Matthew D. Scharff

The polypeptides of adenovirus: I. Evidence for multiple protein components in the virion and a comparison of types 2, 7A, and 12☆

Abstract The adenovirion has been shown to contain at least nine different polypeptides demonstrable by dissociation and acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). Each polypeptide of adenovirus type 2 is chemically distinct by isotopic ratio analysis, and they all contain lysine, arginine, tryptophan, valine, and threonine. The molecular weights of these polypeptides determined by gel electrophoresis range from 120,000 for the largest and most prominent component to 7500 for the smallest. Comparison of nontumorigenic type 2 with tumorigenic types 7A and 12 by double-isotope labeling revealed a generally similar peptide pattern for all types. However, there were distinct differences between the corresponding peptides of all three types. These results imply extensive differences in the genes for most of the capsid proteins.

Activation-induced cytidine deaminase deaminates deoxycytidine on single-stranded DNA but requires the action of RNase.

The expression of activation-induced cytidine deaminase (AID) is prerequisite to a “trifecta” of key molecular events in B cells: class-switch recombination and somatic hypermutation in humans and mice and gene conversion in chickens. Although this critically important enzyme shares common sequence motifs with apolipoprotein B mRNA-editing enzyme, and exhibits deaminase activity on free deoxycytidine in solution, it has not been shown to act on either RNA or DNA. Recent mutagenesis data in Escherichia coli suggest that AID may deaminate dC on DNA, but its putative biochemical activities on either DNA or RNA remained a mystery. Here, we show that AID catalyzes deamination of dC residues on single-stranded DNA in vitro but not on double-stranded DNA, RNA–DNA hybrids, or RNA. Remarkably, it has no measurable deaminase activity on single-stranded DNA unless pretreated with RNase to remove inhibitory RNA bound to AID. AID catalyzes dC → dU deamination activity most avidly on double-stranded DNA substrates containing a small “transcription-like” single-stranded DNA bubble, suggesting a targeting mechanism for this enigmatic enzyme during somatic hypermutation.

A simple method for polyethylene glycol-promoted hybridization of mouse myeloma cells.

A simple method is described for promoting the fusion of mouse myeloma cells in suspension with polyethylene glycol (PEG 1000). By carefully controlling the concentration of PEG and the time of exposure of the cells, it was possible to obtain hybridization frequencies several-hundred-fold higher than those obtained with Sendai virus.

The Biochemistry of Somatic Hypermutation

Affinity maturation of the humoral response is mediated by somatic hypermutation of the immunoglobulin (Ig) genes and selection of higher-affinity B cell clones. Activation-induced cytidine deaminase (AID) is the first of a complex series of proteins that introduce these point mutations into variable regions of the Ig genes. AID deaminates deoxycytidine residues in single-stranded DNA to deoxyuridines, which are then processed by DNA replication, base excision repair (BER), or mismatch repair (MMR). In germinal center B cells, MMR, BER, and other factors are diverted from their normal roles in preserving genomic integrity to increase diversity within the Ig locus. Both AID and these components of an emerging error-prone mutasome are regulated on many levels by complex mechanisms that are only beginning to be elucidated.

Activation-induced cytidine deaminase turns on somatic hypermutation in hybridomas

The production of high-affinity protective antibodies requires somatic hypermutation (SHM) of the antibody variable (V)-region genes. SHM is characterized by a high frequency of point mutations that occur only during the centroblast stage of B-cell differentiation. Activation-induced cytidine deaminase (AID), which is expressed specifically in germinal-centre centroblasts, is required for this process, but its exact role is unknown. Here we show that AID is required for SHM in the centroblast-like Ramos cells, and that expression of AID is sufficient to induce SHM in hybridoma cells, which represent a later stage of B-cell differentiation that does not normally undergo SHM. In one hybridoma, mutations were exclusively in G·C base pairs that were mostly within RGYW or WRCY motifs, suggesting that AID has primary responsibility for mutations at these nucleotides. The activation of SHM in hybridomas indicates that AID does not require other centroblast-specific cofactors to induce SHM, suggesting either that it functions alone or that the factors it requires are expressed at other stages of B-cell differentiation.

Altered somatic hypermutation and reduced class-switch recombination in exonuclease 1-mutant mice.

The generation of protective antibodies requires somatic hypermutation (SHM) and class-switch recombination (CSR) of immunoglobulin genes. Here we show that mice mutant for exonuclease 1 (Exo1), which participates in DNA mismatch repair (MMR), have decreased CSR and changes in the characteristics of SHM similar to those previously observed in mice mutant for the MMR protein Msh2. Exo1 is thus the first exonuclease shown to be involved in SHM and CSR. The phenotype of Exo1−/− mice and the finding that Exo1 and Mlh1 are physically associated with mutating variable regions support the idea that Exo1 and MMR participate directly in SHM and CSR.

Return to the Past: The Case for Antibody-Based Therapies in Infectious Diseases

Abstract In the preantibiotic era, passive antibody administration (serum therapy) was useful for the treatment of many infectious diseases. The introduction of antimicrobial chemotherapy in the 1940s led to the rapid abandonment of many forms of passive antibody therapy. Chemotherapy was more effective and less toxic than antibody therapy. In this last decade of the 20th century the efficacy of antimicrobial chemotherapy is diminishing because of the rapidly escalating number of immunocompromised individuals, the emergence of new pathogens, the reemergence of old pathogens, and widespread development of resistance to antimicrobial drugs. This diminishment in the effectiveness of chemotherapy has been paralleled by advances in monoclonal antibody technology that have made feasible the generation of human antibodies. This combination of factors makes passive antibody therapy an option worthy of serious consideration. We propose that for every pathogen there exists an antibody that will modify the infection to the benefit of the host. Such antibodies are potential antimicrobial agents. Antibody-based therapies have significant advantages and disadvantages relative to standard chemotherapy. The reintroduction of antibody-based therapy would require major changes in the practices of infectious disease specialists.

The polypeptides of adenovirus: II. Soluble proteins, cores, top components and the structure of the virion

Abstract The adenovirion has been shown to have a much more complex structure than was previously assumed. Eight different types of polypeptides have been assigned a place in the particle. Three are present in an outer capsid, three in an inner core, and two others in association with hexons. The hexon capsomere is composed of about three molecules of a single type of peptide of molecular weight (MW) 120,000 which comprises about 50% of the total virion protein. Groups of such capsomeres, released from the virion by 5 M urea, are found to be associated with two minor polypeptides, each of MW 13,000. The penton-base is composed of a single type of peptide of MW 70,000, and the fiber is composed of another peptide of MW 62,000. The internal core released by treatment of the virion with 5 M urea, contains the viral DNA in association with three arginine-rich peptides of MW 44,000, 24,000, and 24,000, comprising some 20% of the total virion protein. The same three peptides are relatively lacking in the “empty” capsids found as the “top components” of cesium chloride gradients of crude virus preparations.

Somatic hypermutation of the AID transgene in B and non-B cells

Somatic hypermutation (SHM) of the Ig genes is required for affinity maturation of the humoral response to foreign antigens. Activation-induced cytidine deaminase (AID), which is specifically expressed in germinal center centroblasts, is indispensable for this process. Expression of AID is sufficient to activate SHM in hybridomas, non-B cells, and Escherichia coli, suggesting that it initiates the mutational process by deaminating DNA. However, the cis-acting sequences that are responsible for targeting AID activity to the variable (V) region of Ig genes are unknown. Here we show that expression of AID in B cell lines (i.e., Burkitts lymphoma Ramos and hybridoma P1–5) not only causes hypermutation of Ig sequences, but also of the AID transgene itself. Because it is possible that B cell-specific transacting factors bind to and recruit the “mutator” to the AID transgene, we tested whether the AID transgene can mutate in non-B cells. Indeed, we show that expression of AID in Chinese hamster ovary cells causes SHM of both the Ig and AID transgenes. These data suggest that high transcription rates alone may predispose any gene to mutation by AID but do not preclude that there are specific B cell factors that account for the extremely high rate of V mutation in vivo and its targeting to the V region.

Serum therapy revisited: Animal models of infection and development of passive antibody therapy

In the preantibiotic era, passively administered immune animal sera, or serum therapy, was the primary mode of treatment for many infectious diseases, including diphtheria, tetanus, scarlet fever, pneumococcal pneumonia, and meningitis caused by Neisseria meningitis and Haemophilus influezae (24). Immune sera contained specific antibodies which mediated therapeutic effects by promoting opsonization, neutralizing toxins, and/or triggering complement-mediated bacterial lysis. Toxicity resulting from the systemic administration of foreign proteins was associated with serum therapy, however, and so serum therapy was abandoned when antibiotics became widely available in the 1940s. Over the past half-century there has been relatively little interest in passive antibody therapy for bacterial and fungal infections because effective antimicrobial drugs have been available. However, several recent developments should renew interest in the use of passive antibody therapy alone or in combination with antimicrobial drugs. First, the emergence of antimicrobial resistance has decreased the efficacy and predictability of antimicrobial chemotherapy. Second, the difficulties of treating infections in immunocompromised individuals, particularly those with AIDS, have revealed the limitations of antimicrobial chemotherapy in the absence of effective immunity. Third, the hybridoma technology introduced in 1975 by Kohler and Milstein (68) provides the means of generating an unlimited supply of homogeneous monoclonal antibodies (MAbs). Technology is now available to reduce the immunogenicity of rodent MAbs in humans by constructing mousehuman chimeric or humanized MAbs (77) or to generate completely human MAbs from either hybridomas or combinatorial libraries (65). Thus, antibody-based therapies no longer depend on heterologous immune sera, with their inherent variations and toxicities, and antibodies can again be considered therapeutic alternatives for a variety of infections. Potentially useful antibodies for the prevention and therapy of infectious diseases are usually identified by demonstrating that they can modify the course of experimental infection. The choice of an animal model for use in the testing of antibody reagents can be a critical decision for demonstrating efficacy. The development of serum therapy in the preantibiotic era relied almost exclusively on animal models in the preclinical testing phase. Here we review serum therapy for pneumococcal and meningococcal infections, with emphasis on the role of the animal models used in their development.