New Possible HIV Treatment reduces HIV-1 Infectivity with the use of GANP to mediate APOBEC3G Packaging in HIV-1 Virions

The road to effective HIV vaccines is littered with many hurdles and blockades that cause vaccines to be ineffective. One of the main reasons why HIV leads to many ineffective drug vaccines is because of its ability to mutate rapidly. The rapid mutation of HIV allows the virus to avoid potential vaccines such as antibody vaccines that could neutralize the virus or help facilitate the death of the virus. As a result of the rapid mutation, many current scientists are looking into new ways to fight off the virus. A couple of the new methods in stopping the virus include targeting specific proteins that make up HIV that allow it to mutate and infect immune cells. Scientists are also looking into methods that enhance the host antiviral proteins found in the immune cells. In a new study from Kumamoto University, researchers have discovered a new method in reducing the infectivity of HIV-1 (the usual HIV strain found in humans). What the researchers discovered could lead to a potentially new development of vaccines in fighting HIV. The scientists discovered the use of a recent protein called GANP (germinal center-associated nuclear protein) that enhances the facilitation of the host antiretroviral protein called A3G (its function causes hypermutations in newly synthesized viral DNA) into the viral capsid in the absence of the HIV accessory protein Vif.

The function of HIV’s accessory protein Vif is to promote the synthesis of proviral DNA by inhibiting a host antiviral protein. The host antiviral protein that Vif inhibits is APOBEC3G where Vif degrades the protein by facilitating the APOBEC3G to a proteasome. In the absence of HIV, APOBEC3G normally functions as an antiviral cytidine deaminase enzyme that converts a cytidine to a uracil (C to U) in viral RNA.

In trying to get a visual understanding of the importance of APOBEC3G, here is a video that will demonstrate the general function of APOBEC3G on HIV:

In an effort to take advantage of APOBEC3G’s ability against viral RNA, the researchers tried to experiment and explore the combination of A3G (a protein of the family of APOBEC proteins that has the same ability) with GANP since GANP has shown in a previous study that it enhances the use of one of the other APOBEC proteins called AID. GANP is a protein that is upregulated when CD4⁺ T cells are activated so the researchers want to see how GANP interacts with A3G in the HIV virion during an HIV infection.  

New Mechanism Found That Adds Another Vector For Anti-Retroviral Therapy!

HIV-1 is one of the most well known viruses in the world. The difficulty in combating infection is the rather prevalent mutagenic nature of the virus, which in turn means that anti-retrovirals are quickly rendered inactive after these mutations occur. In addition to this mutagenic nature, HIV-1 has a wide array of accessory proteins that further aids in successful infection. In a recent study by the Journal of Biological Chemistry, researchers focused on one of these accessory HIV-1 proteins, the curious viral protein, Viral Infectivity Factor (Vif). Vif itself is a rather small 23- kilodalton protein that plays a pivotal role in HIV replication. Vif’s main function is to counteract the antiviral activity of APOBEC3G by tagging it via an E3 ubiquitin ligase and targeting it for proteasomal degradation (Wang 2013). APOBEC3G belongs to a superfamily of APOBEC proteins, which are known to play a large role in innate anti-viral immunity (Harris 2003). APOBEC3G is a certain protein of this family that is responsible for causing hypermutations in the progeny viral DNA. It is a single stranded deoxycytidine deaminase, that, when Vif is not present, incorporates itself into the budding virion through binding of the (+) viral RNA strand. APOBEC3G functions by catalyzing the deamination of cytosine to uracil, thus a deaminase enzyme. Subsequently, this causes reverse transcriptase to transcribe the newly converted uracil as adenine in the viral RNA transcript. This mutation is sufficiently able to mutate the pro viral DNA, and inactivate it (Romani 2009).

Interaction of APOBEC3G/Vif

            The paper focuses on the processivity of APOBEC3G, and how it is affected when in the presence of Vif. Vif was primarily seen to inhibit APOBEC3G by targeting it through proteasomal degradation, as well as down-regulating APOBEC3G mRNA translation, so the ability for it to inhibit its function by another mechanism provides another pathway for anti-retroviral therapy. Past research has shown that APOBEC3G scans its substrate by both sliding and microscopic jumping around the ssDNA, providing a thorough 3D scan that increases its efficiency in locating its deamination target (Chelico 2006). In this paper, the researchers investigate the novel molecular mechanism of degradation-independent Vif mediated inhibition of APOBEC3G. Two Vif variants were used; Vif_IIIB and Vif_HXB2, both are seen to inhibit APOBEC3G deaminase activity, and in this experiment both provide a novel way in which Vif inhibits deaminase activity by successfully altering the scanning mechanism employed by APOBEC3G.

            In the first experiment, the researchers constructed a model HIV-1 replication system to measure any change in APOBEC3G mutagenesis in the presence of the Vif variants. First they performed an assay with only APOBEC3G and saw that in comparison with just reverse transcriptase, APOBEC3G caused a 17-fold increase in population mutation frequency, and a 11-fold increase in the clone mutation frequency. Having a baseline of the amount of mutations that APOBEC3G induces, the researchers then performed two additional assays, this time one of each Vif variant was introduced alongside APOBEC3G. From here they plotted the percentage of clones having a mutation in comparison with their location on the transcript. The histogram presented really makes it easy to understand the data. The histogram, Figure 1, shows that without Vif, APOBEC3G usually has a high range of induced mutations, 30% of recovered clones had 12 mutations or greater per transcript, as well as about 25% of clones showing 6-8 mutations per transcript. In the presence of Vif_HXB2, 55% of clones showed 3-5 mutations per transcript, while there was no more clones with 12 mutations or greater. The Vif_IIIB variant also showed reduction of clone mutations, to a lesser effect, by a shift down to 3-8 mutations per transcript. Together the data shows that both Vif variants are able to inhibit APOBEC3G induced mutagenesis, and their potential for being targeted for therapy.

Figure 1. Population Distribution of Mutations

"Ready or Not, Here I Come!" How Shigella flexneri Hides in our Bodies

            Dysentery is a GI disease with which not many in the industrialized world are familiar, but which posed a severe problem only a hundred years ago and still causes serious illness and death in the developing world. The bacterium that causes dysentery is called Shigella flexneri, which infects the mucosal surfaces of the colon and rectum. Shigella is a gram-negative, intracellular bacterium. This means that it has a cell membrane but no cell wall, and that when it infects a host it establishes itself and replicates inside the cell rather than in the spaces and fluid between cells. Intracellular bacteria can often be more difficult for the immune system to find, especially if they have ways to make a cell look healthy instead of showing the danger signs of infection.

            The innate immune system, or first responder to any infection of your body by a foreign pathogen, is not specific to particular pathogens and relies generally on compounds that are part of bacteria, viruses, or fungi, but are not present in humans¹. A distinguishing characteristic of gram-negative bacteria like Shigella is a compound called lipopolysaccharide, or LPS. LPS is a component of the bacterial cell membrane, and is highly toxic when separated from the membrane². LPS is strongly recognized by the innate immune system as a danger signal, also known as a pathogen-associated molecular pattern (PAMP)¹. LPS is made of a lipid called Lipid A, an oligosaccharide (a type of sugar polymer), and the O-polysaccharide, another sugar located on the bacterial surface³. Immune cells recognize the Lipid A part of bacterial LPS, and this causes the release of certain small molecules called cytokines. The specific cytokines released, particularly IL-1ß , TNF-alpha, IL-18, cause inflammation, which helps alert the rest of the immune system to start combating the infection, but also causes many of the symptoms associated with bacillary dysentery³.

1. Diagram of bacterial LPS

Pathogen Mode of Entry Has a Significant Effect On Host Adaptation

In order for us to properly understand the diseases that make us sick we must examine the entire mechanism the pathogen (disease causing agent) uses to invade our bodies. Much experimental work has been done to understand the pathogen mechanism once it has generated an immune response in the body, however one of the most important steps in the mechanism, which hasn’t really been experimentally studied, is the mode of entry. There are two major ways that a pathogen can enter the body, either through ingestion (orally) or systematically (a prick or needle). When a pathogen enters the body systematically it bypasses much of the innate physiological and anatomical barriers that prevent pathogen entry, like our skin, sweat, oils and mucus we secrete. In contrast, a pathogen must overcome many more barriers when entering the body orally, for instance saliva.

It has been shown in a recent experiment that the path of pathogen entry affects the rate at which the pathogen is eliminated and the way that it responds to the same type of pathogen. In the experiment equal amounts of both male and female Drosophila melanogaster flies were administered with one of two treatments of P. entomophila bacterial strain. Half the male and female flies were introduced to P. entomophila orally (BactOral), whereby food plates were coated in the bacterial strain. The other half of male and female flies was injected in the thoracic (area where the legs and wings attach) region with P. entomophilia (BactSys). Controls for both the injection (ContSys) and food treatments (ContOral) were also observed in the experiment. The mortality of the flies was measured for at least 10 days. It was found that the survival rate of the flies was much higher when the bacterial strain was introduced orally. The evolution of resistance to the particular bacterial strain also developed much faster when P. entomophilia was introduced orally.

These results reaffirm the theoretical notion that oral pathogens have to overcome more physical barriers in order for them to enter the body cavity. This is in contrast to a systematic infection because the physical barriers, like the skin have already been bypassed and the bacteria have been directly injected into the body cavity. So when a pathogen enters systematically the body must rely on other, more active ways of identifying and eliminating the invader, namely through members of the immune system that belong to the adaptive (more-specific) response like plasmatocytes. These more complex and specific ways of eliminating the invader are slower to act because B cells must be activated to produce antibodies, that than must find the pathogen and kill it, whereas much of the pathogen that was introduced to the mice orally didn’t really require the activation of the adaptive immune response. It only had to rely on the innate (more general) immune response and physiologic barriers that were already in place like the mucosa layers of the respiratory and gastrointestinal tracts to prevent pathogen entry into the body cavity. Since these defenses are already localized to the site of pathogen entry and need no further activation, the immune response is much faster when the pathogen is administered orally rather than systematically.

Friend or Foe? The Recognition of Microbiota vs. Pathogens

           The basic function of the human immune system is to detect and combat pathogens and harmful foreign bodies that we encounter on a daily basis. Bacteria, viruses, and fungi contain some very different proteins and nucleic acids than humans do, and it is job of the body’s immune cells to recognize when one of those components has penetrated our physical barriers, such as skin and the mucus in our digestive and urogenital tracts. Some things that trigger the immune system are peptidoglycan (a cell wall component of bacteria that is not present in humans), lipopolysaccharide (a bacterial cell membrane component also not present in humans), and double-stranded RNA (which encodes the genetic information of many viruses) (Mak & Saunders, 2010).

            The basic forms of an immune system are known as the innate immune system. Almost all forms of multicellular life exhibit some properties of an innate immune system, including microscopic organisms such as nematodes. Humans also have an innate branch of their immune system. The job of the innate immune system is to recognize the basic, general components of foreign bodies attacking the cell, such as those described previously. It seems simple, right? If it’s a human protein, leave it alone, and if it isn’t, get rid of it. Research within the past few decades, however, has revealed a gaping hole in this basic principle.

            It has been discovered relatively recently that microbes (aka foreign bodies) are absolutely vital to human life. There are thousands of types of microbes in the different tissues of our bodies, especially our gut, that help us with things like digestion and nutrient absorption (Slonczewski & Foster, 2010). It has been estimated that the number of microbial cells in our bodies is higher than the number of human cells! This estimation makes the hole in immune theory very obvious: if there are so many microbial cells colonizing our bodies, how do our immune cells know what to attack and what to leave alone? In their review article, Hiutung Chu and Sarkis K Mazmanian attempt to provide insight into this problem and perhaps fill in a few pieces of this gap in their 2013 paper “Innate Immune Recognition of the Microbiota Promotes Host-Microbical Symbiosis”.

            The basic argument of this article is that part of the innate immune system, a set of receptors called Pattern Recognition Receptors (PRR’s) evolved specifically to facilitate communication between symbiotic organisms and human cells. PRR’s have thus far been categorized as cell membrane receptors that recognize general features of many different pathogens (Pathogen-Associated Molecular Patterns, or PAMPs). Innate immune cells such as dendritic cells, which engulf and break down foreign bodies, express many PRR’s on their cell membranes. The authors suggest that the evolutionary purpose of PRR’s is not primarily to detect pathogenic cell components and spur phagocytosis of these foreign bodies, but to communicate with symbiotic bacteria and prevent activation of the immune response.

1.A pathogenic component (PAMP) binding to a PRR on aphagocytic immune cell.

         

Tiny Pests Causing Huge Problems

Why do people put on bug spray when they go outside?  The obvious answer is that having itchy mosquito bites all over your arms and legs is one of the most irritating feelings ever.  However, most people forget that these tiny insects can carry extremely dangerous viruses that can cause severe illness or even death.  West Nile virus (WNV), a well-known mosquito-borne virus, is the major cause of encephalitis (inflammation of the brain), which is a disease characterized by flu-like symptoms, and in severe cases, seizures and sensory/movement issues.  Dengue virus (DENV) is another dangerous mosquito-borne virus that is a major cause of viscerotropic disease, which is a disease that is associated with yellow fever and ultimately leads to organ failure and death.  These viruses cause significant problems all over the world, and currently there are no approved vaccines to prevent or combat WNV and DENV.  In order to develop effective vaccines, we need to understand how our bodies’ immune system recognizes the viruses and provide protection.  “Innate Immune Sensing of Flaviviruses” is a recently published paper in PLoS Pathogens, in which Suthar et al. (2013) discusses recent research findings on the way that the innate immune system interacts with WNV and DENV to trigger an antiviral response.¹ Also, understanding how these viruses can evade our immune response is equally important for developing new treatments.  A group mentioned in “Innate Immune Sensing of Flaviviruses,” Aguirre et al. (2012)², investigate one of the main mechanisms that DENV uses to inhibit inducing an immune response.

           

WNV and DENV are part of the flavivirus family, and they contain a single stranded RNA genome.  In order to recognize these viruses, we encode receptor proteins called pattern recognition receptors (PRRs), which recognize and bind to structures on pathogens that are not present in normal, human cells.  These structures are called pathogen-associated molecular patterns (PAMPs).  Once the virus is recognized, antiviral immune defenses are initiated which leads to inflammation.

  

The two PRRs that recognize the viral, non-self RNA structures are called retinoic-acid inducible gene-1 (RIG-1)-like receptors (RLRs) and myeloma differentiation factor 5 (MDA5).  RIG-1 recognizes and binds to double stranded RNA with a triphosphate group on the 5’ end.  MDA5 interacts with long double stranded RNA.  Double stranded RNA does not exist in normal, host cells, and it is a common viral PAMP.  Once the virus is recognized, RIG-1 and MDA5 induce the production of type 1 interferon (IFN) and proinflammatory cytokines that will migrate to the site of infection and recruit/help produce other factors needed to eradicate the virus.  A study done by Fredericksen et al. (2008) demonstrated that RIG-1 and MDA5 function cooperatively in creating an antiviral response to WNV.³ They found that RIG-1 is activated first by WNV, and then MDA5 is activated to sustain type 1 IFN and ISG expression (initiated by RIG-1).³   It is known that the PRRs RIG-1 and MDA5 are involved in pathogen recognition and initiate the immune response, but it is still unclear what the exact PAMP RNA substrate and nucleic acid sequence that RIG-1 and MDA5 interacts with. 

Like a finely fitted suit: IKKε tailors the antiviral response.

Every pathogen is different, so every immune response must be different. There is no “one size fits all” garment in immunology! This tailoring can occur at multiple levels. One systemic response is the development of “Th1” versus “Th2” CD4 T cells, which facilitate immune responses against (generally) intracellular and extracellular pathogens, respectively. On a more localized scale, different cell types, such as cardiac fibroblasts and myocytes, express different basal levels of immune components to dictate how the immune response behaves in particular tissues, such as the heart (1). Recent research by Sze-Ling Ng and colleagues has highlighted another level of tailoring, at the level of how individual cells coordinate their gene expression in response to distinct antiviral cytokines.

            Interferons are central components of the antiviral response. There are several types of these secreted proteins, including type I interferons (IFN-I), which help cells inhibit virus replication, and type II interferons (IFN-II), which are pro-inflammatory: they recruit immune cells to the site of infection. Both types act in a similar manner, by binding to receptors on the cell surface and inducing signals that culminate in a change in gene expression in the cell. Although IFN-I and IFN-II bind to different receptors, they share components of their signal transduction mechanism, including the transcription factor STAT1. Following IFN-I signaling, STAT1 becomes active and binds with two other proteins, STAT2 and IRF-9. Together, this complex binds to specific DNA sequences to mediate transcription of genes (interferon-stimulated genes, or ISGs) that mediate the antiviral state. Following IFN-II signaling, STAT1 also becomes active, but binds to itself to form a STAT1:STAT1 dimer. The STAT1 homodimer binds to DNA to mediate transcription of a distinct, but partially overlapping, set of genes that lead to the inflammatory response. A central question has been how cells balance these two responses, especially since both IFN-I and IFN-II are likely to be produced upon pathogen infection. Ng et al. now demonstrate that a cellular kinase, IKKε, phosphorylates STAT1, to alter its transcriptional response to IFNs.

            IKKε is best known for its role in inducing production of IFN-I in response to viral infection. However, genetic studies in mice have shown that a highly related kinase, TBK-1, is sufficient to induce IFN-I, raising the question of how IKKε contributes to the interferon response. One possibility is that IKKε is only required in a subset of specialized cells. Alternatively, because IKKε is inducible whereas TBK-1 is constitutively present, others speculated that IKKε functioned as a positive feedback mechanism. It was previously shown that IKK<ε could phosphorylate STAT1 on a serine at position 708 (2), and that this modification was necessary for an efficient immune response. Ng et al now show that this phosphorylation alters the capacity of STAT1 to form homodimers, leading to increased formation of the STAT1:STAT2:IRF-9 complex, and altering the balance of IFN-I vs. IFN-II transcription in cells.

Hey Virus: Replicate Here!

The innate immune system, that part of the immune system that responds to non-specific signs of pathogen infection, faces a quandary when mounting a response against a pathogen. On one hand, it must limit the replication of the pathogen to prevent spread throughout the body. On the other, it must facilitate the development of a specific adaptive immune response, which can clear the pathogen from the body and lead to immunologic memory. It might seem like both of these are reasonable tasks, but the quandary lies in the fact that the adaptive immune system must be activated by a certain amount of antigen (proteins derived from the pathogen that can be effectively targeted by the immune system) in order to elicit an effective response. Low levels of antigen lead to weak responses, whereas high amounts of antigen induce strong responses. Thus, if the innate immune system does the first part of its job too well and largely prevents virus replication, there will not be enough viral antigen present to stimulate a robust adaptive response. So how does the immune system get around this predicament? A new paper in Nature Immunology by Nadine Honke and her colleagues sheds some light on this question (1).

            The major weapons in the innate immune system’s arsenal are small, secreted proteins called interferons. When cells in the body detect a pathogen, they produce interferons, which alert the surrounding cells to the impending danger. These “warning flags” bind to interferon receptors found on the surface of all cells, and induce the expression of a large number of genes, termed interferon stimulated genes (ISGs), which can combat the pathogen through various means. Interferons also initiate mechanisms that facilitate the adaptive immune response, by increasing the presentation of specific antigens to the T cells and B cells that comprise the adaptive immune system. To effectively stimulate T cells, however, requires a sufficient dose of antigen: prior studies have indicated that over 20,000 copies of the antigen are required for an antigen-presenting cell to induce a robust T cell response (2). So, how can the innate immune system effectively prevent the spread of a pathogen while still providing enough “grist” to be presented to naïve T cells? Honke et al began to answer this by examining a particular subset of innate immune cells, metallophilic macrophages.

IRF-1 is Essential for Immunity Against West Nile Virus Infection in Mice

The West Nile Virus (WNV) is spread via mosquitoes and has been affecting humans in the United States since 1999 (1). Current research by immunologist further dissects antiviral immunity, in hopes of understanding WNV infectivity and how it can be combated or prevented. Previous research in mice has identified several immune mechanisms of control against WNV including cytokines, chemokines, complement, B CD4+ and CD8+ T cells (reviewed in [1]). These are all various aspects of the innate and adaptive immune system that aid in fighting against pathogens. More specifically, type 1 IFN (IFN-αβ) has been given special attention, as mice deficient in it, rapidly succumb to WNV infection (2). Further, it was determined that IFN induction is dependent on certain transcriptional signals. Past research pointed Brien et al. (2011) in the direction of IRF-1, as it has been reported to contribute to IFN-β induction. For example, it was found that IRF-1 transcription factor activated IFN-β gene transcription and regulated genes that directly impeded replication of several viruses. Further roles of IRF-1 have been found in controlling herpes virus, even though mice lacking IRF-1 did not have defects in their type I IFN response. This would suggest that IRF-1 regulates IFN genes “in a pathogen and cell type-dependent manner” (1). Further research has also identified IRF-1 as a tumor suppressing gene and a regulator of the adaptive immune response (3).
To understand the role of IRF-1, Brien et al. (2011) assessed WNF infectivity in IRF-1 normal and IRF-1 deficient mice (1). As previously mentioned, they chose to manipulate IRF-1 because mice lacking it (IRF-1-/- ) were vulnerable to WNV infection. First, it was confirmed that IRF-1 is required for control of lethal WNV infection. After infection with WNV, Wild-type (WT) mice had a 65% survival rate and a mean time to death of 11 days, whereas IRF-1-/- mice had a 0% survival rate and a mean time to death of 9.5 days.
To better understand how IRF-1 deficiency is a disadvantage for mice with WNV infection, the ‘viral burden’ was measured at various points post infection in serum, several peripheral organs, and the central nervous system (CNS). Indeed, increased levels of viral RNA were found in the serum and lymph nodes in IRF-1-/- mice compared to the WT. Therefore, IRF-1 controls the early stages of WNV infection. Additionally, WNV infected the spleen more rapidly and clearance was delayed in IRF-1-/- mice. WNV was also detected sooner in the kidneys of IRF-1-/- mice, further suggesting that IRF-1 normally functions to control infection in peripheral tissues.

Toll-like Receptor 7 is the Retrovirus Sensing Receptor in Mice

Retroviruses tend to be of great interest to the general public, because of all the media attention given to widely problematic ones, such as the Human Immunodeficiency Virus (HIV). Before effective vaccines against retroviruses like HIV can be produced, a basic understanding of how the immune system responds to them is imperative. When a retrovirus enters the human body, many are known to overcome the immune system, and possibly inhabit the host long-term. However, some animals have developed very efficient immune responses, which allow them to essentially evade or kill the virus. Scientists can study animals with this ability, like mice, to better understand the specificities of their immune systems that facilitate its advantage over the retrovirus, in hopes of relating their findings back to humans.

The first line of defense in the immune response is the innate immune system. The innate immune system is often thought of as “natural,” and provides non-specific protection against foreign invaders (1). Innate defenses are found all over the body, and include physical barriers like skin and mucus. Additionally, the innate immune system responds to pathogens in a generic way and has multiple mechanisms for killing and removing invaders. Most times, the innate defenses are successful. However, if the innate immune system is unsuccessful, it is responsible for triggering the secondary reaction of the adaptive immune system. The adaptive immune response is a specific response to a particular pathogen (foreign invader) and is usually only triggered by those entities that successfully overcome the innate immune response (1). Previous research has shown that in some strains of mice, the innate immune system senses the virus and triggers the appropriate adaptive immune response specific to the virus (2). So although the adaptive immune response is responsible for controlling retroviruses in mice, the first, critical innate virus-sensing step is still not confirmed.

Viruses are unique because they are recognized by endocytic pattern recognition receptors (PRR) on host leukocytes (white blood cells) that detect replication intermediates (nucleic acids) produced by the virus (2). However, it is unknown whether these viral sensors play a vital role in virus sensing in vivo, which is why animal models are very helpful. A recent paper by Kane et al. (2011) is therefore broadly asking whether retroviral replication intermediates are recognized by the innate immune system and further, if they are critical components for the adaptive immune response.