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

Zhang, Qiwei; Zhang, Xiaomin; Wu, Hao; Seto, Donald; Zhang, Haojie; Chen, Zhiwei; Wan, Chengsong; Zheng, Bo‐Jian

doi:10.1371/journal.pone.0031233

Cited by 9 publications

(14 citation statements)

References 55 publications

(50 reference statements)

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“…In comparison, the pXJDC13 and pXJDC17 could display a higher peak p24 antigen over 1500 ng/ml, much higher than the previous reported infectious C/BC clade clones [10], [13], [21].…”

Section: Discussioncontrasting

confidence: 66%

Construction and Characterization of Highly Infectious Full-Length Molecular Clones of a HIV-1 CRF07_BC Isolate from Xinjiang, China

et al. 2013

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Among the various subtypes of the M group of human immunodeficiency virus type 1 (HIV-1), clade CRF07_BC is the most prevalent in China. To date, no strong replicable CRF07_BC infectious clone has been constructed. Here we report on the construction and characterization of highly replicable infectious molecular clones from the isolate XJDC6291 of this HIV-1 subtype. Four full-length clones pXJDC2-7, pXJDC3-7, pXJDC2-6 and pXJDC3-6 were successfully produced, but only pXJDC2-7 presented detectable infectivity and replication capability. To improve the replication capability of pXJDC2-7, a 4.8 kb region spanning from the pol Integrase to nef gene of the clone was replaced by PCR products of the corresponding fragments from the original isolate XJDC6291, which produced two clones pXJDC13 and pXJDC17 that exhibited strong replication capability. The viral stocks obtained by pXJDC-13 and pXJDC-17 transfection into 293T cells replicated efficiently in human PBMCs, human primary CD4+ T cells and displayed CCR5 tropism. Sequence alignment between pXJDC13, pXJDC17 and pXJDC2-7 suggested that polymorphisms in the V1V2 region may influence infectivity, and reverse genetic experiment showed that V1V2 polymorphisms may influence the infectivity of the clones but did not affect the replication capability at a significant level. pXJDC13 and pXJDC17 displayed strong replication capability and are the first full-length infectious clones of HIV-1 CRF07_BC clade in the world. The availability of CRF07_BC infectious clones provides a useful tool for a wide range of studies, including antiretroviral drug and vaccine research as related to this HIV subtype.

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“…In comparison, the pXJDC13 and pXJDC17 could display a higher peak p24 antigen over 1500 ng/ml, much higher than the previous reported infectious C/BC clade clones [10], [13], [21].…”

Section: Discussioncontrasting

confidence: 66%

Construction and Characterization of Highly Infectious Full-Length Molecular Clones of a HIV-1 CRF07_BC Isolate from Xinjiang, China

et al. 2013

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“…16 The patient was symptomatic and diagnosed in the same year. 16 The isolated virus was quantified by a blue focus units (BFU) assay as described previously.…”

Section: Virus Strain and Compoundmentioning

confidence: 98%

“…16 The patient was symptomatic and diagnosed in the same year. 16 The isolated virus was quantified by a blue focus units (BFU) assay as described previously. Briefly, virus stock was 3-to 10-fold serially diluted with DMEM complete culture medium containing 25 lg/ml 2-diethylaminoethanol (DEAE) and inoculated to TZM-bl cells seeded in a 96-well plate.…”

Section: Virus Strain and Compoundmentioning

confidence: 98%

In VitroSelection of HIV-1 CRF08_BC Variants Resistant to Reverse Transcriptase Inhibitors

Zhang

et al. 2015

AIDS Research and Human Retroviruses

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Human immunodeficiency virus type 1 (HIV-1) circulating recombinant form 08_BC (CRF08_BC), carrying the recombinant reverse transcriptase (RT) gene from subtypes B and C, has recently become highly prevalent in Southern China. As the number of patients increases, it is important to characterize the drug resistance mutations of CRF08_BC, especially against widely used antiretrovirals. In this study, clinically isolated virus (2007CNGX-HK), confirmed to be CRF08_BC with its sequence deposited in GenBank (KF312642), was propagated in human peripheral blood mononuclear cells (PBMCs) with increasing concentrations of nevirapine (NVP), efavirenz (EFV), or lamivudine (3TC). Three different resistance patterns led by initial mutations of Y181C, E138G, and Y188C were detected after the selection with NVP. Initial mutations, in combination with other previously reported substitutions (K20R, D67N, V90I, K101R/E, V106I/A, V108I, F116L, E138R, A139V, V189I, G190A, D218E, E203K, H221Y, F227L, N348I, and T369I) or novel mutations (V8I, S134N, C162Y, L228I, Y232H, E396G, and D404N), developed during NVP selection. EFV-associated variations contained two initial mutations (L100I and Y188C) and three other mutations (V106L, F116Y, and A139V). Phenotypic analyses showed that E138R, Y181C, and G190A contributed high-level resistance to NVP, while L100I and V106L significantly reduced virus susceptibility to EFV. Y188C was 20-fold less sensitive to both NVP and EFV. As expected, M184I alone, or with V90I or D67N, decreased 3TC susceptibility by over 1,000-fold. Although the mutation profile obtained in culture may be different from the patients, these results may still provide useful information to monitor and optimize the antiretroviral regimens.

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“…Both 293FT and TZM-bl cells were cultured as described previously. 25 HIV-1 CRF08_BC subtype wild-type (WT) infectious plasmid pBRGX was previously constructed. 25 Drugs, including three NNRTIs [NVP, EFV, and etravirine (ETR)] and five NRTIs [AZT, abacavir (ABC), 3TC, FTC, and TDF], were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH.…”

Section: Cells Plasmids and Drugsmentioning

confidence: 99%

“…Viruses were produced by transfection of constructed infectious plasmids into 293FT cells as described previously. 25,26 Viral titer was detected by the measurement of p24 production using the Vironostika Kit (bioMérieux) according to the manufacturer's instructions and by detection of the 50% tissue culture infective dose (TCID 50 ) using TZM-bl cells as described previously. 27 Only those with p24 production higher than 50 mg/liter were subjected to phenotypic drug resistance assay and RC analysis.…”

Section: Site-directed Mutagenesis and Virus Productionmentioning

confidence: 99%

Novel Mutations L228I and Y232H Cause Nonnucleoside Reverse Transcriptase Inhibitor Resistance in Combinational Pattern

Zhang

et al. 2016

AIDS Research and Human Retroviruses

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The emergence of drug resistance mutations is increasing after the implementation of highly active antiretroviral therapy. To characterize two novel mutations L228I and Y232H in the primer grip of reverse transcriptase (RT) of HIV-1 circulating recombination form 08_BC (CRF08_BC) subtype, both mutant clones were constructed to determine their impacts on viral phenotypic susceptibility and replication capacity (RC). Results showed that the novel mutation, L228I, conferred a low-level resistance to etravirine by itself. L228I in combination with Y188C displayed a high level of cross-resistance to both nevirapine (NVP) and efavirenz (EFV). The copresence of A139V and Y232H induced a moderate level of resistance to NVP and EFV. Mutations Y188C/L228I, A139V, Y232H, and A139V/Y232H reduced more than 55% of viral RC compared with that of the wild-type (WT) reference virus. Modeling study suggested that the copresence of Y188C/L228I or A139V/Y232H might induce conformational changes to RT, which might result in reduced drug susceptibility and viral RC due to abolished hydrogen bonding or complex interaction with vicinal residues. Our results demonstrated that L228I and Y232H were novel accessory nonnucleoside reverse transcriptase inhibitor resistance-related mutations and provided valuable information for clinicians to design more effective treatment to patients infected with HIV-1 subtype CRF08_BC.

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

Cited by 9 publications

References 55 publications

Construction and Characterization of Highly Infectious Full-Length Molecular Clones of a HIV-1 CRF07_BC Isolate from Xinjiang, China

Construction and Characterization of Highly Infectious Full-Length Molecular Clones of a HIV-1 CRF07_BC Isolate from Xinjiang, China

In VitroSelection of HIV-1 CRF08_BC Variants Resistant to Reverse Transcriptase Inhibitors

Novel Mutations L228I and Y232H Cause Nonnucleoside Reverse Transcriptase Inhibitor Resistance in Combinational Pattern

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