Abstract
More Insights Gained into Gag Protein
Researchers at the National Institute of Standards and Technology (NIST) have been able to study the HIV protein known as Gag with more detail than ever before. Gag plays several critical roles in the assembly of HIV. However, persistent difficulties with imaging Gag in a laboratory setting have hampered fully understanding its function.
At different stages during HIV assembly, Gag twists itself into several different shapes inside a host cell. One conformation helps it to drag a piece of HIV genetic material toward the cell membrane, where the viral particles grow. Gag's opposite end becomes anchored there, stretching the protein into a rod-like conformation that eventually helps form a barrier surrounding the infectious genes in the finished virus.
The researchers developed an artificial cell membrane in which Gag can show off its functions for the neutron probes at the NCNR. As a result, new conformations were observed at various stages of HIV replication. The new research technique will also allow scientists to examine large classes of membrane proteins. More details about the research can be found in the October 20 issue of Biophysical Journal 2010;99:2516–2524.
Three-Dimensional Structure of CXCR4 Determined
Structural biologists from the Scripps Research Institute in La Jolla, California, have determined the three-dimensional structure of CXCR4, a molecule involved in HIV infection and in many forms of cancer. The high-resolution structure sheds light on how the molecule functions and could point to ways to control its activity, potentially locking out HIV and stalling cancer's spread.
CXCR4 is part of a large family of proteins called G-protein coupled receptors (GPCRs). These molecules span the cell's membrane and transmit signals from the external environment to the cell's interior. GPCRs help control practically every bodily process, including cell growth, hormone secretion and light perception. Nearly half of all drugs on the market target these receptors. CXCR4 acts as a coreceptor by helping HIV enter cells.
Normally, CXCR4 helps activate the immune system and stimulate cell movement. However, when the signals that activate the receptor aren't properly regulated, CXCR4 can spur the growth and spread of cancer cells. To date, CXCR4 has been linked to more than 20 types of cancer.
Researchers set out to shed light on how CXCR4 functions by capturing snapshots of the protein by using a structure determination method called x-ray crystallography. To understand how natural molecules might bind and signal through the receptor and to see how potential drugs could interact with it, they examined CXCR4 bound to known inhibitors of its activity. Determining the structure of CXCR4 represented a major challenge because membrane proteins are notoriously tricky to coax into the crystal form required for the x-ray technique. After 3 years of optimizing conditions for producing, stabilizing, and crystallizing the molecule, the researchers finally generated five distinct structures of CXCR4. The structures showed that CXCR4 molecules form closely linked pairs, confirming data from other experiments indicating that pairing plays a role in the proper functioning of the receptor. With this knowledge, researchers can delve into how the duos might regulate CXCR4's activity and better understand how CXCR4 functions under normal and disease conditions.
The images also showed that CXCR4 is shaped like two white wine glasses touching in a toast, with the inhibitors bound at the sides of the bowls. By detailing these contacts, the researchers said the pictures suggest how to design compounds that regulate CXCR4 activity or block HIV entry into cells. If developed into drugs, such compounds could offer new ways to treat HIV infection or cancer. More details about the study can be found in the October 7, 2010 online ahead of publication edition of Science.
Antiretroviral Therapy Does Not Reduce HIV Risk in Discordant Couples
For serodiscordant married couples, antiretroviral therapy does not reduce the risk of transmitting HIV to the uninfected partner. The new research from Chinese researchers raises questions as to the real-world effectiveness of the so-called test-and-treat strategy.
Researchers from the Chinese Center for Disease Control & Prevention in Beijing recruited 1927 married couples from the city of Zhumadian, Henan Province, China. The couples were initially serodiscordant. Zhumadian has a high rate of HIV disease related to infected blood products from paid plasma donors during the early 1990s. All infected persons have free access to medical monitoring and treatment, including antiretroviral therapies. The initially seronegative spouses were monitored regularly for seroconversion. Factors associated with a higher or lower risk of HIV transmission were analyzed.
The seroconversion rate was relatively low. Over a median follow-up of 3 years, 4.3% of spouses became HIV positive. However, the seroconversion rate increased over time. The risk of HIV transmission was higher for couples who reported more frequent sexual activity and especially for those who said they did not always use condoms. Risk was also higher for spouses who scored lower on a psychological questionnaire. The results suggest that education—particularly on the need for consistent condom use—may play an important role in preventing transmission from HIV-positive persons to their HIV-negative partners. Efforts to target psychosocial factors may also be helpful. The use of antiretroviral therapy did not lower the risk of HIV transmission to the initially HIV-negative spouse. Risk did appear lower for couples in which the HIV-positive spouse did not switch his or her antiretroviral therapy regimen during follow-up. In previous studies, antiretroviral therapy significantly lowered the rate of HIV transmission in serodiscordant couples, who generally received close medical follow-up. In contrast, the Chinese study shows no reduction in HIV transmission—even though all couples had access to health care services, including ART drugs. A formal research trial is being performed to see if antiretroviral therapy reduces HIV transmission over five years in a large group of serodiscordant couples.
The study was published in the October 2010 issue of Journal of Acquired Immune Deficiency Syndromes 2010;55:232–238.
New Insights Found Regarding Neutralizing Anti-HIV Antibodies
New discoveries about the immune defenses of rare HIV patients who produce antibodies that prevent infection suggest a novel direction for designing new vaccines. Researchers at Rockefeller University and colleagues have now made two fundamental discoveries about neutralizing anti-HIV antibodies, which effectively keep the virus at bay. By detailing the molecular workings of a proven immune response, the researchers hope their work will ultimately enable them to arm those who are not equipped with these benefits.
HIV strains mutate rapidly and evade the immune system. The HIV envelope spike, called gp160, is the site of a host of mutations that obstruct the few elements that all HIV strains share. Prior research has shown that only four super antibodies block the activity of gp160 in a broad range of HIV strains, neutralizing the virus. Earlier research has shown that a diverse group of broadly neutralizing antibodies, cloned from 433 B cells of six slow progressing HIV patients, was as capable of knocking down a broad range of HIV strains as any one of the super antibodies. The ability to isolate and clone antibodies from B cells was first worked out in 2003. Now, having applied that method to the B cells in HIV patients with high titers of broadly neutralizing antibodies, the researchers explore what their antibodies target and how they attack.
The researchers found that most antibodies are traditionally thought to bind to their target, or antigen, in a bivalent fashion. However, HIV virions do not allow for that possibility because the gp160 spikes are too far apart. HIV antibodies can only use one of their two high affinity arms to recognize the viral spike. The researchers found that, on average, 75% of the anti-gp160-HIV antibodies in their large collection were selected by the immune system for polyreactivity, a property that allowed the second free arm of the antibody to enhance overall affinity by binding to the virion nonspecifically. Generally, the immune system weeds out polyreactive antibodies, even though they are naturally produced in significant quantities, because polyreactive antibodies could in theory attack the body itself. Experiments suggest that these sticky antibodies may be an opportunistic adaptation to difficult cases such as HIV, in which homotypic bivalent bonding may not be an option. This particular cadre of polyreactive antibodies takes years to develop in slow progressing HIV patients. The researchers believe that a vaccine designed to elicit antibodies that mimic these properties could be a promising strategy against HIV.
The findings are reported in the September 30 issue of the journal Nature 2010;467:591–595.
Probe Detects HIV Protease and Toxicity of Drugs
A researcher at the University of Arkansas has developed a molecular probe that can simultaneously detect the presence of HIV-1 protease and toxicity levels of chemical compounds used against HIV. The probe can be used to investigate the efficacy and efficiency of HIV drugs.
The research resulted in the creation of a screening system by applying fluorescent proteins, one green and one red, to generate Förster resonance energy transfer signals responsive to HIV-1 protease inhibition and activity. Förster resonance energy transfer is a mechanism of energy transfer between a donor and receptor chromophore, which is the part of a molecule that is responsible for its color. The energy transfer prompts reactions from cells in the form of color. In the absence of protease inhibitors from various compounds, the researchers observed green and yellow cells. When protease inhibitors were added to cells, they showed a red color. It was found that nelfinavir and lopinavir exhibited the most toxicity, followed by indinavir, saquinavir, and ritonavir.
Currently, a cell line is being established that will consistently express the detection system. HeLa cells will be used, the first immortal line of cancer cells kept alive in cell culture. HeLa cells were originally taken from a tissue sample removed from the cervical cancer tumor of Henrietta Lacks, an African American woman at Johns Hopkins Hospital in Baltimore in 1951. Such cells grow rapidly in large quantities. In addition, targeted genes can be easily delivered to HeLa cells.