Archive for May, 2012

Superresolution imaging of HIV in infected cells with FlAsH-PALM

29 May, 2012

See on Scoop.itVirology News

“Imaging protein assemblies at molecular resolution without affecting biological function is a long-standing goal. The diffraction-limited resolution of conventional light microscopy (∼200–300 nm) has been overcome by recent superresolution (SR) methods including techniques based on accurate localization of molecules exhibiting stochastic fluorescence; however, SR methods still suffer important restrictions inherent to the protein labeling strategies. Antibody labels are encumbered by variable specificity, limited commercial availability and affinity, and are mostly restricted to fixed cells. Fluorescent protein fusions, though compatible with live cell imaging, substantially increase protein size and can interfere with their biological activity. We demonstrate SR imaging of proteins tagged with small tetracysteine motifs and the fluorescein arsenical helix binder (FlAsH-PALM). We applied FlAsH-PALM to image the integrase enzyme (IN) of HIV in fixed and living cells under experimental conditions that fully preserved HIV infectivity. The obtained resolution (∼30 nm) allowed us to characterize the distribution of IN within virions and intracellular complexes and to distinguish different HIV structural populations based on their morphology. We could thus discriminate ∼100 nm long mature conical cores from immature Gag shells and observe that in infected cells cytoplasmic (but not nuclear) IN complexes display a morphology similar to the conical capsid. Together with the presence of capsid proteins, our data suggest that cytoplasmic IN is largely present in intact capsids and that these can be found deep within the cytoplasm. FlAsH-PALM opens the door to in vivo SR studies of microbial complexes within host cells and may help achieve truly molecular resolution.”

 

Beautiful pictures, a great technique – and one which may allow ~1 nm resolution imaging inside cells in the near future.  Oh, and incidentally showed that HIV capsids persist all the way to the nuclear membrane.

See on www.pnas.org

Nano Patents and Innovations: Powerful New Approach To Attack Flu Virus

28 May, 2012

See on Scoop.itVirology and Bioinformatics from Virology.ca

An international research team has manufactured a new protein that can combat deadly flu epidemics.

The paper, featured on the cover of the current issue of Nature Biotechnology, demonstrates ways to use manufactured genes as antivirals, which disable key functions of the flu virus, said Tim Whitehead, assistant professor of chemical engineering and materials science at Michigan State University.

See on nanopatentsandinnovations.blogspot.fr

ProMED-mail | MEASLES UPDATE 2012 (22)

28 May, 2012

See on Scoop.itVirology and Bioinformatics from Virology.ca

ProMED Mail is possibly THE premier infectious disease updating service in the world today, having sprung to fame during the Kikwit Ebola outbreak in 1995.  This is one of a series of posts on measles, which is documenting a very disturbing trend: the incidence of the disease is increasing in places where it should have been eradicated, because well-educated and sophisticated communities are not vaccinating their children – or themselves.

 

One very telling quote from the post:

“Measles is highly infectious so we must all do
everything possible to prevent the spread of it, particularly with an outbreak on our doorstep. … MMR vaccination is the only way to prevent
measles. If parents haven’t arranged for their children to be vaccinated – it’s not too late to have the jab. Parents don’t realise that measles is not just a case of a few spots – it can be a very serious illness. Symptoms include fever, cough, soreness of the eyes and a rash which spreads rapidly over the body. Serious complications affect one in 15 children. These include chest infections, fits, encephalitis (swelling of the brain), and brain damage. In very serious cases, measles can kill.”

See on www.promedmail.org

Pandemic 2009 H1N1 vaccination produces antibodies against multiple flu strains

27 May, 2012

See on Scoop.itVirology News

“The pandemic 2009 H1N1 vaccine can generate antibodies in vaccinated individuals not only against the H1N1 virus, but also against other influenza virus strains including H5N1 and H3N2.”

 

And a possible reason for this could be that the H1N1pdm virus’ haemagglutinin is a natural “ancestral” sequence – the kind that HIV vaccine researchers are looking for for gp120/160, which have been shown to elicit a wider spectrum of cross-reacting antibodies than “evolved” proteins, or ones that have been selected for antigenic escape in humans for a good few viral generations.

 

Flu vaccine graphic by Russell Kightley Media

See on www.eurekalert.org

Risk factors for West Nile virus – by Dr Pandula Siribaddana – Helium

27 May, 2012

See on Scoop.itVirology News

“The disease is more common in temperate and tropical geographical areas and usually the disease is said to be found in Africa, Middle East and West Asia. But, from recent times, almost all American states have got affected and from time to time there are outbreaks of large number of patients with West Nile Disease.”

 

Useful little review on the how/why/what of WNV.  Including the fact that we Africans have been living with it forever…B-)

See on www.helium.com

Trends in Intussusception Hospitalizations Among US Infants Before and After Implementation of the Rotavirus Vaccination Program, 2000–2009

27 May, 2012

See on Scoop.itVirology News

“A small increase in intussusception rates was seen among infants aged 8–11 weeks, to whom most first doses of rotavirus vaccine were given, but no sustained population-level change in overall intussusception hospitalizations rates in US infants was observed after implementation of the US rotavirus vaccination program. Although an association between intussusception and rotavirus vaccination cannot be established by this ecologic analysis alone, even if the low risk with the first dose exists, it is outweighed by the well-documented benefits of vaccination of US infants”

This is a big deal- a very important, big deal: human rotavirus kills more than 500 000 people a year (mainly very little), and rotavirus vaccines have been bedevilled with the suspicion that they cause telescoping of the intestine, or intussusception.  Which can be fatal, and is not something you want happening to your healthy baby.

However, and however: I have taught my students for years to be aware of relative risks when talking about vaccines, and there is absolutely no doubt that even the Wyeth vaccine could have been considered “safe” in a developing country environment, where the threat of death due to diarrhoea and dehyderation caused by rotavirus, would have been far greater than any threat from the vaccine.

I thank Rusdsell Kightley Media for the rotavirus graphic

See on jid.oxfordjournals.org

African monkey meat that could be behind the next HIV

25 May, 2012

See on Scoop.itVirology News

Eighty per cent of the meat eaten in Cameroon is killed in the wild and is known as “bushmeat”. The nation’s favoured dishes are gorilla, chimpanzee or monkey because of their succulent and tender flesh. According to one estimate, up to 3,000 gorillas are slaughtered in southern Cameroon every year to supply an illicit but pervasive commercial demand for ape meat .

“Everyone is eating it,” said one game warden. “If they have money they will buy gorilla or chimp to eat.”

Frankie, a poacher in the southern Dja Wildlife reserve who gave a fake name, said he is involved in the trade because he can earn good money from it, charging around £60 per adult gorilla killed. “I have to make a living,” he said. “Women come from the market and order a gorilla or a chimp and I go and kill them.”

 ————————————————————-

This is a big deal – and not because as, in the words of the article, “Britain is at risk from an outbreak caused by the lethal Ebola or Marburg viruses contained in illegal imports of bush meat from Africa”.

 

Because AFRICA is at risk from such diseases – and the death toll will be much higher here, where the standard of care is so much lower than in Europe.

 

And because we are losing the closest relatives that we as humans have – to human greed for meat.  How despicable, and how sad, is that?

See on www.independent.co.uk

Flu shot offers surprising benefits for pregnant women; Vaccine may fight stillbirth, preterm birth, and very low birth weight

25 May, 2012

See on Scoop.itVirology News

A new study announced Tuesday finds the H1N1 flu vaccine not only can protect you from getting sick but can actually benefit your baby.

Researchers from the University of Ottawa in Canada examined data from more than 55,000 child births in Ontario during an outbreak of H1N1, comparing mothers who were vaccinated to those who weren’t.

While prior research has found that pregnant women can safely get the flu shot at any stage of their pregnancies — something many doctors vehemently support — the new findings associate H1N1 vaccinations with a significantly reduced risk of stillbirth, preterm birth, and very low birth weight.

“These are all significant results, but especially interesting is the finding that the vaccinated mothers were one-third less likely to have a stillborn child,” said study researcher Deshayne Fell, a graduate student at McGill University who works with the birth record database. “This is one of the only studies large enough to evaluate the association between maternal flu vaccination and stillbirth — a very rare event.”

 

So much for the disinformation about dangers to pregnant women: in fact, Spanish Flu and the recent H1N1 pandemic were both especially dangerous for unprotected pregnant women.

See on www.nydailynews.com

Muslim leaders enlisted to help stamp out polio

25 May, 2012

See on Scoop.itVirology News

GENEVA (Reuters) – The last three countries where polio is still paralyzing children — Afghanistan, Pakistan and Nigeria — said on Thursday that they have enlisted Muslim women and religious leaders to allay fears of vaccination and wipe out the disease.
Polio cases are at an all-time low worldwide, following its eradication in India last year, raising hopes but also fears about a threat of resurgence especially in sub-Saharan Africa unless remaining reservoirs of polio virus are stamped out.

Conflict and insecurity is preventing health workers from reaching hundreds of thousands of children in Afghanistan and Pakistan with doses of polio vaccine, health ministers said.

See on www.reuters.com

Radical Therapy for HIV-Infected People?

18 May, 2012

To mark HIV Vaccine Awareness Day, 18th May – Journal Club – Lucian Duvenage:

Excision of HIV-1 Proviral DNA by Recombinant Cell Permeable Tre-Recombinase

Mariyanna, L., Priyadarshini, P., Hofmann-Sieber, H., Krepstakies, M., Walz, N., Grundhoff, A., Buchholz, F., Hildt, E., Hauber, J., 2012. Excision of HIV-1 proviral DNA by recombinant cell permeable tre-recombinase. PloS One 7, e31576.

Introduction

HIV Life Cycle. Russell Kightley Media, http://www.rkm.com.au

Highly active antiretroviral therapy (HAART) is a combination of drugs that has significantly elongated the lifespan HIV-infected people. HAART targets viral reverse transcriptase, protease and integrase. There are disadvantages including drug toxicity and the appearance of drug resistant HIV strains in people not adhering to or withdrawing from their treatment. There is a need for new therapies that not only block virus replication but also eliminate HIV from persistent viral reservoirs. An attractive option is Tre-recombinase, which been shown to excise provirus from the genomic DNA of infected cell cultures. The development of Tre recombinase is a previous publication (Sarkar, I., Hauber, I., Hauber, J., Buchholz, F., 2007. HIV-1 proviral DNA excision using an evolved recombinase. Science 316, 1912-5.)

The Tre recombinase was created from the Cre recombinase which is a well-known tool in mouse genetics. The authors were able to alter the specificity of the enzyme by many cycles of directed protein evolution.

The Cre recombinase precursor removes genomic DNA that is flanked by two loxP sites by recombination. The authors were able to alter the specificity for loxP sites to HIV-1 LTR (long terminal repeat) sites. LoxP and the HIV LTR had 50% sequence similarity.

The main problem with the development of antiviral agents is the delivery to infected cells in vivo, without causing adverse side effects.  There are many reported technologies for the delivery of macromolecules such as proteins, nucleic acids or peptides. The most popular currently is the use of protein transduction domains (PTD) also known as cell penetrating peptides (CPP) from different sources. These have been useful for the delivery of various genes and proteins, including site-specific recombinases. The best studied and most applied PTD’s are peptides derived from the basic domain of HIV-1 Tat. But recently a powerful cell permeable translocation motif (TLM) has been described. This TLM is derived from a hepatitis B virus surface antigen. This TLM peptide is able to enter cells very efficiently, without affecting the integrity of the cells, or interfering with intracellular signal transduction cascades.

This paper describes the delivery of Tre-recombinase into cells using these PTD’s including HIV Tat and the HBV TLM. These so-called cell-permeable Tre-recombinases could eventually be useful for antiretroviral therapy, especially for virus eradication.

Results

Proteins

Different protein fusions were created and expressed in E. coli ; Tre-recombinase is fused to His tag, with/without nuclear localisation signal (NLS) and with the PTD (HIV Tat) or TLM (translocation motif derived from hepatitis B) or TLM as an inverted repeat.

They tested the cellular toxicity of the protein at their highest concentration by alarmBlue assay in HeLa cells. The proteins were incubated with the cells for 48 h. None of the proteins had any significant effect on the cellular metabolism

Cell permeability

Interestingly, all of the proteins entered cells, even those without a PTD or nuclear translocation signal. The authors explain that the Cre enzyme precursor to Tre has been shown to transduce into mammalian cells without any help, and therefore it is likely that the Tre enzyme shares this property. The authors did remark that the signal intensities were higher for those proteins with a PTD, indicating higher transduction efficiency.

Analysis of Tre activity in HeLa cells

A transient reporter assay demonstrated the activity of the Tre fusions: The reporter construct contains the target LTR sites that flank a puromycin resistance gene. Tre enzyme activity results in the loss of this gene, and gives a smaller PCR product using primers that anneal to the vector backbone. Cells transfected with the reporter construct were incubated with the 1 µM of the various proteins for 5 hours. The positive control was co-transfected with a construct expressing the Tre enzyme. PCR was performed on DNA extracted from cells after 48 hours. The presence of the smaller PCR product indicated that recombination had happened, as in the positive control. All of the proteins had varying degrees of activity, but notably the protein with the TLM PTD had the highest activity, with no un-recombined product detected by the PCR.

The authors went on to demonstrate that the Tre fusion proteins were active on at the genomic level, i.e. on chromosomal DNA. They used cells with the reporter construct was stably integrated into the genome.

Interaction of proteins with LTR sequences in living T-cells

Co-immunoprecipitation (ChIP) assays were done on using HIV-1 -infected T-cells (CEM-SS) to demonstrate the interaction of two of the Tre fusion proteins with the HIV LTR target sequences. The results showed that the proteins interacted with target LTR sequences in the genome of infected T-cells.

Microarray

The authors performed a transcriptome anaylsis on cells exposed to the Tre fusion proteins, using human whole genome microarrays. They concluded that the proteins were unlikely to have a significant effect on gene expression in the host cells, as very few genes were regulated more than 2.5-fold.

Recombination of the full-length HIV proviral genome:

Up to this point, the Tre fusion proteins had been shown to be capable of excising reporter construct gene flanked by LTR sequences both at the episomal level and the chromosomal level. The authors also showed that the proteins bind to the target sequences in HIV-infected living T cells.

It was essential that the Tre fusions could excise that HIV proviral genome from the chromosomal DNA of HIV-infected cells. The aouthors generated HeLa cells and T cells infected with pseudotype HIV-1. These are cells with the full-length HIV provirus integrated into the genome.They chose one of their proteins (TLM fusion showing highest activity in the reporter assays) for transduction into these cells. After transduction, PCR was performed to detect the HIV circular recombination product. They found that the recombination activity increased in a dose-dependent manner in both the HeLA cells and the T-cells. They also sequenced the PC products and were able to confirm HIV sequences.

Discussion

Some novel therapies for the treatment of HIV focus on the eradication of the virus in infected individuals. These include RNA-based technologies such as RNA aptamers, siRNA and ribozymes, but while these have shown to reduce viral load and viral replication, they have so far failed in virus eradication. A recent approach aimed at virus eradication is the reduction of surface CCR-5 receptors, through the expression of engineered zinc finger nucleases. This results in fewer CCR-5 surface receptors and could prevent new infection by CCR-5 tropic HIV.

The other approach is the use of site specific recombinases like Tre, which can excise the provirus from the host genome, thus potentially eradicating the virus from the individual. Ideally, the gene expressing Tre could be delivered to and expressed in target cells using a viral vector. But there are safety concerns as most of these are derived from pathogenic viruses. Therefore it may be advantageous to deliver the Tre enzyme directly to host cells. One way of doing this is through protein translocation domains (PTD’s). Protein transduction domains (PTD’s) can deliver bioactive molecules, including genes, siRNA, proteins or liposomes into all types of cells in vitro and furthermore into various organs in vivo. But they have not been applied yet for human use. PTD’s are easily fused to any target protein through cloning and expression of the fusion protein. The LTM used in this paper, derived from hepatitis B virus surface antigen, has low immunogenicity and high spreading capacity.

One strategy of using cell-permeable Tre enyme could be to harvest T-cells from the patient by apheresis and transduce them in vitro. They could then potentially be expanded and reinfused into the patient. This could complement or even replace gene transfer procedures.

In this paper the expressed Tre fusion proteins could enter cells and act on the target sequences to excise the HIV provirus from the genome, but the Tat fusion and the TLM fusion had higher activity than those that didn’t have a PTD tag. In particular, fusion to the newly described domain (TLM) from hepatitis B was resulted highest activity. This may explained by the fact that, in contrast to HIV Tat, TLM does not rely on endocytosis to enter cells. This might preserve enzyme activity and protein half-life.

In conclusion, cell permeable Tre enzyme could eventually be useful as an anti-HIV therapy in the post-HAART era.


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