Figure 2. Morphology (left) and genome structure (right)
of influenza virus. The eight RNA segments from
influenza virus are indicated.
Influenza viruses are globally important human pathogens that affect up to 500 million people annually (Figure 2).
During the effective replication cycle of the virus in its hosts, Influenza non-structural protein 1 (NS1) has been demonstrated to perform several important functions. One of the best-characterized functions is the ability of Influenza NS1 to antagonize Type I IFN production, preventing an antiviral state in the host. Dr. Martinez-Sobrido's previous work has shown that Influenza NS1 exerts its inhibitory properties at least in part by its binding to double-stranded RNA (dsRNA), resulting in the sequestration of the retinoic acid-inducible gene I (RIG-I), a cellular sensor of RNA virus infection, therefore inhibiting IRF3 activation. In addition to blocking the activation of IRF3, Influenza NS1 has also been postulated to affect post-transcriptional processing of cellular mRNAs.
He has compared the abilities of NS1 gene products from different human Influenza strains to counteract the antiviral host response and found that Influenza NS1 proteins use different strategies to overcome the IFN-induced antiviral state. Dr. Martinez-Sobrido is focusing his research, now, in studying other aspects required for the anti-IFN function of NS1. To that end, he is using reverse genetics techniques (Figure 3) to rescue recombinant viruses carrying mutations/modifications in the NS1 and study its function.
Figure 3. Influenza virus reverse genetics techniques.
Eight-plasmid-based influenza reverse genetics
techniques influenza viral RNAs cloned into a bidirectional
plasmid vector which contains a polymerase I transcription
unit to encode viral RNA and a polymerase II transcription
unit to encode viral proteins are transfected into cells. Tissue
culture supernatants containing recombinant viruses are used
to study several aspects in the biology of influenza virus.
Generation of Recombinant Influenza Virus From Plasmid DNA
from URMC Microbiology and Immunology on Vimeo.
Figure 4. Generation of an HA-deficient GFP-expressing
influenza virus. A 293T/WSN-HA MDCK co-culture is transfected
with the plasmids required for influenza virus rescue where the
HA gene is substituted with one encoding the green fluorescent
protein (GFP) Tissue culture supernantants are passaged in fresh
HA-expressing MDCK cells and virus replication is detected by
Additionally, he has recently described the rescue of a HA-deficient GFP-expressing influenza virus by using plasmid-based reverse genetics techniques (Figure 4) to establish a simple, sensitive, specific, and safe screening assay for the rapid detection of neutralizing antibodies against influenza virus (including highly pathogenic influenza viruses) under biosafety level 2 (BSL-2) conditions.
« back to all projects