News and Publications

Recognition of DNA by Antibodies

D.R. Burton, H. Wang, K.E. McLane,* J.F. LeBlanc, P. Ghazal

* Genentech, San Francisco, CA

The generation of molecules capable of recognizing double-stranded DNA with high affinity and specificity would have wide implications in chemistry, biology, and medicine. For instance, such reagents could be useful in genome mapping by blocking certain sites to enzyme action, in diagnostic procedures requiring identification of certain DNA sequences, and in therapeutic regimens requiring control of gene expression. Because they recognize a huge number of diverse molecular shapes, antibodies are promising candidates for high-affinity sequence-specific recognition of DNA. However, the generation of monoclonal antibodies to DNA by conventional immunization procedures has not been generally successful.

We have explored a number of strategies, including the affinity selection of high-affinity DNA binders from synthetic antibody libraries displayed on phage and the generation of binders from phage display libraries prepared from tissue from donors with the autoimmune disease systemic lupus erythematosus. In these studies, we have been successful in selecting antibodies that bind double-stranded DNA with high affinity but lack any base-specific recognition properties. In another approach, we have transplanted a DNA-binding recognition domain from a transcription factor into a hypervariable part of the antibody molecule and have shown that the motif confers sequence-dependent DNA recognition on the antibody molecule. Our most recent studies have defined, at the molecular level, contacts made between the designed antibody and DNA (Fig. 1).

Homeostatic Control of Viruses

A. Angulo, F. Garcia del Rey, J.C. Gonzalez, D. Kerry, J.F. LeBlanc, J. Ramirez, H. Wang, J. Wittig, R. Zyla, P. Ghazal

The goal of our work is to understand, from the molecular to the organismal level, the principles that drive and sustain lifelong infection by viruses. These infectious agents live in a dynamic equilibrium (homeostasis) with their hosts in which both immune and nonimmune pathways contribute to viral homeostasis. Disruption of these pathways can have dramatic consequences on pathogenesis. Indeed, those viruses that establish an incurable lifelong infection are often the most devastating in hosts that are immunosuppressed. For instance, human cytomegalovirus (CMV) rarely causes disease in healthy persons but is life threatening in immunodeficient patients and in infants congenitally infected with the virus. Approximately 80% of the adult population is infected with CMV. Our research focuses on novel approaches for control of viral growth, and we use CMV infection as a clinically relevant model. This work primarily involves mechanistic studies on transcriptional control of the expression of viral genes and the application of cell biology and molecular genetics to viral pathogenesis.

CONTROL OF VIRAL GROWTH BY TRANSCRIPTIONAL ENHANCERS

The CMV enhancer is a highly complex regulatory region containing multiple elements that interact with a variety of host-encoded transcription factors. Many of these sequence elements are conserved among the different species strains of CMV, although the arrangement of the various elements and the overall sequence composition of the CMV enhancers differ remarkably. To delineate the importance of this region to a productive infection and to explore the possibility of generating a murine CMV under the control of human CMV genetic elements, we resected the murine CMV enhancer and replaced it with either nonregulatory sequences or paralogous sequences from human CMV.

Mutations in murine CMV that eliminated the enhancer caused a severe deficiency in viral synthesis, and mutants in which the enhancer was replaced with nonregulatory sequences did not grow. This growth defect is effectively complemented by both the homologous murine CMV enhancer and the human CMV enhancer. In the latter case, the chimeric viruses containing the molecularly shuffled human enhancer have infectious kinetics similar to the kinetics of parental wild-type and wild-type revertant murine CMV. These chimeric strains represent murine CMV that is under the control of human CMV genetic elements and thus set the stage for a new model for exploring the in vivo importance of human CMV regulatory elements during acute, reactivated latent infections. This work is providing new insights into growth-control mechanisms of viruses outside immunoregulatory pathways and potentially new ways to control viral disease.

ANALYSIS WITH HIGH-DENSITY OLIGONUCLEOTIDE ARRAYS

In collaboration with the R.W. Johnson Pharmaceutical Institute, microarrays (DNA chips) were prepared by high-speed robotic printing on glass of oligonucleotides to all known (233) open reading frames in the genome of human CMV (one of the largest known viral genomes) for measurements of parallel expression. The expression profiles of known and of many previously uncharacterized open reading frames (~70%) have provided a temporal map of immediate-early, early, and late genes in the entire genome of human CMV. These fabricated microarrays of viral DNA thus allow, in a single hybridization, analysis of gene expression at the level of the whole viral genome (Fig. 1). This capability, coupled with global biochemical and genetic approaches, should greatly speed the functional analysis of both established and newly discovered large viral genomes.

Currently, we are using the CMV DNA microarray to characterize the transcriptional consequences of mutations that affect the activity of regulatory molecules. This work will lead to the dissection and characterization of the principles that drive and sustain lifelong viral infections and the associated regulatory networks. Furthermore, this strategy has important practical applications in the screening of antiviral drugs. DNA microarrays can be used to define the signature pattern of known viral inhibitors and to screen for compounds that develop an alternatively desired signature. Moreover, mutations in specific genes encoding candidate drug targets can serve as surrogates for chemical inhibitors of their activity.

PUBLICATIONS

Angulo, A., Chandararatna, R., LeBlanc, J.F., Ghazal, P. Retinoid modulation of an acute infection by murine cytomegalovirus. J. Virol., in press.

LeBlanc, J.F., McLane, K.E., Parren, P.W.H.I, Burton, D.R., Ghazal, P. Recognition properties of a sequence-specific DNA binding antibody. Biochemistry 37:6015, 1998.