Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695
Performing Department
Microbiology
Non Technical Summary
Our laboratory is seeking to discover new treatments for influenza, also known as the flu; a respiratory disease caused by the influenza A virus. influenza is accompanied by a range of symptoms; from mild to moderate respiratory disease in healthy adults to severe, life-threatening infections in infants and the elderly. Indeed, over 30,000 Americans die annually from infections with influenza A. Vaccines, both inactivated and attenuated, can be used effectively to prevent influenza; however, many individuals are non-compliant and there are several segments of the population who do not derive full benefits from vaccination. infants under six months are not eligible for vaccination (they do not respond) and influenza infections in this group are often complicated by other infections, pneumonia, and hospitalization. the other major susceptible group is the elderly, who respond less well to the vaccine likely due to age-related decline in immune function. Two types of anti-viral drugs are also used to treat influenza; M2 pump inhibitors such as amantadine and rimantidine, and neuraminidase inhibitors including zanamivir and oseltamivir. Several problems are associated with the use of these drugs as well, including; viral resistance, side effects, and limited delivery options. Clearly there is a pressing need for the development of new methods to treat influenza A virus and inhibit the production of the inflammatory alkaloid berberine, which is found in high concentration in goldenseal extracts. Our goal is to define the molecular mechanisms underlying these effects. Understanding these mechanisms will determine, in part, whether berberine should be recommended for use in humans as a treatment of influenza
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The general objective of this research is to define the molecular mechanisms underlying the anti-influenza effects of berberine and berberine-containing extracts of the goldenseal plant. Results from these investigations will be key to determining whether these materials should be recommended for the treatment of influenza in humans. We have divided our objective into three specific aims. Aim 1. Determine the mechanism by which berberine inhibits the growth of influenza A. Our studies to date show that while berberine strongly inhibits the projection of infectious virus, it does not block production of viral RNA'a, including vRNA, cRNA, and viral mRNA. In sedition, the projection of viral proteins is not inhibited by berberine. We have found, however, that in the presence of berberine, the viral HA protein does not localize to its normal position on the plasma membrane. Instead, large "aggregates" of the protein are formed in the perinuclear region o the cell. We hypothesize that berberine is causing this and other viral protein to accumulate in the ER thereby blocking formation of the infectious virions. We anticipate that it will take 1 year to complete this objective. If incorrect, and the aggregates we have observed are not located in the ER, we would seek to identify the nature of the aggregates and their position in the cell. Aim2. Determine the mechanism by which berberine inhibits the influenza A-induced production of the TNF. Our studies have shown that berberine inhibits production of mRNA. Our studies will be focused on determining the mechanism underlying this inhibitory effect. The amount of viral ligands (vRNA and cRNA) are not altered by berberine suggesting that its inhibitory effect targets the signaling pathway between cellular TLR and NLR receptors and transcription factors. We anticipate that these studies will take 2-3 years to complete. Aim 3. Determine the mechanism by which berberine inhibits the influenza A-induced production of PGE2. Our preliminary studies suggest that berberine is exerting both upstream and downstream effects on this pathway. Our data has lead us to hypothesize that a portion of the inhibitory action of berberine stems for effect(s) on one or more on the MAP kinases know to cause cPLA2activation. Experiments focused on the downstream effect of the berberine have failed to reveal an effect on the expression of activity of COX-2 or mPGES-2, the two key enzymes in this pathway. However, for pathway activity, these enzymes must assemble in a complex where product from one is passed directly to the other. Our working hypothesis is therefore that berberine is interfering with complex formation thereby blocking production of PGE@. These studies will be initiated after the completion of Aim 2, and we anticipate that they will take 2-3 years to complete.
Project Methods
Aim 1. Determine the mechanism by which berberine inhibits the growth of influenza A. Immunofluorescence microscopy will be used to determine whether know ER markers colocalize with HA in berberine treated cells. In addition we will use cell fractionation to determine whether HA and other viral proteins are preferentially found in the ER fraction. Aim2. Determine the mechanism by which berberine inhibits the influenza A-induced production of TNF. Inhibitors and interfering RNAs will be used to determine which specific TLRs and MLRs are active in the TNF pathway. Markers on those pathways will then be examined for susceptibility to inhibition by berberine. Reporter and ChlP assays will also be used in these studies. Aim 3. Determine the mechanism by which berberine inhibits the influenza A-induced projection of PGE2. Phosphospcific antibodies will then be used in western blots to estimate kinase activation status. Ratio imaging florescence microscopy to quantify the effects of berberine on levels of intracellular calcium. Immunofluorescience, confocal microscopy, and HPLC will also be used in these studies.