Publications (60) View all
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Article: Visualising single molecules of HIV-1 and miRNA nucleic acids.
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ABSTRACT: BACKGROUND: The scarcity of certain nucleic acid species and the small size of target sequences such as miRNA, impose a significant barrier to subcellular visualization and present a major challenge to cell biologists. Here, we offer a generic and highly sensitive visualization approach (oligo fluorescent in situ hybridization, O-FISH) that can be used to detect such nucleic acids using a single-oligonucleotide probe of 19--26 nucleotides in length. RESULTS: We used O-FISH to visualize miR146a in human and avian cells. Furthermore, we reveal the sensitivity of O-FISH detection by using a HIV-1 model system to show that as little as 1--2 copies of nucleic acids can be detected in a single cell. We were able to discern newly synthesized viral cDNA and, moreover, observed that certain HIV RNA sequences are only transiently available for O-FISH detection. CONCLUSIONS: Taken together, these results suggest that the O-FISH method can potentially be used for in situ probing of, as few as, 1--2 copies of nucleic acid and, additionally, to visualize small RNA such as miRNA. We further propose that the O-FISH method could be extended to understand viral function by probing newly transcribed viral intermediates; and discern the localisation of nucleic acids of interest. Additionally, interrogating the conformation and structure of a particular nucleic acid in situ might also be possible, based on the accessibility of a target sequence.BMC Cell Biology 04/2013; 14(1):21. · 2.59 Impact Factor -
Article: Allosteric Modulation of the HIV-1 gp120-gp41 Association Site by Adjacent gp120 Variable Region 1 (V1) N-Glycans Linked to Neutralization Sensitivity.
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ABSTRACT: The HIV-1 gp120-gp41 complex, which mediates viral fusion and cellular entry, undergoes rapid evolution within its external glycan shield to enable escape from neutralizing antibody (NAb). Understanding how conserved protein determinants retain functionality in the context of such evolution is important for their evaluation and exploitation as potential drug and/or vaccine targets. In this study, we examined how the conserved gp120-gp41 association site, formed by the N- and C-terminal segments of gp120 and the disulfide-bonded region (DSR) of gp41, adapts to glycan changes that are linked to neutralization sensitivity. To this end, a DSR mutant virus (K601D) with defective gp120-association was sequentially passaged in peripheral blood mononuclear cells to select suppressor mutations. We reasoned that the locations of suppressors point to structural elements that are functionally linked to the gp120-gp41 association site. In culture 1, gp120 association and viral replication was restored by loss of the conserved glycan at Asn(136) in V1 (T138N mutation) in conjunction with the L494I substitution in C5 within the association site. In culture 2, replication was restored with deletion of the N(139)INN sequence, which ablates the overlapping Asn(141)-Asn(142)-Ser-Ser potential N-linked glycosylation sequons in V1, in conjunction with D601N in the DSR. The 136 and 142 glycan mutations appeared to exert their suppressive effects by altering the dependence of gp120-gp41 interactions on the DSR residues, Leu(593), Trp(596) and Lys(601). The 136 and/or 142 glycan mutations increased the sensitivity of HIV-1 pseudovirions to the glycan-dependent NAbs 2G12 and PG16, and also pooled IgG obtained from HIV-1-infected individuals. Thus adjacent V1 glycans allosterically modulate the distal gp120-gp41 association site. We propose that this represents a mechanism for functional adaptation of the gp120-gp41 association site to an evolving glycan shield in a setting of NAb selection.PLoS Pathogens 04/2013; 9(4):e1003218. · 9.13 Impact Factor -
Article: Improved quantification of HIV-1-infected CD4+ T cells using an optimised method of intracellular HIV-1 gag p24 antigen detection.
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ABSTRACT: The capacity of CD8+ T cells to inhibit HIV-1 replication in vitro strongly correlates with virus control in vivo. Post-hoc evaluations of HIV-1 vaccine candidates suggest that this immunological parameter is a promising benchmark of vaccine efficacy. Large-scale analysis of CD8+ T cell antiviral activity requires a rapid, robust and economical assay for accurate quantification of HIV-1 infection in primary CD4+ T cells. Detection of intracellular HIV-1 p24 antigen (p24 Ag) by flow cytometry is one such method but it is thought to be less sensitive and quantitative than p24 Ag ELISA. We report that fixation and permeabilisation of HIV-infected cells using paraformaldehyde / 50% methanol / Nonidet P-40 instead of a conventional paraformaldehyde / saponin-based protocol improved their detection across multiplicities of infection (MOI) ranging from 10(-2) - 8x10(-5), and by nearly two-fold (p <0.001) at the optimal MOI tested (10(-2)).. The frequency of infected cells was strongly correlated with p24 Ag release during culture, thus validating its use as a measure of productive infection. We were also able to quantify infection with a panel of HIV-1 isolates representing the major clades. The protocol described here is rapid and cost-effective compared with ELISA and thus could be a useful component of immune monitoring of HIV-1 vaccines and interventions to reduce viral reservoirs.Journal of immunological methods 03/2013; · 2.35 Impact Factor -
Article: The origin of genetic diversity in HIV-1.
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ABSTRACT: One of the hallmarks of HIV infection is the rapid development of a genetically complex population (quasispecies) from an initially limited number of infectious particles. Genetic diversity remains one of the major obstacles to eradication of HIV. The viral quasispecies can respond rapidly to selective pressures, such as that imposed by the immune system and antiretroviral therapy, and frustrates vaccine design efforts. Two unique features of retroviral replication are responsible for the unprecedented variation generated during infection. First, mutations are frequently introduced into the viral genome by the error prone viral reverse transcriptase and through the actions of host cellular factors, such as the APOBEC family of nucleic acid editing enzymes. Second, the HIV reverse transcriptase can utilize both copies of the co-packaged viral genome in a process termed retroviral recombination. When the co-packaged viral genomes are genetically different, retroviral recombination can lead to the shuffling of mutations between viral genomes in the quasispecies. This review outlines the stages of the retroviral life cycle where genetic variation is introduced, focusing on the principal mechanisms of mutation and recombination. Understanding the mechanistic origin of genetic diversity is essential to combating HIV.Virus Research 06/2012; · 2.94 Impact Factor -
Article: HIV taken by STORM: super-resolution fluorescence microscopy of a viral infection.
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ABSTRACT: The visualization of viral proteins has been hindered by the resolution limit of conventional fluorescent microscopes, as the dimension of any single fluorescent signal is often greater than most virion particles. Super-resolution microscopy has the potential to unveil the distribution of proteins at the resolution approaching electron microscopy without relying on morphological features of existing characteristics of the biological specimen that are needed in EM. Using direct stochastic optical reconstruction microscopy (dSTORM) to achieve a lateral resolution of 15-20 nm, we quantified the 2-D molecular distribution of the major structural proteins of the infectious human immunodeficiency virus type 1 (HIV-1) before and after infection of lymphoid cells. We determined that the HIV-1 matrix and capsid proteins undergo restructuring soon after HIV-1 infection. This study provides the proof-of-concept for the use of dSTORM to visualize the changes in the molecular distribution of viral proteins during an infection.Virology Journal 05/2012; 9:84. · 2.34 Impact Factor
