Full-length Infectious Molecular Clone Research Articles

Neutralization Sensitivity of a Novel HIV-1 CRF01_AE Panel of Infectious Molecular Clones.

HIV-1 CRF01_AE is dominant in Thailand where RV144 vaccine trial was conducted. To study immune correlates of protection in ongoing trials, CRF01_AE-derived reagents are essential. Here, we present a panel of 14 HIV-1 infectious molecular clones (IMCs) identified from different stages of infection and characterization of their neutralization sensitivity using 2 standard assays. One full-length IMC was constructed using a transmitted-founder virus to express Renilla luciferase (LucR) reporter gene and full-length envelopes (envs) of exogenous HIV-1. A panel of IMCs was generated, expressing envs of viruses from acute (Fiebig stages I/II and I-IV) and chronic (>Fiebig VI) infection. Neutralization assays were performed using TZM-bl or A3R5 cell lines, and sera or monoclonal antibodies (mAbs). Wilcoxon matched-paired test was used to assess neutralization differences between assays and reagents; correlation coefficients were evaluated by linear regression. Neutralization potency observed was significantly higher in the A3R5 assay when testing mAbs and serum pools (P < 0.0001); the stage of infection from which env was derived did not associate with IMC neutralization sensitivity. Neutralization values from A3R5 and TZM-bl assays were strongly correlated when mAbs were tested (R = 0.7, P < 0.0001), but a weaker association was seen with serum pools (R = 0.17, P = 0.03). This novel panel of CRF01_AE reporter IMC is useful for assessing vaccine-induced neutralizing antibodies in multiple assays, including those using primary cell targets. The significant differences in TZM-bl and A3R5 neutralization sensitivity, as well as the poor association when using polyclonal sera indicates the need for caution in choosing one specific platform.

Construction and characterization of a full-length infectious clone from a fast-replicating, X4-tropic HIV-1 subtype B′ isolate

In HIV-1 epidemics in China, HIV-1 subtype B' is the most predominant subtype circulating in intravenous drug users. In this study, we constructed an HIV-1 full-length infectious molecular clone based on the primary virus LWJ, which was isolated from an HIV-infected patient in Fujian Province, China. Phylogenetic and bootscanning analysis of the viral sequence revealed that the isolate LWJ belonged to HIV-1 subtype B'. The infectious clone was designated as "pLWJ". The virus (LWJ-c) produced from this infectious clone by in vitro transfection of 293T cells could infect both human peripheral blood mononuclear cells (PBMCs) and human the T cell line MT4. Interestingly, the cloned LWJ-c virus utilized CXCR4 as its co-receptor and could replicate in vitro with similar efficiency and kinetics compared to its parental primary isolate LWJ as well as the clade B reference virus NL4-3. The LWJ-c virus could also cause cytopathic effects in both PBMCs and MT cells. Sequence analysis of the envelope glycoprotein of pLWJ showed that a conserved GPGR motif and an arginine at position 11 were present in the V3 loop, which was consistent with previous reports regarding CXCR4 co-receptor usage and syncytium-inducing (SI) phenotype. Thus, the infectious clone represents a fast-replicating, high-producing, CXCR4-tropic and syncytium-inducing isolate. Given the prevalence of HIV-1 subtype B' in China, this infectious clone can be a very useful tool to provide a versatile molecular model for research focusing on the biological properties of this subtype.

P19-41. Full-length IMC expressing Renilla luciferase for neutralization assay using PBMC

Background Neutralizing antibody is a crucial parameter in the evaluation of vaccine candidates and therefore, generating a relevant neutralization assay has been a major focus of research. Four critical variables are the viruses, the cells they were grown in, the target cells, and the mode of detection used in the assay. In order to standardize the PBMC-based assay, we are generating primary isolatederived full-length infectious molecular clones (IMC) from HIV+ individuals representing subtypes A, B, C, D, CRF01_AE, and CRF02_AG. Each of these primary isolate IMCs is then modified to express the Renilla luciferase gene.

Construction and Characterization of a Full-Length Infectious Molecular Clone from the HIV Type 1 Subtype Thai-B Isolated in Henan Province, China

Among the various subtypes of the M group of human immunodeficiency virus type 1 (HIV-1), subtype Thai-B is the most prevalent in China, particularly in the country's central region. Here we report on the construction of an infectious molecular clone (CNHN24) of this HIV-1 subtype. We show that the viral stock obtained after transfection of CHNH24 could replicate efficiently in PBMC and MT4 cells. Unlike other previously reported HIV infectious clones, CNHN24 was constructed with the low copy plasmid pLG338, allowing for the HIV genome to be very stable during the process of molecular manipulation. Given the prevalence of subtype Thai-B in China's HIV epidemic, the availability of pCNHN24 as the first infectious molecular clone of this subtype provides a useful tool for a wide range of studies including antiviral drug and vaccine research as related to this subtype of viruses.

Derivation of infectious HIV-1 molecular clones with LTR mutations: Sensitivity to the CD8 + cell noncytotoxic anti-HIV response

CD8 + cells from healthy, asymptomatic HIV-1-infected individuals can inhibit HIV-1 replication in naturally or acutely infected CD4 + cells in the absence of cell killing. This CD8 + cell noncytotoxic anti-HIV response (CNAR) is mediated by a soluble CD8 + cell antiviral factor (CAF). CNAR/CAF inhibits HIV-1 replication by blocking viral RNA transcription. HIV transcription is regulated by a variety of cis-acting DNA sequence elements within the proviral long terminal repeat (LTR). We hypothesized that one of the HIV-1 LTR proviral DNA sequence elements that binds host cell transcriptional factors is involved in this antiviral activity. To assess this possibility, we constructed full-length infectious HIV-1 molecular clones with mutations in the LTR elements NFAT, AP-1, IL-2 homology region, and the downstream ISRE. We also tested full-length infectious molecular clones that had deletions of either the NF-κB or Sp1 sites of the LTR or lacked functional Tat and TAR elements. Viruses generated from these molecular clones were used to acutely infect CD4 + cells that subsequently were either co-cultured with CD8 + cells from individuals that exhibited strong CNAR or cultured with CAF-containing fluids. The replication of all of the mutant HIV-1 viruses tested was substantially reduced in the presence of CNAR/CAF. These findings suggest that other regions in the viral LTR or other host cell processes are involved in the transcriptional block elicited by CNAR/CAF.

Construction and characterization of an infectious molecular clone of HIV-1 subtype A of Indian origin

India has the second highest number of HIV-1 infected people next to South Africa. The predominant proportion of HIV-1 circulating in India is of subtype C origin, with a small fraction made up of subtypes A and B. In this report, we describe the construction and characterization of the first full-length infectious molecular clone p1579A-1 HIV-1, from an HIV-1 subtype A infected person from India, using long PCR and successive ligation of the amplimers. Phylogenetic analysis of the sequence of the entire proviral DNA and LTR confirmed p1579A-1 to be an HIV-1 subtype A. Analysis of the env gene of p1579A-1 showed a conserved GPGQ motif and the absence of basic amino acids at positions 11 and 25 suggesting CCR5 coreceptor usage. Analysis of env N-linked glycosylation sites revealed fewer sites in the V1 region of envelope compared to other subtype A. Transcription factor binding site analysis of the LTR sequences identified conserved as well as unique transcription factor binding sites (TFBS) in p1579A-1. This infectious clone of HIV-1 can be useful to study the molecular mechanism of dominance of subtype C in India.

Construction and characterization of an HIV-1 group O infectious molecular clone and analysis of vpr- and nef-negative derivatives

In this report, we describe the construction and characterization of the first full-length infectious molecular clone from the Cameroonian HIV-1 group O primary isolate MVP8913. Virus obtained after transfection of the proviral clone pCMO2.3 replicated to levels comparable to its parental isolate in the human T-cell line PM-1, although replication was reduced by fivefold in peripheral blood mononuclear cells (PBMC) and was barely detectable in primary monocyte-derived macrophages (MDM). Phylogenetic analysis of the complete proviral sequence revealed a closer relationship to ANT70 than to MVP5180, the two prototypic group O primary isolates. All reading frames for structural and accessory genes were open except for vpr that contained an in-frame stop codon. In the nef gene, a mutation disrupting the functionally important myristoylation signal was observed. Repairing the defect in nef enhanced replication in PBMC and MDM, although repairing the vpr defect only affected replication in MDM, consistent with the known phenotypes of vpr and nef mutants in HIV-1 group M viruses. Repairing both vpr and nef showed an additive effect, but the resulting virus was still impaired compared to the parental isolate. This defect was overcome when the gag-pol coding region was exchanged for that from another O-type isolate giving rise to the proviral clone pCMO2.5. Virus obtained from pCMO2.5 replicated with similar kinetics as the parental O-type isolate in both PBMC and MDM, making this proviral clone a valuable tool for further studies on functional characteristics of HIV-1 group O viruses.

Induction of disease by a molecularly cloned highly pathogenic simian immunodeficiency virus/human immunodeficiency virus chimera is multigenic.

One of three full-length infectious molecular clones of SHIV(DH12R), designated SHIV(DH12R-CL-7) and obtained from productively infected rhesus monkey peripheral blood mononuclear cells, directed rapid and irreversible loss of CD4+ T cells within 3 weeks of its inoculation into Indian rhesus monkeys. Induction of complete CD4+ T-cell depletion by SHIV(DH12R-CL-7) was found to be dependent on inoculum size. The acquisition of this pathogenic phenotype was accompanied by the introduction of 42 amino acid substitutions into multiple genes of parental nonpathogenic SHIV(DH12). Transfer of the entire SHIV(DH12R-CL-7) env gene into the genetic background of nonpathogenic SHIV(DH12) failed to confer the rapid CD4+ T-lymphocyte-depleting syndrome; similarly, the substitution of gag plus pol sequences from SIV(smE543) for analogous SIV(mac239) genes in SHIV(DH12R-CL-7) attenuated the pathogenic phenotype. Amino acid changes affecting multiple viral genes are necessary, but insufficient by themselves, to confer the prototypically rapid and irreversible CD4+ T-cell-depleting phenotype exhibited by molecularly cloned SHIV(DH12R-CL-7).

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

Based on our previous analysis of HIV-1 isolates from Cameroon, we constructed a full-length infectious molecular clone from a primary isolate belonging to the CRF02.AG group of recombinant viruses which dominate the HIV-epidemic in West and Central Africa. The virus derived by transfection of the proviral clone pBD6-15 replicated with similar efficiency compared to its parental isolate and used CXCR4 as coreceptor as well. Furthermore, HIV-1 BD6-15 exhibited similar replication properties and virus yield as the reference B-type HIV-1 strain NL4-3. Sequence analysis revealed open reading frames for all structural and accessory genes apart from vpr. Phylogenetic and bootscanning analyses confirmed that BD6-15 clusters with CRF02.AG recombinant strains from West and Central Africa with similar cross-over points as described for the CRF02.AG prototype strain lbNG. Thus, pBD6-15 represents the first non-subtype B infectious molecular clone of a fast replicating, high producer, X4-tropic primary HIV-1 isolate, which had only been briefly passaged in primary cells.

Mutations that abrogate transactivational activity of the feline leukemia virus long terminal repeat do not affect virus replication

The U3 region of the LTR of oncogenic Moloney murine leukemia virus (Mo-MuLV) and feline leukemia viruses (FeLV) have been previously reported to activate expression of specific cellular genes in trans, such as MHC class I, collagenase IV, and MCP-1, in an integration-independent manner. It has been suggested that transactivation of these specific cellular genes by leukemia virus U3-LTR may contribute to the multistage process of leukemogenesis. The U3-LTR region, necessary for gene transactivational activity, also contains multiple transcription factor-binding sites that are essential for normal virus replication. To dissect the promoter activity and the gene transactivational activity of the U3-LTR, we conducted mutational analysis of the U3-LTR region of FeLV-A molecular clone 61E. We identified minimal nucleotide substitution mutants on the U3 LTR that did not disturb transcription factor-binding sites but abrogated its ability to transactivate the collagenase gene promoter. To determine if these mutations actually have altered any uncharacterized important transcription factor-binding site, we introduced these U3-LTR mutations into the full-length infectious molecular clone 61E. We demonstrate that the mutant virus was replication competent but could not transactivate cellular gene expression. These results thus suggest that the gene transactivational activity is a distinct property of the LTR and possibly not related to its promoter activity. The cellular gene transactivational activity-deficient mutant FeLV generated in this study may also serve as a valuable reagent for testing the biological significance of LTR-mediated cellular gene activation in the tumorigenesis caused by leukemia viruses.

Characterization of a full-length infectious molecular clone for human foamy virus (HFV)

Characterization of a full-length infectious molecular clone for human foamy virus (HFV)

Distinguishing features of an infectious molecular clone of the highly divergent and noncytopathic human immunodeficiency virus type 2 UC1 strain.

A full-length infectious molecular clone was derived from the noncytopathic human immunodeficiency virus type 2 UC1 strain (HIV-2UC1) that was originally recoverd from an individual from the Ivory Coast. Like the parental isolate, the molecularly cloned virus (HIV-2UC1mc or UC1 mc) demonstrates a reduced ability to induce syncytium formation, to kill cells, and to down-modulate the cell surface CD4 receptor in infected cells. Phylogenetic analysis of the DNA sequence of UC1mc revealed that it is the first full-length infectious molecular clone in the second HIV-2 subgroup previously identified by partial sequence analysis of the HIV-2D205 and HIV-2GH-2 strains. These highly divergent HIV-2 strains appear to be genetically equidistant from other HIV-2 and simian immunodeficiency virus SIVmac/sm strains. UC1mc is unlike any other HIV-2 or SIVmac/sm strain in that it lacks a cysteine residue at the proposed signal peptide cleavage site in Env. However, site-directed mutagenesis experiments indicate that this missing cysteine is not alone important in the noncytopathic phenotype of UC1mc. Like other HIV-2 and SIV strains, the UC1mc Env transmembrane protein (gp43) is mutated to a truncated form (gp34) after passage in certain T-cell lines. The UC1 molecular clone should be helpful in determining the genetic sequences associated with HIV-2 cytopathicity.