Pierre Sonigo

Neutralization sensitivity and accessibility of continuous B cell epitopes of the feline immunodeficiency virus envelope

Antibodies elicited during natural infection of domestic cats by the feline immunodeficiency virus (FIV) recognize continuous epitopes in nine domains of the virus envelope glycoproteins. Whereas antibodies directed against the V3 envelope region can neutralize laboratory-adapted virus, neutralization of FIV has been shown to depend upon cellular substrate, and virus adaptation to laboratory cell lines may alter sensitivity to neutralizing antibodies. We therefore undertook a systematic analysis of the continuous B cell epitopes of the envelope of a primary FIV isolate, Wo. The capacity of feline antisera elicited against nine envelope domains to neutralize primary and laboratory-adapted virus was evaluated in feline peripheral blood mononuclear cells (PBMC). The laboratory-adapted strain Petaluma was used to compare neutralization in PBMC and Crandell feline kidney cells (CrFK). Antibodies specific for the V3 region neutralized both primary and laboratory-adapted virus whether residual infectivity was measured in CrFK or in feline PBMC. However, a large discrepancy in the efficiency of neutralization was observed in these ex vivo models of infection, perhaps reflecting diversity in the interaction between virus and different cellular targets. We also examined the accessibility of epitopes on the functional oligomeric envelope complex of FIV. Most of the epitopes were poorly exposed on native envelope glycoproteins at the surface of live infected cells. The most accessible domain was the only domain sensitive to neutralizing antibodies. These results suggest that inaccessibility on oligomeric envelope glycoproteins may frequently underlie the insensitivity of diverse lentivirus B cell epitopes to neutralization.

B-cell epitopes of the envelope glycoprotein of caprine arthritis-encephalitis virus and antibody response in infected goats.

Goats infected with caprine arthritis-encephalitis virus (CAEV) develop high titres of antibodies to Env. Not only is no consistent neutralizing response found but anti-Env antibodies have even been associated with disease in infected goats. To identify the continuous antigenic determinants involved in this atypical anti-Env response, we mapped CAEV-CO Env by screening an epitope expression library with infected goat sera. In addition to the four previously described epitopes, seven novel antigenic sites were identified, of which five were located on the surface (SU) and two in the transmembrane (TM) subunits of Env. The SU antibody-binding domains located in the variable regions of the C-terminal part of the molecule (SU3 to SU5) showed the strongest reactivity and induced a rapid seroconversion in six experimentally infected goats. However, the response to these immunodominant epitopes did not appear to be associated with any neutralizing activity. The pattern of serum reactivity of naturally infected goats with these epitopes was restricted, suggesting a type-specific reaction. Interestingly, the reactivity of peptides representing SU5 sequences derived from CAEV field isolates varied with the geographical and/or breeding origin of the animals. This suggests that peptides corresponding to the immunodominant SU epitopes may well be useful in the serotyping of CAEV isolates. Furthermore, the identification of the CAEV Env epitopes will permit us to functionally dissect the antibody response and to address the role of anti-Env antibodies either in the protection from or in the pathogenesis of CAEV infection.

Comparison of serological tests for the diagnosis of feline immunodeficiency virus infection of cats

A new enzyme-linked-immunosorbent assay (ELISA) for the detection of antibodies to feline immunodeficiency virus was compared with previously described ELISAs. Serum samples from 184 infected or uninfected cats were tested using a whole virus lysate kit and ELISAs based on recognition of one of two synthetic peptides (P237 and P253) localized in the transmembrane domain of the viral envelope. The whole virus lysate commercial kit led to the detection of 6% false positive and 4.3% false negative sera. The ELISA based on peptide P253 gave no false positive result and failed to detect only one serum that was subsequently shown to be positive by radio-immunoprecipitation assay. A sandwich-ELISA test using Galanthus nivalis agglutinin, a lectin that specifically binds terminal mannose groups of the envelope proteins was used as a confirmatory test for equivocal results with peptide ELISA and gave similar results. This study indicates that recognition of P253 could serve as a sensitive and specific test for the diagnosis of seropositivity to feline immunodeficiency virus, and moreover that the Galanthus nivalis ELISA could be useful in equivocal cases as a confirmatory test.

Reverse transcriptase jumps and gaps

Introduction. Retroviruses are single-stranded RNA viruses of eukaryotes. Different subfamilies have been described. Avian or murine oncoviruses induce neoplasms, whereas lentiviruses, typified by human immunodeficiency virus and spumaviruses, produce persistent infections. Lentiviral infections may cause chronic disease whereas spumaviruses are apparently non-pathogenic. The retroviral life cycle is characterized by reverse transcription of their single-stranded plus (i.e. coding) RNA genome into a double-stranded DNA intermediate that integrates into the host genome. Over the last 25 years the mechanism of reverse transcription has been studied in great detail and these studies have led to the model shown in Fig. I. The result of reverse transcription is a linear double-stranded DNA molecule with a long terminal repeat (LTR) at each extremity. Synthesis of each DNA strand by the virus-encoded reverse transcriptase requires one template switch, also called a jump: the first jump is needed for synthesis of a minus DNA strand complementary to the viral RNA, the second jump for plus DNA strand synthesis.

CONVERSION OF THYMIDYLATE SYNTHASE INTO AN HIV PROTEASE SUBSTRATE

Thymidylate synthase (TS) is an essential enzyme of DNA metabolism. We have carried out an extensive insertional mutagenesis of the Escherichia coli TS gene (thyA) using three different methods. Insertion of exogenous sequences at unique restriction sites or at random positions produced defective mutants, whereas comparison of TS sequences from different species allowed us to identify six zones permissive for insertions of exogenous sequences. The insertion of Human immunodeficiency virus type 1 (HIV-1) protease substrate sequences into the permissive sites converted TS to an HIV-1 protease substrate, and the in vivo cleavage of these insertions by the cloned HIV-1 protease conferred a thymidylate synthase-deficient phenotype in some of our E. coli mutant strains. In agreement with crystallographic data, these results show that the permissive sites are located in regions of the TS protein not essential for enzyme activity and accessible to cleavage by HIV protease. These results also show that it is possible to control a growth phenotype in E. coli through the protease-mediated destruction of an essential metabolic enzyme. Because both wild type and thymidylate synthase-deficient phenotypes are selectable on the appropriate growth medium, these thyA mutants could be used for genetic selections of protease inhibitors and analysis of protease specificities.