Understanding Viral Pathogenesis
As recent years revealed, new pathogens regularly emerge in the human population, and viral infections such as SARS and the swine-origin H1N1 influenza are still vividly remembered for their medical, social, and economic impact. Understanding viral pathogenesis represents an important task for years to come.
While viruses have relatively few genes, polymorphisms and sequence variations are abundant among different viruses and often have pathological significance. Thus, gaining insight into the viral genomes and capturing sequence variations represent challenging but important tasks with medical and public health significance.
“We used the Gateway® technology [Life Technologies], which is a fantastic system to express proteins in different expression vectors,” explains Pierre-Olivier Vidalain, Ph.D., project leader at the laboratory of viral genomics and vaccinations at the Pasteur Institute. One challenge with respect to viral sequences is that many viruses were first isolated in the 50s or 60s and have been propagated in the laboratory since then.
As a result, the sequence from a pathogenic strain might not always correspond to the wild-type virus any longer, and in some instances virulence in certain animal models might have been lost. These small sequence variations are important to understand viral biology and host-pathogen interaction.
“Another significant challenge that we sometimes have been facing is that several of these viruses generate quasi-species,” explains Dr. Vidalain. This means that when the genomes of hundreds of different viral particles are sequenced, each of them will contain a slightly different sequence, with specific mutations. Cloning the coding sequence of such a virus sometimes requires many different isolates to be sequenced, until the one corresponding to the average sequence from GenBank is obtained.
“We have generated a database to manage this collection of viral sequences.” Dr. Vidalain and colleagues recently developed ViralORFeome 1.0, an open-access database with integrated bioinformatics tools that allows viral ORFs to be cloned by using the Gateway technology and makes them available for reverse proteomics experiments. This platform is particularly suitable to keep track of the diversity and sequence variation among viral strains.
Partly as a result of the enormous diversity and high mutagenesis rate of viruses, each sequence obtained from a clinical sample may be different from sequences found in GenBank. “I think that this is one aspect where we are original and different from similar projects conducted in the past in terms of mass cloning of yeast, humans, or C. elegans sequences.”
While the main objective in Dr. Vidalain’s group is to study the viral proteins individually, this database has many other potential uses. One of them is the ability to perform comparisons, across the same virus family, for a specific protein that has subtle variations, and learn about the function.
For example, viruses that are closely related to Chikungunya, such as the O’nyong’nyong, Sindbis, and Venezuelan equine encephalitis viruses, induce quite different diseases, but the molecular mechanisms that explain these differences are still not fully understood. “We believe that although these viruses have closely related proteins, the functions of these proteins are sufficiently different so that the disease, at the end, is different,” says Dr. Vidalain.
Dr. Vidalain and colleagues developed the idea of comparative interactomics, a strategy that can be used to compare different viruses—for example, vaccine strains with wild-type strains, or oncogenic strains with nononcogenic ones.
“We think that by comparing the function, and in particular the ability to interact with cellular proteins, we could explain, at least in part, differences in pathogenesis,” reveals Dr. Vidalain. For example, research in Dr. Vidalain’s group revealed that both the type I interferon and the NFκBs signaling pathways are inhibited by the nsP2 protein from the Chikungunya and Semliki Forest viruses, but this was not the case for a laboratory-adapted Sindbis virus strain.
Furthermore, the investigators found that the V protein from mumps, measles, and Nipah viruses blocked IFN-β signaling, but this effect was not observed for the Tioman virus, suggesting that this virus did not adapt to humans yet. “Small variations from one species to another are sufficient to change pathogenicity,” explains Dr. Vidalain.
“The major topic that we have been working on, over the past few years, is the search for genes encoding virulence factors from the Burkholderia cepacia complex,” says Jorge H. Leitão, Ph.D., assistant professor at Instituto Superior Técnico from Lisbon, Portugal. The Burkholderia cepacia complex encompasses a group of over 17 closely related bacterial species that have emerged, in recent decades, as important opportunistic pathogens, particularly among individuals suffering from cystic fibrosis and in immunocompromised patients.
Dr. Leitão and colleagues have used a random mutagenesis strategy based on plasposons, which are a class of transposons that facilitate the recovery and identification of interrupted genes. This approach relies on the generation of mutant libraries, followed by the selection of mutants that are attenuated for virulence. The phenotype is subsequently tested in a Caenorhabditis elegans model of infection.
By using this strategy, researchers in Dr. Leitão’s group recently identified several new virulence factors, including a gene cluster involved in the bacterial exopolysaccharide biosynthesis and a pleiotropic regulator involved in stress resistance and virulence.
An important finding was that this pleiotropic regulator, Pbr, appeared to be involved in biofilm formation and in shaping the fitness of the pathogen under adverse environmental condition, a finding that promises to unveil previously unknown facets of the host-pathogen interaction. More recently, this strategy led the researchers to the identification of two RNA chaperones, Hfq and Hfq2, which are believed to be major players in post-transcriptional regulation among bacteria of the Burkholderia cepacia complex.
These and many other examples reveal that developing new cloning strategies, and optimizing the existing ones, are emerging as increasingly important facets of applications such as constructing unbiased libraries, elucidating the biology of toxic or unstable genes, and understanding host-pathogen interaction.
As a technology with implications that extend far beyond the mere manipulation of genes and gene fragments, cloning assumes an active role in the process of experimental inquiry and becomes instrumental in addressing some of the most intriguing and challenging scientific questions.