Fighting HIV-1, Marked for Death!

Based on a recent article published in January of this year in theProceedings of the Natural Academy of Science (http://www.pnas.org/content/110/11/4351.full.pdf), there may be advances in how we can mark target sites on HIV-1 virions for destruction. A main way that the body fights infection is by marking a cell for death, and then its recognition by the immune system that follows up with an antiviral response. This function of our bodies works well against most infections that we acquire through life, however HIV-1 is a true nuisance, why? Well HIV-1 belongs to a class of viruses called retroviruses. Retroviruses are different than many other viruses due to their genome being encoded with RNA. Reverse Transcriptase is essential for these viruses to produce DNA from their RNA genome, which then can be further translated to proteins. In the process of creating the DNA strand from the RNA template there is a lot of error that goes on. Because of this frequent coding error, there are usually many mutant types of protein that can either lead to increased virulence or cause no functional change at all¹. 

          In the case of cell targeting, there is usually a defined cell region that a known protein can bind to, its binding site. Once these sites are confirmed, steps can be taken to attach immunogen proteins to them, mimicking the same structure that would allow binding to the binding site. This works, however, when the defined binding region is known and readily accessible by the protein. The problem with HIV-1 is that due to its high error-prone reverse transcriptase, the coding for this binding site or the region of the virus envelope frequently changes^2,3. 

     This leads to the problem of accurately marking these sites and triggering them for destruction. Thus the researchers of this article have discovered an antibody protein that shows stable binding to the viral envelope, which can lead to engineering a vaccine, preventing viral infection.

SO WHATS THE PROTEIN AND WHAT MAKES IT SO GOOD AS A MARKER?

            Well the protein discussed is actually an antibody, and it is apart of a large family of proteins called broadly neutralizing antibodies, or bnABs. Recently these antibodies have been shown to bind to the nuclear envelope of the HIV-1 virus and effectively neutralizing it. Binding the envelope is very important since the glycoprotein found on the envelope is the mechanism by which the virus particle gains entry to the target cell. If the glycoprotein can be changed or constricted in any way (i.e. binding to it), then the virus particle is hampered from entering the cell and causing infection¹. 

        The problem is that these antibodies have mediocre binding potential due to the difficulty of binding to the conserved region on the viral envelope; with other proteins blocking the site as well as steric hindrance. However PG9, a unique bnAB, showed a stable and repetitive binding site conserved on the viral envelope. PG9 bound to the envelope in a way that was most curious, it bound with the trimers of the glycoprotein of the virus and formed a quaternary structure with the binding site. This type of binding leads to high stability, which was confirmed by the heat points recorded during the PG9-Envelope glycoprotein interaction¹. Using different viral viewing methods, such as electron microscopy and crystal structures, the researchers were able to identify the sites that PG9 binds to. The research showed that there was a high affinity in binding between PG9 and the HIV-1 envelope glycoprotein. The PG9 antibody binds to two out of the three monomers of the envelope glycoprotein, a trimer complex. This dual binding is important but other antibodies have this characteristic as well, so what is it about PG9 that makes it better? The researchers found that PG9 has a second interaction site beside the three monomers of the envelope glycoprotein, a monomer on an adjacent glycoprotein. This three monomer binding complex that PG9 forms with the envelope glycoprotein has shown to be thermally stable and readily able to neutralize virus due to its interactions at the monomers of the glycoprotein⁴. By blocking these monomers, the overall integrity of the trimers in the glycoprotein is compromised, possibly leading to its inability to enter the cell.

NOW WHAT?!

Drug Resistant HSV, Soon not to be a Problem!

HERPES!

Herpes is nothing new to society and has been a constant nuisance for many years. But a recent article published by the Proceedings of the National Academy of Sciences claims that they have engineered a mutant antibody that is able to effectively neutralize HSV and drug resistant HSV…in mice. Granted not the best news in the world but hey it’s a start!

Before that, a little bit more about the Herpes virus. The Herpes virus can be broken up into two types, HSV-1 which normally causes oral sores and HSV-2, which usually affects the genital region.  Herpes is an enveloped, double strand DNA virus, that is particularly good at causing long-term infection, or latent infections. Once it enters the body it usually persists for the remainder of ones lifetime, with the possibility of the infection becoming active from time to time. Studies show that usually a very minimal amount of people, with a functioning immune system, reactivate the virus, than those who are immunocomprised.

Various antiviral drugs have been developed and have been shown to decrease virus reactivity, such as Acyclovir. However, as attributed to many viruses, they become drug resistant. This drug resistance is particularly seen in individuals with compromised immune systems, and a better treatment for them is continually being researched. Thus we arrive at what the researchers did!

Although still in its early trials, researchers have engineered an antibody known as mAb hu2c. The antibody works by negating the mechanism through which HSV is able to spread from cell to cell. This is a huge step in defeating the virus, being that HSV is able to successfully avoid the immune system through the cell-to-cell spread mechanism. A quick summary of this mechanism: most viruses infect target cells by releasing virions that ultimately infect target cells. In order for this to occur the virus has to face the extracellular environment and in doing so triggers an immune response. Cell-to-cell spread is a mechanism in which a virus can infect other surrounding cells without ever having to face the extracellular environment, and thus can essentially be “hidden” from our immune system. HSV’s ability to cause infection from cell to cell is mediated by viral glycoproteins.

Interferon, Always Messing Stuff Up

Persistent viral infection such as those from HIV, Hepatitis B and Hepatitis C can lead to continuous pain and suffering in infected individuals. Persistent viruses are able to avoid clearance by down-regulating the immune system and suppressing the activation of antiviral CD4 and CD8 T cells. After persistent infection, a hyperimmune response is often observed characterized by elevated pro-inflammatory mediators and a constant interferon signature. Type 1 interferon (IFN-1) signaling is upstream of many immune responses such as activation of T cells, B cells and natural killer cells as well as inflammatory genes. Interferon is a protein that cells display during the presence of pathogens to communicate to other cells and initiate the innate immune system. Interferon signaling is necessary for host defense against viral infection and viral immunity during acute infections.(1) Because of this it is suspected that IFN-1 is responsible for the hyperimmune activity in persistent viral infection.

         To understand the role of IFN-1 in persistent viral infection the authors used the Armstrong (Arm) and clone 13 (Cl13) strains of lymphocytic choriomeningitis virus. The Arm strain is characterized by an acute infection, while the Cl13 strain is characterized by a systemic viral infection. Through the use of IFN-β-yellow fluorescent protein tagging in plasmacytoid dendritic cells (pDCs) it was determined that IFN-β was produced during Cl13 infection and not during Arm infection. Fluorescent protein tagging works by attaching an antibody to a cell surface protein receptor. Then an additional molecule that contains the fluorescent tag binds to the antibody attached to the protein of interest thereby allowing you to observe the expression of the target protein, in this case IFN-β .By replacing the glycoproteins of Arm and Cl13 viruses with green fluorescent protein, the authors confirmed that Cl13 infection preferentially targets pDCs. Green fluorescent protein is a protein that is found in jellyfish. We can insert the gene for the green fluorescent protein into the coding sequence of our target protein, then when the protein of interest gets transcribed the green fluorescent protein will be transcribed as well and we can measure target protein levels by measuring the level of green fluorescent protein. pDCs showed increased green fluorescent protein expression when infected with the Cl13 viruses marked with the protein compared to when infected with the Arm viruses marked with the green fluorescent protein.

Just Sitting Back, Biding Its Time. Waiting for the Moment to Pounce

     Human cytomegalovirus (HCMV) is a type of herpesvirus. HCMV infects the majority of individuals, but a healthy immune system is able to suppress a primary infection. Upon initial infection HCMV inhibits an antiviral response through the up-regulation of myeloid cell leukemia-1 protein (MCL-1). The induction of MCL-1 expression is dependent on the activation of the ERK-MAPK pathway rather than viral gene expression.(1) The ERK-MAPK pathway induces proteins such as mitogen-activated protein kinase and extracellular signal-regulated kinase. After the primary infection is controlled HCMV establishes a latent infection in undifferentiated bone marrow precursor cells and monocytes cells.(2) The activation of the ERK-MAPK pathway impacts long-term latency in progenitor cells by priming them from the initial virus encounter and enabling them for future reactivation.(1) When the individual with the latent infection becomes immunocompromised such as during transplants, however, the latent HCMV can become reactivated and have detrimental effects. During viral latency there is limited viral gene expression and no detectable virion production. One viral protein expressed during latency is latency unique natural antigen (LUNA) encoded by the latency associated transcript UL81-82ast. Previous studies have suggested that LUNA is involved in the regulation of HCMV reactivation by suppressing lytic transcription and without LUNA may not be able to enter into latency in order to be reactivated later.(2,3) Another viral protein present during latency is UL138. The UL133-138 is required for HCMV infection in endothelial cells.(4) The function of UL138 in productive and lytic infection is understood, but not much is known about the function of UL138 in latent infection.

     Using plasma membrane profiling the authors determined which plasma membrane protein levels were affected by the presence of UL138 during HCMV latency. Multidrug resistance-associated protein-1 (MRP1) was the most dramatically down-regulated of the three proteins affected by the presence of UL138. In fact, in the presence of UL138 MRP1 was undetectable in cells, suggesting that it might be getting degraded.  MRP1 is important in multidrug resistance and handling organic anions. It is found in bone marrow progenitor calls and monocytes, as well as mature leukocytes(5). In infected cells, the deletion of UL138 restored the expression of MRP1, indication that UL138 is necessary for MRP1 down-regulation. UL138 is also known to be present during lytic infection in addition to latent infection and high levels of UL138 48 hours after lysis coincides with decreased levels of MRP1. It was determined that MRP1 mRNA levels were not decreased from UL138 expression, so the down-regulation of MRP1 is probably post-transcriptional. MRP1 produces many cytotoxic agents including vincristine. When vincristine was added to HCMV-latent monocytes the number of latent cells decreased as well as the expression of UL138 RNA. This indicates that vincristine caused the death of latently infected cells. In addition to reducing the number of latently infected cells through killing, vincristine also reduced the number of cells in which reactivation of HCMV occurred after differentiation of monocytes to dendritic cells.

     What is not understood is why UL138 targets MRP1 for degradation in latent cells. One possible reason is that the reduction of MRP1 also reduced LTC4 which is a substrate of MRP1 and therefore inhibited migration of infected cells and impaired the activation of an immune response. The decreased expression of MRP1 could also inhibit the differentiation of precursor cells until the environment for reactivation was sufficient, therefore maintaining latent infection.

Knocking Out Breakbone Fever: New Mammalian Transmission Model for Dengue Virus

        

A recent study uses several varieties of knockout mice to develop a dengue fever transmission model. This approach could greatly contribute to knowledge of transmission between vertebrate hosts and the dengue virus vector, the Aedes mosquito. 

         Agonizing muscle and joint pains. Measles-like skin rashes. Sudden-onset high fever. Severe headaches. With symptoms such as these there is little wondering why dengue fever is so feared. Even more concerning is that cases of dengue fever have been spreading into new geographic regions; a result of increased population density, international travel, habitat development, environmental change, and a plethora of other factors. Additionally, with no approved vaccine for dengue virus currently on the market, it has become of utmost importance to gain a better knowledge of the entomological, virological and immunological components of infection establishment and transmission to prevent its spread. 

          Dengue fever and the more severe conditions it can cause –like dengue shock syndrome and dengue hemorrhagic fever - are a result of infection by dengue virus, a single positive-stranded RNA flavivirus. It is the most common arboviral (viruses that are transmitted by arthropods) infection of humans. The dengue virus vector - the organism that transmits the virus to another organism - is the mosquito Aedes aegypti, a species that originated in Africa. However, the range of this vector has dramatically spread over the past few decades and can now be found in most tropical and sub-tropical regions. The ability of this vector to move into new regions is one of the primary reasons that dengue fever epidemics are now regularly occurring in southeast Asia, India, the western Pacific and much of South America. 

         Even with dengue fever becoming a severe global threat, the study of the disease has been neglected. This is partially a result because of how difficult dengue virus is to study, as result of the lack of a good vertebrate transmission model. While it has been found that mice can get infected in lab settings, they don't get sick in any way which limits the usefulness of mouse models to the study of dengue virus replication. As a result, much research into dengue vaccines has been limited as few are willing to jump into clinical trials with a product that has yet to be animal tested. 

         However, a lab at the Louisiana State University in Baton Rouge might have found the solution to this problem. Using an array of genetic technologies it is possible to target and "knock out" specific mouse genes, allowing researchers to replace existing genes with altered versions. Using this technique, several varieties of knockout mice deficient in type I and II interferon receptors have been developed. By decreasing the response of interferons, the proteins that drive transcription to activate an antiviral response, researchers suspected that they could increase the permissiveness of cells to dengue fever and allow for increased virus transmission. The knock-out mice were inoculated with a non-mouse adapted dengue virus and tested for viral load and cytokine production daily. In addition, the mice were exposed to mosquitoes that were fed the same virus strain via an artificial membrane feeder. Finally, uninfected mosquitos were allowed to forage on dengue virus infected mice to determine if the mosquitos could contract the virus from the mammalian host.