Research Summary
Paul Bieniasz explores the mechanisms underlying retrovirus-host interactions, focusing on how HIV and related viruses replicate in cells. His research advances knowledge of the molecular interplay between viruses and their hosts, as well their coevolution.
Our research focuses on the molecular biology of retroviruses. Although the retrovirus life cycle is conceptually simple, there is much complexity in the way that retroviruses interact with host cells. Retrovirus-host interactions occur in two ways: First, retroviruses extensively parasitize host cell processes in order to replicate. Second, cells have evolved functions to specifically inhibit colonization of their genomes by retroviruses.
Our overall goal is to understand the mechanics of biological processes that accompany retrovirus replication and how the evolution of hosts and retroviruses has been influenced by their interactions, and to exploit this knowledge to develop better animal models of human retroviral disease. Most of our work centers on human and simian immunodeficiency viruses (HIV, SIV), but we also study other model retroviruses (e.g., murine leukemia virus [MLV]) and retroviral "fossils"—extinct, defective "endogenous" retroviruses that are preserved in mammalian DNA because they infected germline cells in the ancestors of modern organisms.
Retrovirus Assembly and Budding
Among the most complex processes in retrovirus replication is the construction of new infectious retrovirus particles from their component parts. The retroviral Gag protein has intrinsic particle-forming ability, and expression of Gag in permissive cells, in the absence of other viral proteins, is sufficient to generate immature virus-like particles. Specific cellular functions are also required to generate extracellular virions. For example, we and others have shown that short peptide motifs encoded within the Gag protein of HIV-1 and other retroviruses recruit an array of host proteins, including ubiquitin ligases and components of the class E vacuolar protein-sorting pathway. These factors induce the fission of virion and cellular membranes, allowing the release of nascent virus particles. One focus of our research is to determine how these proteins are recruited and how they induce particle release. Additionally, we have identified novel host proteins within virus particles, and we are investigating their role in virion assembly and other phases of the viral life cycle.
We also devote significant effort to imaging the process of virion assembly, in collaboration with Sanford Simon (Rockefeller University). To enable these studies, we demonstrated that HIV-1 particles are generated at the plasma membrane, thereby resolving a controversy over where within cells HIV-1 assembly is initiated and completed. Armed with this knowledge, we are developing techniques to visualize the genesis of individual HIV-1 particles in real time, using fluorescent fusion proteins in living cells. Total internal reflection fluorescence microscopy (TIR-FM) is uniquely suited to imaging events at the plasma membrane, and we are applying this technique to generate an unprecedented view of HIV-1 particle assembly. We are developing approaches derived from the basic technique to understand how and when various virion components and cellular proteins that facilitate and inhibit HIV-1 particle assembly and release are recruited to sites of virion assembly.
Intrinsic and Innate Immunity to Retroviruses
Another major interest is the identification and characterization of cellular factors that govern retrovirus host range. For many years, the host range of any given retrovirus was thought to be governed solely by its ability to parasitize the particular array of host molecules (e.g., receptors, transcription factors) provided by a candidate host cell. We now know, partly through our own work, that mammalian host cells are equipped with several proteins that specifically inhibit retrovirus replication; these proteins are equally critical determinants of host range. Among these antiviral factors are the TRIM5 proteins that inactivate incoming retroviral capsids and the APOBEC3 family of cytidine deaminases that infiltrate assembling virions and hypermutate nascent viral DNA during the subsequent cycle of reverse transcription. HIV-1 and SIVs avoid or antagonize TRIM5 and APOBEC3 through capsid sequence variation or, in the case of APOBEC3, by encoding an antagonist, Vif. We work both on the molecular mechanisms by which these antiretroviral proteins act and on how they impact retroviral host range and evolution.
Recently we discovered a new host defense molecule, which we have termed tetherin. This antiviral protein can be constitutively expressed or induced by interferon-α, and its expression results in the formation of protein-based tethers that cause retention of fully formed HIV-1 particles on infected cell surfaces. We also discovered that the HIV-1 accessory protein, Vpu, is an antagonist of tetherin and thereby promotes the release and dissemination of HIV-1 particles from tetherin-expressing cells. We are interested in precisely how tetherin functions. Remarkably, it appears to be active against a range of diverse enveloped viruses. We are also attempting to determine how Vpu and other viral antagonists of tetherin that we have recently discovered counteract its antiviral activity.
Paleovirology, Viral and Host Evolution
Because of their role as an antiretroviral viral defense, TRIM5, APOBEC3, and tetherin genes exhibit hallmarks of genes that have evolved under strong diversifying selection pressure. Specifically, interspecies sequence comparisons reveal unexpectedly high rates of nonsynonymous mutation, suggesting that these genes have been selected for greater antiviral efficiency against different viruses in different host species. Additionally, although these proteins were discovered as a result of their activity against HIV and SIV, it is likely that they evolved, in part, to antagonize ancient retroviruses that are now extinct.
To try to understand how retroviruses and host defenses have coevolved, we have begun mining genome sequences that contain a fossil record of ancient retroviruses for evidence of reciprocal coevolution of retroviruses and their hosts, and instances of retroviral extinction by host defenses. For example, we completely synthesized a consensus, pseudoancestral genome of an extinct human retrovirus, HERV-K(HML-2). This reconstructed virus, unlike any of the fossilized remnants that are present in modern human genomes, is capable of infection in cultured cells. We have undertaken a number of investigations of this heretofore extinct virus, including an analysis of sensitivity to host antiviral proteins, and we have shown that some copies of HERV-K were inactivated by APOBEC3 proteins.
In other studies of variation of host resistance factors, we uncovered a striking example of convergent evolution in the TRIM5 gene in primates, in which a chimeric TRIM5 gene arose through retrotransposition of a cyclophilin A cDNA into the TRIM5 locus. Remarkably, similar retrotransposition events happened on two separate occasions in the germlines of two different primate species. These occurrences illustrate how seemingly unlikely evolutionary events can be less improbable than they might intuitively appear. Moreover, these drastic changes in TRIM5-coding potential impact the sensitivity of the primate host species to ancient and modern retroviruses and can have important practical consequences for the development of animal models of retroviral diseases.
Better Animal Models of Human AIDS
Our work on intrinsic and innate immunity to retroviruses, particularly on species-specific variation in TRIM5 and APOBEC3 proteins, has suggested that it might be possible to engineer HIV-1 strains with expanded tropism. Specifically, the generation of HIV-1 strains that can replicate in monkeys would be of enormous practical benefit and would likely revolutionize preclinical studies of HIV-1 drugs and vaccines. By engineering and adapting specific components of HIV-1 (the capsid and Vif proteins) to avoid or inactivate TRIM5 and APOBEC3 host resistance factors, we successfully derived simian cell tropic HIV-1 strain (stHIV-1). Unlike wild-type HIV-1, which cannot replicate in macaque cells, stHIV-1 replicates robustly in macaque cells, in vitro. With our colleague, Theodora Hatziioannou (Aaron Diamond AIDS Research Center), and collaborators Vineet KewalRamani and Jeffrey Lifson (National Cancer Institute, Frederick, MD), we are developing stHIV-1 variants and testing them for their ability to replicate in macaques in vivo. Using these approaches, it may be possible to derive an animal model that recapitulates the characteristics of human HIV-1 infection.
Grants from the National Institutes of Health, the Elizabeth Glaser Pediatric AIDS Foundation, and the American Foundation for AIDS Research provided support for this work.
As of May 30, 2012
