MicrobiologyBytes

The human JC polyomavirus is a bit of a mystery. Many people are infected with it, but few become ill as a result. This virus bides its time, waiting for your immune systen to let its guard down, then wham! People infected with HIV, those who have AIDS, or those receiving immunomodulatory therapies for autoimmune diseases are at serious risk for progressive multifocal leukoencephalopathy (PML), where the virus can spread from the kidney to the central nervous system and cause a fatal, demyelinating disease.

Recent reports have shown that virus isolates from PML patients often have distinct changes within the major capsid protein. This paper shows that that these mutations result in abolished engagement of the carbohydrate receptor motif necessary for infection. Viruses with PML-associated mutations are not infectious in glial cells, suggesting that they may play an alternative role in PML. Interesting stuff, suggesting that interaction with cell surface receptors is an important determinant of tissue tropism and JC virus pathogenesis for PML, even though the best defence remains a healthy immune system.

Progressive Multifocal Leukoencephalopathy-Associated Mutations in the JC Polyomavirus Capsid Disrupt Lactoseries Tetrasaccharide c Binding. (2013) mBio 4(3): e00247-13 doi: 10.1128/mBio.00247-13
The human JC polyomavirus (JCPyV) is the causative agent of the fatal, demyelinating disease progressive multifocal leukoencephalopathy (PML). The Mad-1 prototype strain of JCPyV uses the glycan lactoseries tetrasaccharide c (LSTc) and serotonin receptor 5-HT2A to attach to and enter into host cells, respectively. Specific residues in the viral capsid protein VP1 are responsible for direct interactions with the α2,6-linked sialic acid of LSTc. Viral isolates from individuals with PML often contain mutations in the sialic acid-binding pocket of VP1 that are hypothesized to arise from positive selection. We reconstituted these mutations in the Mad-1 strain of JCPyV and found that they were not capable of growth. The mutations were then introduced into recombinant VP1 and reconstituted as pentamers in order to conduct binding studies and structural analyses. VP1 pentamers carrying PML-associated mutations were not capable of binding to permissive cells. High-resolution structure determination revealed that these pentamers are well folded but no longer bind to LSTc due to steric clashes in the sialic acid-binding site. Reconstitution of the mutations into JCPyV pseudoviruses allowed us to directly quantify the infectivity of the mutants in several cell lines. The JCPyV pseudoviruses with PML-associated mutations were not infectious, nor were they able to engage sialic acid as measured by hemagglutination of human red blood cells. These results demonstrate that viruses from PML patients with single point mutations in VP1 disrupt binding to sialic acid motifs and render these viruses noninfectious.

The eradication of HIV-1 from infected individuals is prevented by the persistence of the virus in a stable reservoir of latently infected CD4+ T cells. Latently infected cells can be found in all HIV-1 infected individuals at a very low frequency and allow the virus to persist despite antiretroviral therapy for the lifetime of an infected patient. Current efforts are focused on identifying small molecules or immune strategies to eliminate these latently infected cells. To assess the efficacy of these elimination strategies in HIV-1 infected patients, we must be able to measure the size of the remaining latent reservoir. While a previous assay can measure the size of this latent reservoir, it is too laborious and costly to be utilized in large-scale HIV-1 eradication trials. A new paper in PLoS Pathogens describes a rapid assay to measure the size of the HIV-1 latent reservoir more amenable to eradication trials.

Rapid Quantification of the Latent Reservoir for HIV-1 Using a Viral Outgrowth Assay. (2013) PLoS Pathog 9(5): e1003398. doi:10.1371/journal.ppat.1003398
HIV-1 persists in infected individuals in a stable pool of resting CD4+ T cells as a latent but replication-competent provirus. This latent reservoir is the major barrier to the eradication of HIV-1. Clinical trials are currently underway investigating the effects of latency-disrupting compounds on the persistence of the latent reservoir in infected individuals. To accurately assess the effects of such compounds, accurate assays to measure the frequency of latently infected cells are essential. The development of a simpler assay for the latent reservoir has been identified as a major AIDS research priority. We report here the development and validation of a rapid viral outgrowth assay that quantifies the frequency of cells that can release replication-competent virus following cellular activation. This new assay utilizes bead and column-based purification of resting CD4+ T cells from the peripheral blood of HIV-1 infected patients rather than cell sorting to obtain comparable resting CD4+ T cell purity. This new assay also utilizes the MOLT-4/CCR5 cell line for viral expansion, producing statistically comparable measurements of the frequency of latent HIV-1 infection. Finally, this new assay employs a novel quantitative RT-PCR specific for polyadenylated HIV-1 RNA for virus detection, which we demonstrate is a more sensitive and cost-effective method to detect HIV-1 replication than expensive commercial ELISA detection methods. The reductions in both labor and cost make this assay suitable for quantifying the frequency of latently infected cells in clinical trials of HIV-1 eradication strategies.

Microbiology Today: HIV and the ‘functional’ cure

http://www.sgm.ac.uk/en/publications/microbiology-today/current-issue.cfm

There is intense interest in developing a cure for HIV. How such a cure will be quantified and defined is not known. Researchers applied a series of measurements of HIV persistence to the study of an HIV+ adult who has exhibited evidence of cure after a stem cell transplant.

Samples from blood, spinal fluid, lymph node, and gut were analyzed in multiple laboratories using different approaches. No HIV was detected in blood cells, spinal fluid, lymph node, or small intestine, and no infectious virus was recovered from blood. However, HIV was detected in plasma (2 laboratories) and HIV DNA was detected in the rectum (1 laboratory) at levels considerably lower than those expected in antiretroviral treated patients. The occasional, low-level HIV signals might be due to persistent HIV or might reflect false positives. The sensitivity of the current generation of assays to detect HIV RNA, HIV DNA, and infectious virus are close to the limits of detection. Improvements in these tests will be needed for future curative studies.

The lack of rebounding virus after five years without therapy, the failure to isolate infectious virus, and the waning HIV-specific immune responses all indicate that the Berlin Patient has been effectively cured.

Challenges in Detecting HIV Persistence during Potentially Curative Interventions: A Study of the Berlin Patient. (2013) PLoS Pathog 9(5): e1003347. doi:10.1371/journal.ppat.1003347
There is intense interest in developing curative interventions for HIV. How such a cure will be quantified and defined is not known. We applied a series of measurements of HIV persistence to the study of an HIV-infected adult who has exhibited evidence of cure after allogeneic hematopoietic stem cell transplant from a homozygous CCR5Δ32 donor. Samples from blood, spinal fluid, lymph node, and gut were analyzed in multiple laboratories using different approaches. No HIV DNA or RNA was detected in peripheral blood mononuclear cells (PBMC), spinal fluid, lymph node, or terminal ileum, and no replication-competent virus could be cultured from PBMCs. However, HIV RNA was detected in plasma (2 laboratories) and HIV DNA was detected in the rectum (1 laboratory) at levels considerably lower than those expected in ART-suppressed patients. It was not possible to obtain sequence data from plasma or gut, while an X4 sequence from PBMC did not match the pre-transplant sequence. HIV antibody levels were readily detectable but declined over time; T cell responses were largely absent. The occasional, low-level PCR signals raise the possibility that some HIV nucleic acid might persist, although they could also be false positives. Since HIV levels in well-treated individuals are near the limits of detection of current assays, more sensitive assays need to be developed and validated. The absence of recrudescent HIV replication and waning HIV-specific immune responses five years after withdrawal of treatment provide proof of a clinical cure.

HIV may not always want to go unnoticed. It is known that HIV replicates more efficiently in TH cells that have been activated, and this presents the virus with a bit of a dilemma – it certainly doesn’t pay to be recognized by CTLs, the hired assassins of the immune system. But there’s also a possible benefit in triggering the activation of the TH cells that they’ve infected. Where does the balance lie? Is it conceivable that there are some conditions where it is better for HIV epitopes to be recognized than to be ignored?

To answer this question, scientists have built a complex mathematical model of interactions between TH cells, CTLs, antigen-presenting cells and viruses. They set up two different versions of the model – one with a virus that infects non-immune cells (like HCV infecting hepatocytes), and one with a virus that infects T cells (like HIV). The results are quite striking – in the case of HCV, evasion always pays off. But in the case of HIV, the dependence on TH activation seems to sometimes favor virus epitopes that are strongly recognized by cellular immune system. The authors propose that recognition of HIV epitopes by TH cells can be beneficial for the long-term survival of the virus within the body. Another key variable is how many TH cells are activated in the body by pathogens other than HIV. If this “background activation” is strong, HIV obtains no payoffs from being recognized. Studying the sequence data confirms that in the capsid genes (Gag) it is TH epitopes, rather than CTL epitopes, that are constrained and that epitopes stay the same within patient viral populations rather than between patients, again implicating chronic internal transmission between cells.

There is an important practical implication of these findings. If this model is correct, then HIV vaccines based on TH epitopes might prove counterproductive, playing into the hands of the virus. Instead, vaccines should target only CTL-specific viral epitopes.

HIV Plays (and Wins) a Game of T Cell Brinkmanship. (2013) PLoS Biol 11(4): e1001521. doi:10.1371/journal.pbio.1001521

Technical developments in imaging-based techniques have greatly improved our understanding of HIV–host cell interactions. HIV-1 virions labeled with fluorophores were pivotal in shedding light onto multiple aspects of the virus–host interplay during all steps of HIV-1 replication cycle. Nevertheless, few optical approaches have been so far developed to visualize viral particles within the nuclear compartment, which limits our comprehension of the interaction between HIV-1 and the nuclear architecture. Moreover, the existing detection tools are based on the visualization of the viral protein complexes or envelope but not of the viral DNA with the only exception of the fluorescence in situ hybridization (FISH) technique. Even though FISH is a powerful technique, it is not very sensitive for HIV-1 detection and moreover disrupts the native architecture of the nuclear compartment as it requires harsh denaturation conditions. In addition, this technique does not allow the discrimination between integrated and nonintegrated viral DNA.

A new paper describes a fluorescent approach to visualize HIV-1 DNA in the nuclear compartment of infected cells. A 3D topological analysis demonstrated that integrated viral DNA localizes at the periphery of the nuclei revealing important insights in the nuclear biology of HIV-1.

Single-Cell Imaging of HIV-1 Provirus (SCIP). PNAS USA 19 March 2013, doi: 10.1073/pnas.1216254110
Recent advances in fluorescence microscopy provided tools for the investigation and the analysis of the viral replication steps in the cellular context. In the HIV field, the current visualization systems successfully achieve the fluorescent labeling of the viral envelope and proteins, but not the genome. Here, we developed a system able to visualize the proviral DNA of HIV-1 through immunofluorescence detection of repair foci for DNA double-strand breaks specifically induced in the viral genome by the heterologous expression of the I-SceI endonuclease. The system for Single-Cell Imaging of HIV-1 Provirus, named SCIP, provides the possibility to individually track integrated-viral DNA within the nuclei of infected cells. In particular, SCIP allowed us to perform a topological analysis of integrated viral DNA revealing that HIV-1 preferentially integrates in the chromatin localized at the periphery of the nuclei.

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HIV, the virus that causes AIDS, has an extensive repertoire of tricks that it uses to evade, manipulate, and subvert the human immune system. Perhaps most insidious is the virus’s ability to turn the immune system’s defensive tactics to its own advantage. For example, HIV-1 uses a type of immune cell, the dendritic cell (DC), to spread infection.
Mature DCs recognize sialic acid–containing glycolipids (gangliosides) present in the HIV’s lipid envelope. New research show that the sialic acid–containing molecule Siglec-1 promotes both virus uptake and transmission to T cells. Future work may point the way towards therapeutic interventions against HIV and viruses that use similar infection strategies.
PLOS Biology: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001454

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