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Martinez-Sobrido Laboratory

Luis Martinez-Sobrido (right), 2007 Milstein Young Investigator Award recipient, during the International Society for Interferon and Cytokine Research (ISICR) meeting at Oxford, UK (September 2007) celebrating the 50th anniversary of the discovery of interferon by Alick Isaacs and Jean Lindenmann (left).

Research Focus

Evasion of the innate immune response by viruses

Interferon

Figure 1. Virus against host. IFN

induction, signaling and action

pathway (left) and viral gene

products from different virus

families that have been

described to inhibit IRF-3 (right).

Originally discovered by the British virologist Alick Isaacks and the Swiss researcher Jean Lindenmann (Picture, above) , at the National Institute for Medical Research in London, 1957, Interferon (IFN) plays an important key role in the host antiviral innate immune response (Figure 1, The IFN pathway). For that reason, viruses have developed a plethora of strategies to disrupt the IFN mediated antiviral defense of the host, and viral gene products responsible for these disruptions are often major virulence determinants (Figure 1, Virus IFN-antagonists).

Influenza Virus

Influenza viruses are globally important human pathogens that affect up to 500 million people annually (Figure 2).

Figure 2. Morphology (left)

and genome structure (right)

of influenza virus. The eight

RNA segments from influenza

virus are indicated.

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.

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.

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 GFP expression.

Arenavirus

Arenaviruses merit significant interest as clinically important human pathogens, including several causative agents of hemorrhagic fever disease (Figure 5).

Figure 5. Morphology (A), Genome

structure (B) andreplication (C) of

A renaviruses.

Using Lymphocytic Choriomeningitis Virus (LCMV), the prototype member in the family, Dr. Martinez-Sobrido have found that the viral nucleoprotein (NP) inhibits the IFN response in infected cells by interfering with the activation of the Interferon Regulatory Factor-3 (Figure 6, Inhibition of IRF-3). This was the first nucleoprotein described to block production of Type I interferon during viral infection as well as the first member of the Arenaviridae family described to counteract the Type I IFN response. Additionally, he found that the nucleoprotein encoded by other members in the family (with the exception of Tacaribe virus) is able to counteract the IFN response. By creating several LCMV-NP mutants, Dr. Martinez-Sobrido have been able to undercover the LCMV-NP domain involve in inhibition of type I IFN, including amino acids at positions 382-386 (Figure 6, NP functional domains).

Figure 6. Inhibition of IRF-3 by influenza virus NS1 and arenavirus NPs (left): HA-tagged influenza NS1 and NPs from the indicated members of the Arenaviridae family (red) were tested for inhibiting nuclear translocation of a GFP-tagged IRF-3 (green), after Sendai Virus infection. NP functional domains (right): A domain in the C-terminal region of LCMV-NP (residues 370-553) was identified as critical for the anti-IFN function of the viral protein. Amino acid residues at positions 382, 385, and 386 on LCMV-NP were identified as critical for counteracting the IFN response. All primary structure of the viral protein, with the exception of the last 5 amino acid residues is required for the replication and transcription activity of the viral polypeptide.

He is currently using reverse genetics techniques (Figure 7) to rescue recombinant viruses carrying mutations on NP that affect its ability to counteract the IFN response and determine its contribution in viral pathogenesis.

Figure 7. Arenavirus plasmid-based reverse genetics techniques: Plasmids encoding the viral proteins required for replication and transcription and the two viral RNA segments are co-transfected into BHK21 cells. Tissue culture supernatants containing recombinant viruses are used to study different aspects of the biology of arenaviruses.

Vaccine Development

Figure 8. Influenza virus as a

vaccine vector: Foreign influenza

virus epitopes and polypeptides

expressed from recombinant i

nfluenza virus can be used as

vaccines for influenza viral

infections as well as for the

treatment of other human diseases.

Another important aspect in Dr. Martinez-Sobrido research is the development of vaccines against viral infections. To that end, he is using attenuated forms of influenza virus that can be used as life attenuated vaccines for use in humans. Additionally, he is planning to use recombinant influenza viruses expressing epitopes or/and polypeptides sequences from other human pathogens to develop new viral vectors for the treatment of different human diseases (Figure 8). In the same direction, he is developing potential vaccine vectors for the treatment of arenaviruses that cause hemorrhagic fever in humans, including Lassa Virus.