mutations in the NS3, NS5A, and/or NS5B proteins are required for efficient replication of these RNAs and increase by orders of magnitude the numbers of G418-resistant colonies selected following transfection of Huh7 cells. Here, we demonstrate that a subgenomic replicon (NNeo/3-5B) derived from an infectious molecular clone of a second genotype 1b virus, HCV-N (Beard et al., Hepatology 30:316-324, 1999) is also capable of efficient replication in Huh7 cells. G418-resistant cells selected following transfection with NNeo/3-5B RNA contained abundant NS5A antigen and HCV RNA detectable by Northern analysis. Replicon RNA in one of three clonally isolated cell lines contained no mutations in the NS3-NS5B polyprotein, confirming that adaptive mutations are not required for efficient replication in these cells. However, the deletion of a unique 4-amino-acid insertion that is present within the interferon sensitivity-determining region (ISDR) of the NS5A protein in wild-type HCV-N drastically decreased the number of G418-resistant colonies obtained following transfection of Huh7 cells. This effect could be reversed by inclusion of a previously described Con1 cell culture-adaptive mutation (S2005→I), confirming that this natural insertion has a controlling role in determining the replication capacity of wild-type HCV-N RNA in Huh7 cells. Additional selectable, dicistronic RNAs encoding NS2-NS5B, E1-NS5B, or the full-length HCV polyprotein were also capable of replication and gave rise to G418-resistant cell clones following transfection of Huh7 cells. We conclude that RNA derived from this documented infectious molecular clone has a unique capacity for replication in Huh7 cells in the absence of additional cell culture-adaptive mutations.

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Isolation and characterization of an infectious molecular clone of the MN strain of HIV-1

Biochemical and Biophysical Research Communications ◽

10.1016/0006-291x(91)91725-r ◽

1991 ◽

Vol 179 (3) ◽

pp. 1377-1383 ◽

Cited By ~ 3

Author(s):

Kesh Prakash ◽

Richard L. Hodinka ◽

Diana M. Hullihen ◽

Stanley A. Plotkin

Keyword(s):

Infectious Molecular Clone ◽

Molecular Clone ◽

Isolation And Characterization ◽

Hiv 1

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Construction and characterisation of a full-length infectious molecular clone from a fast replicating, X4-tropic HIV-1 CRF02.AG primary isolate

Virology ◽

10.1016/s0042-6822(03)00381-7 ◽

2003 ◽

Vol 313 (2) ◽

pp. 645-652 ◽

Cited By ~ 13

Author(s):

Denis M. Tebit ◽

Léopold Zekeng ◽

Lazare Kaptué ◽

Hans-Georg Kräusslich ◽

Ottmar Herchenröder

Keyword(s):

Full Length ◽

Primary Isolate ◽

Infectious Molecular Clone ◽

Molecular Clone ◽

Hiv 1

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Infectious Molecular Clone of a Recently Transmitted Pediatric Human Immunodeficiency Virus Clade C Isolate from Africa: Evidence of Intraclade Recombination

Journal of Virology ◽

10.1128/jvi.78.24.14066-14069.2004 ◽

2004 ◽

Vol 78 (24) ◽

pp. 14066-14069 ◽

Cited By ~ 26

Author(s):

Ricky D. Grisson ◽

Agnès-Laurence Chenine ◽

Lan-Yu Yeh ◽

Jun He ◽

Charles Wood ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Vaccine Development ◽

Infectious Molecular Clone ◽

Molecular Clone ◽

Development Tool ◽

Immunodeficiency Virus ◽

Clade C ◽

Hiv 1

ABSTRACT Although human immunodeficiency virus type 1 (HIV-1) clade C continues to dominate the pandemic, only two infectious clade C proviral DNA clones have been described (N. Mochizuki, N. Otsuka, K. Matsuo, T. Shiino, A. Kojima, T. Kurata, K. Sakai, N. Yamamoto, S. Isomura, T. N. Dhole, Y. Takebe, M. Matsuda, and M. Tatsumi, AIDS Res. Hum. Retrovir. 15:1321-1324, 1999; T. Ndung'u, B. Renjifo, and M. Essex, J. Virol. 75:4964-4972, 2001). We have generated an infectious molecular clone of a pediatric clade C strain, HIV1084i, which was isolated from a Zambian infant infected either intrapartum or through breastfeeding. HIV1084i is an R5, non-syncytium-inducing isolate that bears all known clade C signatures; gag, pol, and env consistently mapped within clade C. Interestingly, gag resembled Asian isolates, whereas pol and env resembled African isolates, indicating that HIV1084i probably arose from an intraclade recombination. As a recently transmitted clade C strain, HIV1084i will be a useful vaccine development tool.

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