MicrobiologyBytes

One of the first attempts to use gene therapy to treat HIV has produced promising results in clinical trials. When the therapy was tested on 74 patients, it was shown to be safe and appeared to reduce the effect of the virus on the immune system. In theory, one treatment should be enough to replace the need for a lifetime of antiretroviral therapy. The latest therapy involves giving patients blood stem cells modified to carry a molecule called OZ1, which is designed to stop HIV reproducing itself by targeting two key proteins. The patients in the trial either received the therapy, or a dummy treatment. After 48 weeks the researchers found there was no statistically significant difference in the amount of HIV circulating in the blood of the two groups of patients. However, after 100 weeks the patients who received the gene therapy had higher levels of CD4+ cells – the key cells of the immune system which are specifically destroyed by HIV.

BBC News

Phase 2 gene therapy trial of an anti-HIV ribozyme in autologous CD34+ cells. Nature Medicine, 15 February 2009
Gene transfer has potential as a once-only treatment that reduces viral load, preserves the immune system and avoids lifetime highly active antiretroviral therapy. This study, which is to our knowledge the first randomized, double-blind, placebo-controlled, phase 2 cell-delivered gene transfer clinical trial, was conducted in 74 HIV-1–infected adults who received a tat-vpr–specific anti-HIV ribozyme (OZ1) or placebo delivered in autologous CD34+ hematopoietic progenitor cells. There were no OZ1-related adverse events. There was no statistically significant difference in viral load between the OZ1 and placebo group at the primary end point (average at weeks 47 and 48), but time-weighted areas under the curve from weeks 40–48 and 40–100 were significantly lower in the OZ1 group. Throughout the 100 weeks, CD4+ lymphocyte counts were higher in the OZ1 group. This study indicates that cell-delivered gene transfer is safe and biologically active in individuals with HIV and can be developed as a conventional therapeutic product.

Related:

• HIV “can never be cured”
• Retroviruses
• HIV-2 – a kinder AIDS virus?
• HIV mutation and the immune response
• Immune exhaustion in HIV infection
• How does HIV cause AIDS?

It has been estimated that around 2% of the world’s population, approximately 170 million people, are infected with hepatitis C virus (HCV). Over 4 million people in the USA are infected with HCV, a prevalence rate of 1.6%. The peak prevalence of HCV infection occurs among people of 40 to 49 years of age and a history of injection drug use is the strongest risk factor. The UK Health Protection Agency’s annual reports on HCV estimate that in England and Wales around 4,500 people are suffering from severe liver disease due to chronic HCV infection, and that this could rise to around 7,000 by 2010. Over 200,000 UK residents have chronic HCV infection, but five out of six are unaware of this. It is believed that around 80% of UK HCV infections are linked to the use of injected drugs, and 50% of injecting drug users are infected with HCV.

The majority of cases of HCV infection give rise to an acute illness, but up to 80% may then develop into chronic hepatitis. Almost all patients develop a vigorous antibody and cell-mediated immune response which fails to clear the virus infection but may contribute to liver damage. Spontaneous resolution of chronic liver disease is very rare (<2%) and patients with chronic disease are at risk of developing hepatocellular carcinoma (HCC).

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So HCV is a pretty important virus, but it has not been the easiest virus to study. The virus only infects only humans and chimpanzees and effectively cannot be grown in the laboratory. Consequently, until now studies of this virus in animal models have been restricted to chimpanzees and to immunodeficient mice transplanted with human hepatocytes hampering understanding of the virus and drug development. A paper in the most recent edition of Nature identifies a host protein that is essential for HCV entry into cells (Human occludin is a hepatitis C virus entry factor required for infection of mouse cells. Nature 457: 882-886, 12 February 2009). If this protein could be introduced into cells by genetic manipulation, this might produce new models for the study of HCV infection, and manipulating the protein in humans might be a route to protection against the virus.

The newly-identified protein which plays a role in HCV cell entry is occludin (OCLN), a protein involved in forming the tight junctions between cells. The researchers showed that adding this protein to mouse cells makes them susceptible to infection with HCV. Several other HCV cell entry factors had previously been identified, including CD81, scavenger receptor class B type I and claudin-1 (CLDN1). The mouse versions of SR-BI and CLDN1 function at least as well as the human proteins in promoting HCV entry, but OCLN and CD81 must be of human origin to allow efficient infection of mouse cells. The identification of the essential cell entry factors for HCV is an important advance in efforts to develop new models to allow us to study and fight this virus.

Related:

• Hepatitis C Virus: a mountain to climb
• Viruses and Human Cancer
• Does the outcome of HCV infection vary with the infecting virus type?

GlaxoSmithKline, the world’s second biggest pharmaceutical company, is to provide cheap drugs to millions of people in the developing world. Andrew Witty, the new head of the company, has said he will cut prices on all medicines, including HIV treatments, in the 50 poorest countries to no more than 25 per cent of the levels in Britain and the US. The company will also give back 20 per cent of profits to be spent on hospitals and clinics and share knowledge about potential drugs currently protected by patents. Drug companies have been repeatedly criticised for failing to drop prices for HIV drugs as millions have died in Africa and Asia.

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Dengue fever and dengue hemorrhagic fever are major global public health burdens, with up to 100 million cases occurring annually, yet no vaccines or specific preventative medicines are currently available. Dengue viruses, globally the most prevalent arboviruses, are transmitted to humans by persistently infected Aedes aegypti mosquitoes. However, although DENVs can cause severe disease in humans, mosquito infections are non-pathogenic and persistent. Determining how the virus evades the mosquito’s defense is an important next step in research that aims to fight disease by interrupting the growth of dengue virus within the mosquito before it can be transmitted. Researchers have discovered that mosquitoes that transmit deadly viruses such as dengue avoid becoming ill by mounting an immediate, potent immune response. Because their immune system does not eliminate the virus however, they are able to pass it on to a new victim. The researchers showed that RNA interference – a mosquito immune response – is initiated immediately after they ingest blood containing dengue virus, but the virus multiplies in the mosquitoes nevertheless.

RNA interference is an evolutionarily ancient antiviral defense used by mosquitoes and other invertebrates to destroy the RNA of many invading arthropod-borne viruses. This team of researchers previously showed that ramping up the RNA interference response in mosquitoes prevented dengue infection, and now they show that temporarily impairing this immune response increased virus transmission. The investigators analyzed RNA from adult mosquitoes, finding that both the trigger and initiator molecules for RNA interference were formed after infection, yet viral RNA could readily be detected in the same mosquitoes. They also measured infectious virus rates in the mosquitoes’ saliva, which revealed levels whereby the mosquitoes could transmit the disease to humans. These findings indicate that genetic manipulation of RNA interference could be a significant weapon in stopping dengue virus transmission by Aedes aegypti.

Understanding the mechanisms mosquitoes use to modulate infections by these agents of serious human diseases should give us critical insights into virus–vector interactions leading to transmission. RNA interference (RNAi) is an innate defense mechanism used by invertebrates to inhibit RNA virus infections; however, little is known about the antiviral role of RNAi in mosquitoes. RNAi is triggered by double-stranded RNA, leading to degradation of RNA with sequence homology to the dsRNA trigger. Dengue virus type 2 (DENV2) infection of Ae. aegypti by the natural route generates dsRNA and DENV2-specific small interfering RNAs, hallmarks of the RNAi response; nevertheless, persistent infection of mosquitoes occurs, suggesting that DENV2 circumvents RNAi. DENV2 infection is also modulated by RNAi, since impairment by silencing expression of genes encoding important sensor and effector proteins in the RNAi pathway increases virus replication in the vector and decreases the incubation period before virus transmission.

Dengue Virus Type 2 Infections of Aedes aegypti Are Modulated by the Mosquito’s RNA Interference Pathway. PLoS Pathog 5(2): e1000299
A number of studies have shown that both innate and adaptive immune defense mechanisms greatly influence the course of human dengue virus (DENV) infections, but little is known about the innate immune response of the mosquito vector Aedes aegypti to arbovirus infection. We present evidence here that a major component of the mosquito innate immune response, RNA interference (RNAi), is an important modulator of mosquito infections. The RNAi response is triggered by double-stranded RNA (dsRNA), which occurs in the cytoplasm as a result of positive-sense RNA virus infection, leading to production of small interfering RNAs (siRNAs). These siRNAs are instrumental in degradation of viral mRNA with sequence homology to the dsRNA trigger and thereby inhibition of virus replication. We show that although dengue virus type 2 (DENV2) infection of Ae. aegypti cultured cells and oral infection of adult mosquitoes generated dsRNA and production of DENV2-specific siRNAs, virus replication and release of infectious virus persisted, suggesting viral circumvention of RNAi. We also show that DENV2 does not completely evade RNAi, since impairing the pathway by silencing expression of dcr2, r2d2, or ago2, genes encoding important sensor and effector proteins in the RNAi pathway, increased virus replication in the vector and decreased the extrinsic incubation period required for virus transmission. Our findings indicate a major role for RNAi as a determinant of DENV transmission by Ae. aegypti.

Related:

• MicrobiologyBytes: Dengue Virus
• Dengue Virus
• Clustering of dengue virus infections
• Wolbachia could combat dengue fever
• Dissecting the Cell Entry Pathway of Dengue Virus

The process of bacterial pathogenesis involves complex and dynamic responses from both pathogen and host. While the host can mount an array of defense mechanisms to counteract an infection, bacterial pathogens utilize a number of virulence mechanisms to help them in their quest to invade, colonize, and infect. The expression pattern of virulence factors such as toxins, adhesions factors, enzymes, and others within the tissue determines the progression and outcome of infection. Deciphering the roles of bacterial effector proteins in the in vivo situation is essential to fully understand the infection process. The last decade has provided major technological developments, enabling dynamic live single cell imaging with high spatio-temporal resolution in vitro as well as in vivo. This review outlines current in vivo imaging techniques with primary focus on multiphoton microscopy, which was only recently adapted for real-time studies of bacterial infections within the host. Only few examples have been published to date, but they illustrate the huge potential real-time analysis of bacterial infections has to identify previously unknown aspects of tissue responses linked to bacterial pathogenesis.

Real-time live imaging to study bacterial infections in vivo. Curr Opin Microbiol. Jan 7 2009
In vitro studies have been essential to describe the molecular details of bacteria-host cell interactions in general and the functions of bacterial effector proteins in particular. Recent advancements in in vivo imaging techniques are facilitating the next logical step to visualize the dynamic infection process as it happens within the living host while analyzing the role of bacterial effector proteins in vivo. Data obtained from this emerging field of ’tissue microbiology’, combined with the massive knowledge base generated from research in ‘cellular microbiology’ will eventually provide a complete picture of the complex infection process.

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• Tracking Lyme Disease in Living Hosts
• Imaging Poliovirus Entry in Live Cells

A retrovirus has a lipoprotein envelope lined with a layer of matrix protein (MA), surrounding a nucleoprotein core. In the core, the diploid RNA genome in complex with nucleocapsid protein (NC) and the replication enzymes is enclosed within the capsid – a shell of CA protein. MA, CA and NC are derived from a common precursor, the Gag polyprotein, which assembles into a thick-walled spherical shell in the immature virus. After it buds off from the host cell, the viral protease is activated, releasing CA subunits that assemble into capsids. Capsids of a given retrovirus vary in structure, and the predominant types vary among retroviruses; for instance, those of HIV are conical, those of Rous sarcoma virus (RSV) are irregular polyhedra and those of murine leukemia virus are round. Some virions contain more than one capsid, and nested (multilayer) capsids are also observed.

For a retrovirus such as HIV to be infectious, a properly formed capsid is needed; however, unusually among viruses, retrovirus capsids are highly variable in structure. According to the fullerene conjecture, they are composed of hexamers and pentamers of capsid protein (CA), with the shape of a capsid varying according to how the twelve pentamers are distributed and its size depending on the number of hexamers. Hexamers have been studied in planar and tubular arrays, but the predicted pentamers have not been observed. Here we report cryo-electron microscopic analyses of two in-vitro-assembled capsids of Rous sarcoma virus. Both are icosahedrally symmetric: one is composed of 12 pentamers, and the other of 12 pentamers and 20 hexamers. Fitting of atomic models of the two CA domains into the reconstructions shows three distinct inter-subunit interactions. These observations substantiate the fullerene conjecture, show how pentamers are accommodated at vertices, support the inference that nucleation is a crucial morphologic determinant, and imply that electrostatic interactions govern the differential assembly of pentamers and hexamers.

Visualization of a missing link in retrovirus capsid assembly. Nature 457: 694-698 (5 February 2009)

Related:

• Retroviruses
• Retrovirus infection of resting cells

In bacterial cells, the peptidoglycan cell wall is the stress-bearing structure that dictates cell shape. Although many molecular details of the composition and assembly of cell-wall components are known, how the network of peptidoglycan subunits is organized to give the cell shape during normal growth and how it is reorganized in response to damage or environmental forces have been relatively unexplored. In this work, we introduce a quantitative physical model of the bacterial cell wall that predicts the mechanical response of cell shape to peptidoglycan damage and perturbation in the rod-shaped Gram-negative bacterium Escherichia coli. To test these predictions, we use time-lapse imaging experiments to show that damage often manifests as a bulge on the sidewall, coupled to large-scale bending of the cylindrical cell wall around the bulge. Our physical model also suggests a surprising robustness of cell shape to peptidoglycan defects, helping explain the observed porosity of the cell wall and the ability of cells to grow and maintain their shape even under conditions that limit peptide crosslinking. Finally, we show that many common bacterial cell shapes can be realized within the same model via simple spatial patterning of peptidoglycan defects, suggesting that minor patterning changes could underlie the great diversity of shapes observed in the bacterial kingdom.

Cell shape and cell-wall organization in Gram-negative bacteria. PNAS USA December 2, 2008

Related:

• Getting bacteria into shape
• Peptidoglycan – the strength and weakness of bacteria

Retroviruses have been studied for over 100 years since the discovery by Ellerman and Bang in 1908 that cell-free tissue filtrates could transmit leukaemia in chickens. The first pathogenic human retrovirus (HTLV) was discovered in 1981 and HIV, the causative agent of AIDS was discovered in 1983, but this presentation concentrates on the basic biology of retroviruses.

Retrovirus particles consist of a core, which contains the RNA genome of the virus plus the nucleocapsid (NC) protein and reverse transcriptase (RT), integrase (IN) and protease (PR) enzymes. The core lies inside an icosahedral capsid (CA protein) which is surrounded by the matrix (MA) which links the capsid to the lipid envelope. The transmembrane protein (TM) and surface glycoprotein (SU) are associated with the envelope.

All retrovirus genomes consist of two molecules of RNA, which are equivalent to mRNA. These range in size from ~7-11kb. Retrovirus genomes have four unique features:

1. They are the only viruses which are fully diploid.
2. They are the only RNA viruses whose genome is produced by cellular transcriptional machinery (without any participation by a virus-encoded polymerase).
3. They are the only viruses whose genome requires a specific cellular RNA (tRNA) for replication.
4. They are the only plus-sense RNA viruses whose genome does not serve directly as mRNA immediately after infection.

The gene order in all retroviruses is the same:

5′ – gag – pol – env – 3′

Some retroviruses have additional genes, such as the tax and rex genes in HTLV and tat and rev in HIV.

To initiate infection, the SU envelope glycoprotein binds to a specific receptor on the surface of the host target cell. The specificity of this interaction does much to determine the cell-tropism of different retroviruses, or even different isolates of the same virus (e.g. in HIV). Receptor binding results in conformational changes in the glycoprotein spike, revealing the (previously masked) fusion domain in the TM protein and resulting in fusion of the virus envelope with the cell membrane. Penetration and uncoating are poorly understood, but it is now known that uncoating is only partial, resulting eventually in a core (nucleocapsid) particle within the cytoplasm. Reverse transcription occurs inside the ordered structure of this core particle. During reverse transcription, the two single-stranded genome RNA molecules are converted into one double-stranded DNA version of the virus genome, which has the addition of long terminal repeats (LTRs).

The double-stranded DNA form of the virus genome is integrated into the chromatin of the host cell by the integrase (IN) enzyme, where it is known as a provirus. Promoter sequences in the upstream LTR direct expression of virus genes using host cell RNA polymerase. Alternative splicing is used to express virus genes gag, pol and env. In addition to encoding the gag proteins, the full length mRNA transcript also forms new genomes which are packaged into virus particles.

The genetics of retroviruses are complex:

• High mutation rate – reverse transcription is an error-prone process.
• Recombination – occurs during reverse transcription, promoted by the combination of two strands of RNA into one double-stranded DNA provirus.
• Interactions with the host cell – insertional mutagenesis, transduction.

In addition to infectious viruses, retrotransposons are endogenous retrovirus-like genetic elements which make up much of the human genome.

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So what have retroviruses ever done for us?
Apart from forming much of our genome: Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis. 2000 Nature 403: 785-9.

Retroviruses are responsible for a wide range of diseases:

• Paralysis
• Wasting
• Ataxia
• Arthritis
• Dementia
• Neuropathy
• Transformation
• Immunodeficiency

Retroviruses have been studied intensively for over 100 years as causes of disease and more recently as gene vectors.

Related:

• Latest News: Retroviruses

Joe DeRisi talks about amazing new ways to diagnose viruses (and treat the illnesses they cause) using DNA. His work may help us understand malaria, SARS, avian flu – and the 60 percent of everyday viral infections that go undiagnosed.