Jenny L. Anderson

An in-depth comparison of latent HIV-1 reactivation in multiple cell model systems and resting CD4+ T cells from aviremic patients

The possibility of HIV-1 eradication has been limited by the existence of latently infected cellular reservoirs. Studies to examine control of HIV latency and potential reactivation have been hindered by the small numbers of latently infected cells found in vivo. Major conceptual leaps have been facilitated by the use of latently infected T cell lines and primary cells. However, notable differences exist among cell model systems. Furthermore, screening efforts in specific cell models have identified drug candidates for “anti-latency” therapy, which often fail to reactivate HIV uniformly across different models. Therefore, the activity of a given drug candidate, demonstrated in a particular cellular model, cannot reliably predict its activity in other cell model systems or in infected patient cells, tested ex vivo. This situation represents a critical knowledge gap that adversely affects our ability to identify promising treatment compounds and hinders the advancement of drug testing into relevant animal models and clinical trials. To begin to understand the biological characteristics that are inherent to each HIV-1 latency model, we compared the response properties of five primary T cell models, four J-Lat cell models and those obtained with a viral outgrowth assay using patient-derived infected cells. A panel of thirteen stimuli that are known to reactivate HIV by defined mechanisms of action was selected and tested in parallel in all models. Our results indicate that no single in vitro cell model alone is able to capture accurately the ex vivo response characteristics of latently infected T cells from patients. Most cell models demonstrated that sensitivity to HIV reactivation was skewed toward or against specific drug classes. Protein kinase C agonists and PHA reactivated latent HIV uniformly across models, although drugs in most other classes did not.

Proteasome Inhibition Reveals that a Functional Preintegration Complex Intermediate Can Be Generated during Restriction by Diverse TRIM5 Proteins

ABSTRACT The primate TRIM5 proteins constitute a class of restriction factors that prevent host cell infection by retroviruses from different species. The TRIM5 proteins act early after virion entry and prevent viral reverse transcription products from accumulating. We recently found that proteasome inhibitors altered the rhesus monkey TRIM5α restriction of human immunodeficiency virus type 1 (HIV-1), allowing reverse transcription products to accumulate even though viral infection remained blocked. To assess whether sensitivity to proteasome inhibitors was a common feature of primate TRIM5 proteins, we conducted a similar analysis of restriction mediated by owl monkey TRIM-cyclophilin A (CypA) or human TRIM5α. Similar to rhesus monkey TRIM5α restriction, proteasome inhibition prevented owl monkey TRIM-CypA restriction of HIV-1 reverse transcription, even though HIV-1 infection and the output of 2-LTR circles remained impaired. Likewise, proteasome inhibition alleviated human TRIM5α restriction of N-tropic murine leukemia virus reverse transcription. Finally, HIV-1 reverse transcription products escaping rhesus TRIM5α restriction by proteasome inhibition were fully competent for integration in vitro, demonstrating that TRIM5α likely prevents the viral cDNA from accessing chromosomal target DNA. Collectively, these data indicate that the diverse TRIM5 proteins inhibit retroviral infection in multiple ways and that inhibition of reverse transcription products is not necessary for TRIM5-mediated restriction of retroviral infection.

Establishment of HIV-1 latency in resting CD4+ T cells depends on chemokine-induced changes in the actin cytoskeleton

Eradication of HIV-1 with highly active antiretroviral therapy (HAART) is not possible due to the persistence of long-lived, latently infected resting memory CD4+ T cells. We now show that HIV-1 latency can be established in resting CD4+ T cells infected with HIV-1 after exposure to ligands for CCR7 (CCL19), CXCR3 (CXCL9 and CXCL10), and CCR6 (CCL20) but not in unactivated CD4+ T cells. The mechanism did not involve cell activation or significant changes in gene expression, but was associated with rapid dephosphorylation of cofilin and changes in filamentous actin. Incubation with chemokine before infection led to efficient HIV-1 nuclear localization and integration and this was inhibited by the actin stabilizer jasplakinolide. We propose a unique pathway for establishment of latency by direct HIV-1 infection of resting CD4+ T cells during normal chemokine-directed recirculation of CD4+ T cells between blood and tissue.

Visualization of a proteasome-independent intermediate during restriction of HIV-1 by rhesus TRIM5α

TRIM5 proteins constitute a class of restriction factors that prevent host cell infection by retroviruses from different species. TRIM5α restricts retroviral infection early after viral entry, before the generation of viral reverse transcription products. However, the underlying restriction mechanism remains unclear. In this study, we show that during rhesus macaque TRIM5α (rhTRIM5α)–mediated restriction of HIV-1 infection, cytoplasmic HIV-1 viral complexes can associate with concentrations of TRIM5α protein termed cytoplasmic bodies. We observe a dynamic interaction between rhTRIM5α and cytoplasmic HIV-1 viral complexes, including the de novo formation of rhTRIM5α cytoplasmic body–like structures around viral complexes. We observe that proteasome inhibition allows HIV-1 to remain stably sequestered into large rhTRIM5α cytoplasmic bodies, preventing the clearance of HIV-1 viral complexes from the cytoplasm and revealing an intermediate in the restriction process. Furthermore, we can measure no loss of capsid protein from viral complexes arrested at this intermediate step in restriction, suggesting that any rhTRIM5α-mediated loss of capsid protein requires proteasome activity.

Intracellular trafficking of retroviral vectors: obstacles and advances

Retroviruses are efficient vehicles for delivering transgenes in vivo. Their ability to integrate into the host genome, providing a permanent imprint of their genes in the host, is a key asset for gene therapy. Furthermore, the lentivirus subset of retroviruses can infect nondividing as well as dividing cells. This expands the cell types capable of gene therapy, driving the development of lentiviral vectors. However, the precise mechanisms used by different retroviruses to efficiently deliver their genes into cell nuclei remains largely unclear. Understanding these molecular mechanisms may reveal features to improve the efficacy of current retroviral vectors. Moreover, this knowledge may expose elements pliable to other gene therapy vehicles to improve their in vivo performance and circumvent the biosafety concerns of using retroviral vectors. Therefore, the mechanisms underlying the early trafficking of retroviral vectors in host cells are reviewed here, as understood from studying the native retroviruses. Events after virus entry up to nuclear delivery of the viral cDNA are discussed. Cellular obstacles faced by these retroviral vectors and how they advance beyond these barriers is emphasized.

APOBEC3G restricts early HIV-1 replication in the cytoplasm of target cells

Cellular APOBEC3G (A3G) protein is packaged into human immunodeficiency virus type 1 (HIV-1) virions in producer cells yet restricts viral replication in target cells. To characterize this restriction in target cells, the effect of A3G on generating various HIV-1 cDNA products was measured by quantitative real-time PCR. A3G decreased cDNA products from Vif-deficient HIV-1, with minor effects on early reverse transcripts and larger declines in late reverse transcripts. However, the greatest decline was typically observed in nuclear 2-LTR circles. Moreover, the magnitude of these declines varied with A3G dose. Adding integration inhibitor did not stop the A3G-mediated loss in 2-LTR circles. Moreover, obstructing HIV-1 nuclear entry using vesicular stomatitis virus matrix protein did not stop the A3G-mediated decline in late reverse transcripts. Collectively, these data suggest that A3G has important restriction activity in the cytoplasm and progressively diminishes viral cytoplasmic and nuclear cDNA forms with increasing magnitude during restriction.

Both Linear and Discontinuous Ribosome Scanning Are Used for Translation Initiation from Bicistronic Human Immunodeficiency Virus Type 1 env mRNAs

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) generates 16 alternatively spliced isoforms of env mRNA that contain the same overlapping open reading frames for Vpu and Env proteins but differ in their 5′ untranslated regions (UTR). A subset of env mRNAs carry the extra upstream Rev initiation codon in the 5′ UTR. We explored the effect of the alternative UTR on the translation of Vpu and Env proteins from authentic env mRNAs expressed from cDNA constructs. Vpu expression from the subset of env mRNA isoforms with exons containing an upstream Rev AUG codon was minimal. However, every env mRNA isoform expressed similar levels of Env protein. Mutations that removed, altered the strength of, or introduced upstream AUG codons dramatically altered Vpu expression but had little impact on the consistent expression of Env. These data show that the different isoforms of env mRNA are not redundant but instead regulate Vpu production in HIV-1-infected cells. Furthermore, while the initiation of Vpu translation conforms to the leaky ribosome-scanning model, the consistent Env synthesis infers a novel, discontinuous ribosome-scanning mechanism to translate Env.

A new way of measuring apoptosis by absolute quantitation of inter-nucleosomally fragmented genomic DNA

Several critical events of apoptosis occur in the cell nucleus, including inter-nucleosomal DNA fragmentation (apoptotic DNA) and eventual chromatin condensation. The generation of apoptotic DNA has become a biochemical hallmark of apoptosis because it is a late ‘point of no return’ step in both the extrinsic (cell-death receptor) and intrinsic (mitochondrial) apoptotic pathways. Despite investigators observing apoptotic DNA and understanding its decisive role as a marker of apoptosis for over 20 years, measuring it has proved elusive. We have integrated ligation-mediated PCR and qPCR to design a new way of measuring apoptosis, termed ApoqPCR, which generates an absolute value for the amount (picogram) of apoptotic DNA per cell population. ApoqPCR’s advances over current methods include a 1000-fold linear dynamic range yet sensitivity to distinguish subtle low-level changes, measurement with a 3- to 4-log improvement in sample economy, and capacity for archival or longitudinal studies combined with high-throughput capability. We demonstrate ApoqPCR’s utility in both in vitro and in vivo contexts. Considering the fundamental role apoptosis has in vertebrate and invertebrate health, growth and disease, the reliable measurement of apoptotic nucleic acid by ApoqPCR will be of value in cell biology studies in basic and applied science.

Persistence of integrated HIV DNA in CXCR3 + CCR6 + memory CD4+ T cells in HIV-infected individuals on antiretroviral therapy.

Background: HIV latent infection can be established in vitro by treating resting CD4+ T cells with chemokines that bind to chemokine receptors (CKR), CCR7, CXCR3, and CCR6, highly expressed on T cells. Objective: To determine if CKR identify CD4+ T cells enriched for HIV in HIV-infected individuals receiving suppressive antiretroviral therapy (ART). Design: A cross-sectional study of CKR expression and HIV persistence in blood from HIV-infected individuals on suppressive ART for more than 3 years (n = 48). A subset of 20 individuals underwent leukapheresis and sorting of specific CD4+ T-cell subsets. Methods: We used flow cytometry to quantify CCR5, CCR6, CXCR3, and CXCR5 expression on CD4+ T cells. HIV persistence was quantified using real-time Polymerase Chain Reaction to detect total, integrated HIV DNA, 2-long terminal repeat circles and cell-associated unspliced (CA-US) HIV RNA in total CD4+ T cells from blood or sorted T-cell subsets. Associations between CKR and HIV persistence in CD4+ T cells in blood were determined using regression models and adjusted for current and nadir CD4+ T-cell counts. Results: The frequency of cells harbouring integrated HIV DNA was inversely associated with current CD4+ T-cell count and positively associated with CCR5+ CD4+ T cells, CXCR3+CCR6+ and CXCR3+CCR6– expression on total memory CD4+ T cells (P < 0.001, 0.048, 0.015, and 0.016, respectively). CXCR3+CCR6+ CM CD4+ T cells contained the highest amount of integrated HIV DNA and lowest ratio of CA-US HIV RNA to DNA compared to all T-cell subsets examined. Conclusion: CXCR3 and CCR6 coexpression defines a subset of CD4+ T cells that are preferentially enriched for HIV DNA in HIV-infected individuals on ART.

Stepping up: a nurse-led model of care for insulin initiation for people with type 2 diabetes

BACKGROUND Most people with type 2 diabetes (T2D) have glycaemic levels outside of target. Insulin is effective in improving glycaemia and most people with T2D eventually need this. Despite this, transition to insulin therapy is often delayed in primary care. OBJECTIVE To develop a model of care (Stepping Up) for insulin initiation in routine diabetes care in Australian general practice. To evaluate the model for feasibility of integration within routine general practice care. METHODS Drawing on qualitative work and normalisation process theory, we developed a model of care that included clarification of roles, in-practice systems and simple clinical tools. The model was introduced in an educational and practice system change intervention for general practitioners (GPs) and practice nurses (PNs). Five practices (seven GPs and five PNs) and 18 patients formed the feasibility study. Evaluation at 3 and 12 months explored experiences of GPs, PNs and patients. RESULTS Fourteen patients commenced insulin, with average HbA1c falling from 8.4% (68.3 mmol/mol) to 7.5% (58.5 mmol/mol) at 3 months. Qualitative evaluation highlighted how the model of care supported integration of the technical work of insulin initiation within ongoing generalist GP care. Ensuring peer support for patients and issues of clinical accountability and flexibility, managing time and resources were highlighted as important. CONCLUSIONS The Stepping Up model allowed technical care to be embedded within generalist whole-person care, supported clinicians and practice system to overcome clinical inertia and supported patients to make the timely transition to insulin. Testing of the models effectiveness is now underway.