Major Areas of Research
- The multiple roles of the envelope glycoproteins during the flavivirus lifecycle
- Mechanisms of antibody-mediated neutralization of viruses
- Humoral immunity to flavivirus infection
Flaviviruses are a group of RNA viruses responsible for considerable morbidity and mortality worldwide. Members of this genus with a significant impact on public health include dengue virus (DENV), yellow fever virus, Japanese encephalitis virus, tick-borne encephalitis virus, and West Nile virus (WNV). Flaviviruses are small spherical virions composed of a single copy of an ~11 kb genomic RNA of positive polarity, a capsid protein (C), a lipid envelope derived from the endoplasmic reticulum, and two envelope glycoproteins: envelope (E) and pre-membrane/membrane (prM/M). The E proteins are incorporated into virions as a dense symmetrical array that orchestrates the process of virus entry, assembly, and budding from infected cells. These proteins are also the primary targets of neutralizing antibodies elicited in vivo. The goal of our laboratory is to understand the function of flavivirus E proteins and their interactions with the host.
Above: The flavivirus lifecycle
Flaviviruses bind to cells via interactions between the envelope (E) proteins of the virion and a diverse group of cellular factors on the target cell. Cellular receptors/attachment factors absolutely required for infection have not yet been identified; molecules implicated in the entry of flaviviruses into cells include several lectins (DC-SIGN, DC-SIGNR, and mannose receptor), heparin sulfate, and molecules of the TIM/TAM family. Additionally, viruses may bind to and enter cells as an immune complex in a process called antibody-dependent enhancement of infection; this pathway may play a role in severe clinical manifestations of dengue virus infection. Once bound, flaviviruses are internalized into cells via clathrin-mediated endocytosis and traffic into a late endosomal compartment, in which viral membrane fusion occurs in a pH-dependent fashion. The positive-stranded genomic RNA is then released into the cytoplasm, in which translation occurs. Replication of the viral RNA occurs in the context of complex three-dimensional networks of membranes induced by the viral non-structural proteins. Virus assembly occurs on membranes derived from the endoplasmic reticulum (ER). Virions bud into the ER as immature virus particles that incorporate sixty trimeric spikes of the pre-membrane (prM) and E proteins. During egress, prM is cleaved by the cellular serine protease furin. A relatively smooth, infectious, mature virus particle is released into the extracellular space.
Understanding the molecular basis of antibody-mediated neutralization and enhancement of flavivirus infection
Humoral immunity is a critical aspect of host protection against flaviviruses; eliciting protective antibodies is a primary focus of ongoing vaccine development efforts for several of these viruses, including DENV. Complicating these efforts is the potential for antibodies elicited by natural infection or vaccination to modulate DENV pathogenesis and enhance disease. The ability of antibodies to enhance the efficiency of infection is called antibody-dependent enhancement of infection (ADE). The biochemical and functional properties of a protective antibody response are not known. A major focus of our laboratory is to understand the mechanisms of humoral immunity against flaviviruses in biochemical and structural detail. We are working to define the factors that govern the potency of neutralizing antibodies, their potential for ADE, and their mechanism of action using WNV and DENV as model systems. Using quantitative functional methods developed by our laboratory, we have defined the stoichiometric requirements for neutralization and ADE of WNV. This estimate of the number of antibodies required to neutralize and enhance WNV infection has provided a useful foundation to explore the genetic and biochemical complexity of factors that modulate antibody function. Our findings highlight the importance of the accessibility of epitopes on the virion as a key parameter that shapes the epitope occupancy requirements for neutralization. Ongoing studies are focused on how the structural dynamics of the virion (virus breathing) modulates antibody-mediated neutralization.
Investigating the impact of the structural heterogeneity and dynamics of the virion on the biology of flaviviruses
Insights into the biology of flaviviruses have been dramatically accelerated by advances in the structural biology of these viruses. Structures of the E protein and how they are arranged on the virus particle at multiple stages of the virus lifecycle have been reported. These have significantly influenced the development of our models of antibody-mediated neutralization. However, recent studies indicate that populations of flaviviruses are much more complex than suggested by existing structural models. Several lines of evidence suggest that the cleavage of prM during the virion maturation process can be quite inefficient, resulting the release of "partially mature" virus particles that incorporate uncleaved prM in unknown quantities. Our lab is investigating several aspects of the biology of partially mature virions, including how uncleaved prM markedly impacts the sensitivity of virions to neutralization by antibodies. Our understanding of the structure of the virion is complicated further by the fact that the E proteins on virions appear to sample an ensemble of conformations or arrangements at equilibrium. This presents a moving target for antibody recognition.
Investigating the functional complexity of the humoral response to flavivirus vaccination
Eliciting a protective neutralizing antibody response is a major goal of ongoing vaccine development efforts for DENV. Several tetravalent DENV vaccines are in clinical development. Flavivirus infection results in the production of antibodies that bind to a variety epitopes on the prM and E proteins. We are developing functional approaches to map the composition and dynamics of the polyclonal response to flavivirus infection, with the goal of identifying epitopes recognized by antibodies that are important for neutralization and protection.
Dr. Pierson received his Ph.D. from The Johns Hopkins University School of Medicine in 2001. While training in the laboratory of Dr. Robert F. Siliciano, Dr. Pierson investigated the molecular biology of the pre-integration state of HIV-1 latency and the contribution of this relatively labile reservoir toward the persistence of HIV-1 in the face of aggressive antiretroviral therapy. After completing these studies, Dr. Pierson took a postdoctoral fellowship in the laboratory of Dr. Robert W. Doms in the department of microbiology at the University of Pennsylvania. While training there, Dr. Pierson initiated a new research program to study the cell biology of the envelope proteins of flaviviruses, with a focus on West Nile virus and dengue viruses. In 2005, Dr. Pierson was recruited to initiate the Viral Pathogenesis Section of the Laboratory of Viral Diseases and to continue his work on flaviviruses.
Postdoctoral Fellows: Swati Mukherjee, Ph.D.; Kimberly Dowd, Ph.D.
Graduate Students: Christopher Obara, B.S.; Laura Vanblargan, B.S.
Post-Baccalaureate Fellow: Phong Lee, B.S.
Lin TY, Dowd KA, Manhart CJ, Nelson S, Whitehead SS, Pierson TC. A novel approach for the rapid mutagenesis and directed evolution of the structural genes of west nile virus. J Virol. 2012 Apr;86(7):3501-12.
Mukherjee S, Lin TY, Dowd KA, Manhart CJ, Pierson TC. The infectivity of prM-containing partially mature West Nile virus does not require the activity of cellular furin-like proteases. J Virol. 2011 Nov;85(22):12067-72.
Dowd KA, Jost CA, Durbin AP, Whitehead SS, Pierson TC. A dynamic landscape for antibody binding modulates antibody-mediated neutralization of West Nile virus. PLoS Pathog. 2011 Jun;7(6):e1002111.
Dowd KA, Pierson TC. Antibody-mediated neutralization of flaviviruses: a reductionist view. Virology. 2011 Mar 15;411(2):306-15.
Mehlhop E, Nelson S, Jost CA, Gorlatov S, Johnson S, Fremont DH, Diamond MS, Pierson TC. Complement protein C1q reduces the stoichiometric threshold for antibody-mediated neutralization of West Nile virus. Cell Host Microbe. 2009 Oct 22;6(4):381-91.