MicrobiologyBytes

HIV is evolving rapidly to escape the human immune system. Researchers have shown HIV is able to adapt rapidly to counter human genes controlling immune system molecules that can target it for destruction. Progression to AIDS is tied to genes which control production of key immune system molecules called human leucocyte antigens (HLAs). Humans differ in the HLA genes they have, and even small differences can have a big impact on how quickly AIDS develops. Researchers found mutations that enabled HIV effectively to neutralise the effect of a particular HLA gene were more frequent in populations with a high prevalence of that specific gene.

BBC News

Adaptation of HIV-1 to human leukocyte antigen class I. Nature, 25 February 2009
The rapid and extensive spread of the human immunodeficiency virus (HIV) epidemic provides a rare opportunity to witness host–pathogen co-evolution involving humans. A focal point is the interaction between genes encoding human leukocyte antigen (HLA) and those encoding HIV proteins. HLA molecules present fragments (epitopes) of HIV proteins on the surface of infected cells to enable immune recognition and killing by CD8+ T cells; particular HLA molecules, such as HLA-B*57, HLA-B*27 and HLA-B*51, are more likely to mediate successful control of HIV infection1. Mutation within these epitopes can allow viral escape from CD8+ T-cell recognition. Here we analysed viral sequences and HLA alleles from >2,800 subjects, drawn from 9 distinct study cohorts spanning 5 continents. Initial analysis of the HLA-B*51-restricted epitope, TAFTIPSI (reverse transcriptase residues 128–135), showed a strong correlation between the frequency of the escape mutation I135X and HLA-B*51 prevalence in the 9 study cohorts (P = 0.0001). Extending these analyses to incorporate other well-defined CD8+ T-cell epitopes, including those restricted by HLA-B*57 and HLA-B*27, showed that the frequency of these epitope variants (n = 14) was consistently correlated with the prevalence of the restricting HLA allele in the different cohorts (together, P < 0.0001), demonstrating strong evidence of HIV adaptation to HLA at a population level. This process of viral adaptation may dismantle the well-established HLA associations with control of HIV infection that are linked to the availability of key epitopes, and highlights the challenge for a vaccine to keep pace with the changing immunological landscape presented by HIV.

Related:

• Why is HIV a pathogen?
• How does HIV cause AIDS?
• Immune exhaustion in HIV infection
• HIV-2 – a kinder AIDS virus?

Genome recombination is important for its role in unlinking deleterious mutations from those that may be neutral or beneficial, allowing populations at least partial escape from the negative fitness effects of accumulating deleterious mutations. Similarly, recombination allows otherwise asexual organisms such as viruses and bacteria to avoid the evolution-retarding effects of “clonal interference”, which results from competition among distinct beneficial mutations that reside concurrently in multiple genomes – eventually one dominates within the population at the expense of the rest.

In viruses, where genetic exchange between different species or even unrelated taxa is possible, recombination is also capable of generating spectacular genetic diversity. While natural recombination between distantly related genomes has only rarely been shown to occur in double-stranded DNA and RNA viruses, it is apparently quite common amongst most reverse-transcribing, positive-sense single-stranded RNA and single-stranded DNA viruses.

Maize streak virus (MSV), the type strain of the genus Mastrevirus in the family Geminiviridae, has a simple genome consisting of virion sense movement protein (mp) and coat protein (CP) (cp) genes, and a complementary sense replication-associated protein (rep) gene that is expressed in two alternatively spliced isoforms. Separating the complementary and virion sense genes are a long intergenic region (LIR), containing the v-ori and transcriptional promoter elements, and a short intergenic region (SIR), containing the complementary sense ori and transcription termination elements. A recent paper describes a conceptually simple but powerful new experimental system to demonstrate how recombination in geminiviruses such as MSV can be a remarkably efficient mechanism capable of rapidly generating progeny genomes with increased fitness.

Rapid host adaptation by extensive recombination. 2009 J Gen Virol 90: 734-746
Experimental investigations into virus recombination can provide valuable insights into the biochemical mechanisms and the evolutionary value of this fundamental biological process. Here, we describe an experimental scheme for studying recombination that should be applicable to any recombinogenic viruses amenable to the production of synthetic infectious genomes. Our approach is based on differences in fitness that generally exist between synthetic chimaeric genomes and the wild-type viruses from which they are constructed. In mixed infections of defective reciprocal chimaeras, selection strongly favours recombinant progeny genomes that recover a portion of wild-type fitness. Characterizing these evolved progeny viruses can highlight both important genetic fitness determinants and the contribution that recombination makes to the evolution of their natural relatives. Moreover, these experiments supply precise information about the frequency and distribution of recombination breakpoints, which can shed light on the mechanistic processes underlying recombination. We demonstrate the value of this approach using the small single-stranded DNA geminivirus, maize streak virus (MSV). Our results show that adaptive recombination in this virus is extremely efficient and can yield complex progeny genomes comprising up to 18 recombination breakpoints. The patterns of recombination that we observe strongly imply that the mechanistic processes underlying rolling circle replication are the prime determinants of recombination breakpoint distributions found in MSV genomes sampled from nature.

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• A feeling for the molechism
• Rolling down the road

Measles virus (MV) is one of the most contagious human pathogens. It is transmitted by aerosols, infecting a new host via the upper respiratory tract. Eventually, infection can spread to many organs of the body. The host cell receptors for MV are well defined: CD46, a member of the human complement regulatory proteins and a ubiquitous cellular receptor found on all nucleated cells, and CD150 or SLAM (signalling lymphocyte activation molecule), a membrane glycoprotein present on activated B cells, T cells and monocytes. It is generally believed that laboratory and vaccine strains of MV use both CD150 and CD46 as their cellular receptors, but wild-type MV strains mainly use CD150.

Oncolytic viruses have been selected or engineered to replicate in tumour cells. Approaches towards targeting cancer cells frequently exploit antigens that are unique to or are over expressed on the surface of tumour cells. As cell surface recognition and virus entry is the key first step for specific targeting, engineering oncolytic viruses in order to recognize exclusively the tumour cell-surface is important. Therefore, retargeting of oncolytic viruses a promising approach to exploit the potential of virotherapy.

In a recently published paper (Genetically engineered attenuated measles virus specifically infects and kills primary multiple myeloma cells. J Gen Virol. 2009 90: 693-701), researchers describe use of a mouse monoclonal antibody Wue-1, which is specific for B cells and their malignant counterparts, to retarget a CD46- and CD150-blinded recombinant MV towards Wue-1+ cells. This engineered virus with altered receptors specifically and efficiently infected primary multiple myeloma cells and induced apoptosis.

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Although virotherapy of hard-to-treat tumours with engineered viruses is a tantalizing prospect, this approach is still limited to laboratory studies at present. In order to develop a practical oncolytic treatments, successful tests in vitro using susceptible cells must lead to evaluation of the efficacy of tumour reduction in vivo in animal models with objective measurable parameters of safety and efficacy. Only then will we be ready to begin consideration of human trials of these potent new weapons in the fight against cancer.

Related:

• How Measles Virus Infects Cells
• Measles vaccination and SSPE
• Measles and Mumps in the USA
• Measles virus: cellular receptors, tropism and pathogenesis. J Gen Virol. 2006 87: 2767-2779

The pathogenesis of HIV begins with a profound depletion of CD4+ T cells in the gut followed by a long period of clinically silent but dynamic virus replication and diversification with high host cell turnover before the onset of AIDS. The AIDS-defining opportunistic infections and tumors mark the end-point of a long balancing act between virus and host that occurs when CD4+ T cell numbers fall below a level that can sustain immunity. Comparative studies of lentivirus infections in other species show that AIDS is not an inevitable outcome of infection because simian immunodeficiency virus in natural hosts seldom causes disease. What distinguishes pathogenic from ‘passenger’ infection is a systemic activation of immune responses followed by destruction of the integrity of lymphoid follicles. Macrophage and dendritic cell infection also contribute to pathogenesis. Maedi-Visna virus infection in sheep, which targets these cells but not T lymphocytes, also leads to progressive disease and death that resembles the wasting and brain diseases of HIV without the T cell immunodeficiency. Thus, lessons from pathogenic and nonpathogenic lentivirus infections provide insight into the complex syndrome called AIDS.

Why is HIV a pathogen? Trends Microbiol. 2008 16(12):555-60

Related:

• How does HIV cause AIDS?
• Immune exhaustion in HIV infection
• HIV “can never be cured”
• HIV-2 – a kinder AIDS virus?

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?

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.

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• MicrobiologyBytes: Dengue Virus
• Dengue Virus
• Clustering of dengue virus infections
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• Dissecting the Cell Entry Pathway of Dengue Virus

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