HESI/FDA workshop on immunomodulators and cancer risk assessment: Building blocks for a weight-of-evidence approach.

Profound immunosuppression (e.g., AIDS, transplant therapy) is epidemiologically associated with an increased cancer risk, and often with oncogenic viruses. It is currently unclear how broadly this association translates to therapeutics that modulate immunity. A workshop co-sponsored by the FDA and HESI examined how perturbing the immune system may contribute to carcinogenesis, and highlighted priorities for improving non-clinical risk assessment of targeted immunomodulatory therapies. Conclusions from the workshop were as follows. 1) While profound altered immunity can promote tumorigenesis, not all components of the immune system are equally important in defense against or promotion of cancer and a similar cancer risk for all immunomodulatory molecules should not be assumed. 2) Rodent carcinogenicity studies have limitations and are generally not reliable predictors of cancer risk associated with immunosuppression. 3) Cancer risk needs to be evaluated based on mechanism-based weight-of-evidence, including data from immune function tests most relevant to tumor immunosurveillance or promotion. 4) Information from nonclinical experiments, clinical epidemiology and immunomodulatory therapeutics show that immunosurveillance involves a complex network of cells and mediators. To support a weight-of-evidence approach, an increased focus on understanding the quantitative relationship between changes in relevant immune function tests and cancer risk is needed.

Transduction of cellular sequence by a human immunodeficiency virus type 1-derived vector.

During studies examining the rate of human immunodeficiency virus type 1 (HIV-1) mutation in a single cycle of replication, the 5' long terminal repeat of one progeny provirus was found to contain an insertion of 147 bp including an entire tRNA sequence as well as an additional 66 bp insertion of nonviral origin. Database searches revealed that 65 of 66 bp aligned with the human CpG island sequence found on chromosomes 6, 14, and 17. Therefore it seems probable that it is of human cellular sequence origin and was transduced by HIV-1. This is the first demonstration that HIV-1 can capture a cellular sequence. The site of integration of the parental provirus was mapped to chromosome 1p32.1. Sequence with homology to the transduced CpG island was not found on chromosome 1, suggesting that the transduced cellular sequence was not linked to the site of viral integration.

Site-specific incorporation of nucleoside analogs by HIV-1 reverse transcriptase and the template grip mutant P157S. Template interactions influence substrate recognition at the polymerase active site.

Studies of drug-resistant reverse transcriptases (RTs) reveal the roles of specific structural elements and amino acids in polymerase function. To characterize better the effects of RT/template interactions on dNTP substrate recognition, we examined the sensitivity of human immunodeficiency virus type 1 (HIV-1) RT containing a new mutation in a "template grip" residue (P157S) to the 5'-triphosphates of (-)-beta-2',3'-dideoxy-3'-thiacytidine (3TC), (-)-beta-2',3'-dideoxy-5-fluoro-3'-thiacytidine (FTC), and 3'-azido-3'-deoxythymidine (AZT). A primer extension assay was used to monitor quantitatively drug monophosphate incorporation opposite each of multiple target sites. Wild-type and P157S RTs had similar catalytic activities and processivities on heteropolymeric RNA and DNA templates. When averaged over multiple template sites, P157S RT was 2-7-fold resistant to the 5'-triphosphates of 3TC, FTC, and AZT. Each drug triphosphate inhibited polymerization more efficiently on the DNA template compared with an RNA template of identical sequence. Moreover, chain termination by 3TC and FTC was strongly influenced by template sequence context. Incorporation of FTC and 3TC monophosphate varied up to 10-fold opposite 7 different G residues in the DNA template, and the P157S mutation altered this site specificity. In summary, these data identify Pro(157) as an important residue affecting nucleoside analog resistance and suggest that interactions between RT and the template strand influence dNTP substrate recognition at the RT active site. Our findings are discussed within the context of the HIV-1 RT structure.

High rate of recombination throughout the human immunodeficiency virus type 1 genome.

The diploid nature of human immunodeficiency virus type 1 (HIV-1) indicates that recombination serves a central function in virus replication and evolution. Previously, while examining the nature of obligatory primer strand transfers during reverse transcription, a high rate of recombination was observed at the ends of the viral genome within the viral long terminal repeats, prompting the following question: does recombination occur at a high rate throughout the genome? To address this question, two vectors based upon different strains of HIV-1 were utilized. The vectors were comprised predominantly of autologous HIV-1 sequence and were approximately the same size as the parental genome. The proviral progeny of heterodimeric virions were analyzed after a single cycle of replication, and the sequence heterogeneity between the two strains allowed direct examination of recombination crossovers. The results obtained indicate that HIV-1 undergoes approximately two to three recombination events per genome per replication cycle. These results imply that both HIV-1 RNAs are typically utilized during reverse transcription and that recombination is an important aspect of HIV-1 replication.

A new point mutation (P157S) in the reverse transcriptase of human immunodeficiency virus type 1 confers low-level resistance to (-)-beta-2',3'-dideoxy-3'-thiacytidine.

A P157S mutation in the reverse transcriptase (RT) of human immunodeficiency virus type 1 conferred fivefold resistance to (-)-beta-2',3'-dideoxy-3'-thiacytidine in cell culture. Interestingly, the P157S mutation resulted in increased sensitivity (two- to threefold) to 3'-azido-3'-deoxythymidine (AZT) and to (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA). A similar increase in susceptibility to AZT and to PMPA was also conferred by the M184V mutation in RT.

The nature of human immunodeficiency virus type 1 strand transfers.

The diploid nature of human immunodeficiency virus type 1 (HIV-1) suggests that recombination serves a central function in virus replication and evolution. A system was developed to examine HIV-1 strand transfers, including the obligatory DNA primer strand transfers as well as recombinational crossovers during reverse transcription. Sequence heterogeneity between different strains of HIV-1 was exploited for examining primer transfer events. Both intra- and intermolecular primer transfers were observed at similar frequencies during minus-strand DNA synthesis, whereas primer transfers during plus-strand DNA synthesis were primarily intramolecular. Sequence analysis of long terminal repeats from progeny proviruses also revealed a high rate of homologous recombination during minus-strand synthesis, corresponding to an overall rate of approximately three crossovers per HIV-1 genome per cycle of replication. These results imply that both viral genomic RNAs serve as templates during HIV-1 reverse transcription and that primer strand transfers and recombination may contribute substantially to the rapid genetic variation of HIV-1.

A novel point mutation at position 156 of reverse transcriptase from feline immunodeficiency virus confers resistance to the combination of (-)-beta-2',3'-dideoxy-3'-thiacytidine and 3'-azido-3'-deoxythymidine.

Mutants of feline immunodeficiency virus (FIV) resistant to (-)-beta-2',3'-dideoxy-3'-thiacytidine (3TC) were selected by culturing virus in the presence of increasing stepwise concentrations of 3TC. Two plaque-purified variants were isolated from the original mutant population, and both of these mutants were resistant to 3TC. Surprisingly, these mutants were also phenotypically resistant to 3'-azido-3'-deoxythymidine (AZT) and to the combination of 3TC and AZT. Purified reverse transcriptase (RT) from one of these plaque-purified mutants was resistant to the 5'-triphosphates of 3TC and AZT. DNA sequence analysis of the RT-encoding region of the pol gene amplified from the plaque-purified mutants revealed a Pro-to-Ser mutation at position 156 of RT. A site-directed mutant of FIV engineered to contain this Pro-156-Ser mutation was resistant to 3TC, AZT, and the combination of 3TC and AZT, confirming the role of the Pro-156-Ser mutation in the resistance of FIV to these two nucleoside analogs. This represents the first report of a lentiviral mutant resistant to the combination of AZT and 3TC due to a single, unique point mutation.

Increased activity and fidelity of DNA polymerase beta on single-nucleotide gapped DNA.

DNA polymerase beta (pol beta) is an error-prone polymerase that plays a central role in mammalian base excision repair. To better characterize the mechanisms governing rat pol beta activity, we examined polymerization on synthetic primer-templates of different structure. Steady-state kinetic analyses revealed that the catalytic efficiency of pol beta (kcat/Km,dNTPapp) is strongly influenced by gap size and the presence of a phosphate group at the 5'-margin of the gap. pol beta exhibited the highest catalytic efficiency on 5'-phosphorylated 1-nucleotide gapped DNA. This efficiency was >/=500 times higher than on non-phosphorylated 1-nucleotide and 6-nucleotide (with or without PO4) gapped DNAs and 2,500 times higher than on primer-template with no gaps. The nucleotide insertion fidelity of pol beta, as judged by its ability to form G-N mispairs, was also higher (10-100 times) on 5'-phosphorylated single-nucleotide gapped DNA compared with the other DNA substrates studied. These data suggest that a primary function of mammalian pol beta is to fill 5'-phosphorylated 1-nucleotide gaps.

Discontinuous plus-strand DNA synthesis in human immunodeficiency virus type 1-infected cells and in a partially reconstituted cell-free system.

Human immunodeficiency virus type 1 (HIV-1) replication requires conversion of viral RNA to double-stranded DNA. To better understand the molecular mechanisms of this process, we examined viral DNA synthesis in a simple cell-free system that uses the activities of HIV-1 reverse transcriptase to convert regions of single-stranded HIV-1 RNA to double-stranded DNA in a single incubation. This system recapitulated several of the required intermediate steps of viral DNA synthesis: RNA-templated minus-strand polymerization, preferential plus-strand initiation at the central and 3' HIV-1 polypurine tracts, and DNA-templated plus-strand polymerization. Secondary sites of plus-strand initiation were also observed at low frequency both in the cell-free system and in cultured virus. Direct comparison of viral and cell-free products revealed differences in the precision and selectivity of plus-strand initiation, suggesting that the cell-free system lacks one or more essential replication components. These studies provide clues about mechanisms of plus-strand initiation and serve as a starting point for the development of more complex multicomponent cell-free systems.

A genetic system to identify DNA polymerase beta mutator mutants.

DNA polymerase beta (pol beta) is a 39-kDa protein that functions in DNA repair processes in mammalian cells. As a first step toward understanding mechanisms of polymerase fidelity, we developed a genetic method to identify mammalian pol beta mutator mutants. This screen takes advantage of a microbial genetics assay and the ability of rat pol beta to substitute for Escherichia coli DNA polymerase I in DNA replication in vivo. Using this screen, we identified 13 candidate pol beta mutator mutants. Three of the candidate mutator mutants were further characterized in vivo and shown to confer an increased spontaneous mutation frequency over that of wild-type pol beta to our bacterial strain. Purification and subsequent analysis of one of our putative mutator proteins, the pol beta-14 protein, showed that it possesses intrinsic mutator activity in four different assays that measure the fidelity of DNA synthesis. Therefore, residue 265, which is altered in pol beta-14 and another of our mutant proteins, pol beta-166, is probably critical for accurate DNA synthesis by pol beta. Thus, our genetic method of screening for pol beta mutator mutants is useful in identifying active mammalian DNA polymerase mutants that encode enzymes that catalyze DNA synthesis with altered fidelity compared with the wild-type pol beta enzyme.

Human immunodeficiency virus Type 1 nucleocapsid protein (NCp7) directs specific initiation of minus-strand DNA synthesis primed by human tRNA(Lys3) in vitro: studies of viral RNA molecules mutated in regions that flank the primer binding site.

Retroviral reverse transcription starts near the 5' end of unspliced viral RNA at a sequence called the primer binding site (PBS), where the tRNA primer anneals to the RNA template for initiation of DNA synthesis. We have investigated the roles of NCp7 in annealing of primer tRNA(Lys3) to the PBS and in reverse transcriptase (RT) activity, using a cell-free reverse transcription reaction mixture consisting of various 5' viral RNA templates, natural primer tRNA(Lys3) or synthetic primer, human immunodeficiency virus type I (HIV-1) nucleocapsid protein (NCp7), and HIV-1 RT. In the presence of tRNA(Lys3), NCp7 was found to stimulate synthesis of minus-strand strong-stop DNA [(-)ssDNA], consistent with previous reports. However, specific DNA synthesis was observed only at a NCp7/RNA ratio similar to that predicted to be present in virions. Moreover, at these concentrations, NCp7 inhibited the synthesis of nonspecific reverse-transcribed DNA products, which are initiated because of self-priming by RNA templates. In contrast to results obtained with tRNA(Lys3) as primer, NCp7 inhibited the synthesis of (-)ssDNA products primed by an 18-nucleotide (nt) ribonucleotide (rPR), complementary to the PBS, even though rPR can initiate synthesis of such material in the absence of preannealing with NCp7. Primer placement band shift assays showed that NCp7 was necessary for efficient formation of the tRNA-RNA complex. In contrast, NCp7 was found to prevent formation of the rPR-RNA complex. Since NCp7 appears to exert opposite effects (annealing versus dissociation) on tRNA(Lys3) and rPR substrates, the non-PBS binding regions of the tRNA(Lys3) molecule may play a role in the annealing of tRNA to the template. We also investigated the roles of an A-rich loop upstream of the PBS, a 7-nt region immediately downstream of the PBS, and a 54-nt deletion further downstream of the PBS in interactions with tRNA(Lys3). We found that deletions in the 54-nt region that may prevent formation of the U5-leader stem prevented tRNA(Lys3) placement and priming, while deletions in the A-rich loop or the 7-nt sequence had relatively minor effects in this regard.

Effect of human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein on HIV-1 reverse transcriptase activity in vitro.

Conversion of human immunodeficiency virus type 1 (HIV-1) genomic RNA to viral DNA is a requisite step in the virus life cycle. This conversion is catalyzed by reverse transcriptase (RT) associated with a large nucleoprotein complex composed of several viral proteins including nucleocapsid (NC). To better characterize the biochemical mechanisms of viral DNA synthesis, we overexpressed and purified recombinant HIV-1 NC and studied its effect on the activity and processivity of HIV-1 RT during polymerization of HIV-1 template sequences in vitro. The effect of NC on steady-state RT activity was dependent on the order of addition of reaction components. Addition of NC prior to formation of RT-primer.template-dNTP ternary complexes inhibited primer extension and reduced total product yields by slowing steady-state RT turnover. In contrast, addition of NC to preformed ternary complexes resulted in efficient primer extension and increased RT processivity at specific DNA template sites. NC stimulated polymerization (2-4 times) through eight of 13 sites examined in the cRRE region of HIV-1 env and increased the rate of polymerization through the D3/CTS region of HIV-1 pol 10 times. The data suggest that NC affects RT processivity by facilitating polymerization through regions of template secondary structure. Thus, NC functions as a single-strand binding (SSB)-like accessory replication factor for RT in vitro and may be part of a multicomponent retroviral replication complex.

Mechanisms of retroviral mutation.

Retroviruses, like other RNA viruses, mutate at very high rates (0.05-1 mutations per genome per replication cycle) and exist as complex genetically heterogeneous populations ('quasispecies') that are ever changing. De novo mutations are generated by inherently error-prone steps in the retroviral life cycle that introduce base substitutions, frame shifts, genetic rearrangements and hypermutations.

Genetic rearrangements occurring during a single cycle of murine leukemia virus vector replication: characterization and implications.

Retroviruses evolve at rapid rates, which is presumably advantageous for responding to selective pressures. Understanding the basic mutational processes involved during retroviral replication is important for comprehending the ability of retroviruses to escape immunosurveillance and antiviral drug treatment. Moreover, since retroviral vectors are important vehicles for somatic cell gene therapy, knowledge of the mechanism of retroviral variation is critical for anticipating untoward mutational events occurring during retrovirus-medicated gene transfer. The focus of this report is to examine the spectrum of genomic rearrangements arising during a single cycle of Moloney murine leukemia virus (MoMLV) vector virus replication. An MoMLV vector containing the herpes simplex virus thymidine kinase (tk) gene was constructed. MoMLV vector virus was produced in packaging lines, and target cells were infected. From a total of 224 mutant proviruses analyzed, 114 had gross rearrangements readily detectable by Southern blotting. The remaining proviruses were of parental size. PCR and DNA sequence analysis of 73 of the grossly rearranged mutant proviruses indicated they resulted from deletions, combined with insertions, duplications, and complex mutations that were a result of multiple genomic alterations in the same provirus. Complex hypermutations distinct from those previously described for spleen necrosis virus and human immunodeficiency virus were detected. There was a correlation between the mutation breakpoints and single-stranded regions in the predicted viral RNA secondary structure. The results also confirmed that the tk gene is inactivated at an average rate of about 8.8% per cycle of retroviral replication, which corresponds to a rate of mutation of 3%/kbp.

Marked infidelity of human immunodeficiency virus type 1 reverse transcriptase at RNA and DNA template ends.

Human immunodeficiency virus type 1 (HIV-1) is genetically highly variable. This is attributed to the error-prone nature of HIV-1 replication and its proclivity for recombination. During replication and recombination, reverse transcriptase (RT) must polymerize DNA to the 5' ends of multiple RNA and DNA template termini while converting HIV-1 RNA to double-stranded DNA. We have determined the fidelity of HIV-1 RT in vitro during polymerization to the 5' ends of HIV-1 long terminal repeat DNA template sequences and to the end of a partial HIV-1 genomic RNA template that mimics a recombination intermediate. HIV-1 RT readily extended recessed DNA primers to form full-length blunt-end DNA-DNA and DNA-RNA duplexes. In addition, HIV-1 RT catalyzed high yields of products with one to four extra nucleotides at the 3' ends of the nascent DNAs. These products were formed processively via a nontemplated mechanism that is highly specific for the addition of purine nucleotides (A > G >> T > or = C). Thus, HIV-1 RT is extremely unfaithful at both DNA and RNA template ends, introducing errors (extra nucleotides) in one out of every two or three nascent strands processively polymerized. This error rate is 1000 times higher than for HIV-1 RT-catalyzed errors at internal template positions. Blunt-end additions were also catalyzed by other retroviral RTs at relative rates of HIV-1 approximately Moloney murine leukemia virus > avian myeloblastosis virus. These data suggest a potentially important mechanism for retroviral mutation mediated by nontemplated blunt-end addition of purines prior to forced copy-choice recombination.