Denise Guetard

HIV-1 Genome Nuclear Import Is Mediated by a Central DNA Flap

HIV-1 and other lentiviruses have the unique property among retroviruses to replicate in nondividing cells. This property relies on the use of a nuclear import pathway enabling the viral DNA to cross the nuclear membrane of the host cell. In HIV-1 reverse transcription, a central strand displacement event consecutive to central initiation and termination of plus strand synthesis creates a plus strand overlap: the central DNA flap. We show here that the central DNA flap acts as a cis-determinant of HIV-1 DNA nuclear import. Wild-type viral linear DNA is almost entirely imported into the nucleus where it integrates or circularizes. In contrast, mutant viral DNA, which lacks the DNA flap, accumulates in infected cells as unintegrated linear DNA, at the vicinity of the nuclear membrane. Consistently, HIV-1 vectors devoid of DNA flap exhibit a strong defect of nuclear import, which can be corrected to wild-type levels by reinsertion of the DNA flap sequence.

Human Immunodeficiency Virus Type 2 Infection Associated with AIDS in West Africa

We recently reported the isolation of a new retrovirus, termed human immunodeficiency virus type 2 (HIV-2), from two West African patients with the acquired immunodeficiency syndrome (AIDS). This virus is related to but distinct from the well-characterized AIDS retrovirus, human immunodeficiency virus type 1 (HIV-1). We report here evidence of infection with HIV-2 in 30 patients, almost all from West Africa. Seventeen of them had a clinical syndrome indistinguishable from AIDS (7 of these 17 died). Others had either the AIDS-related complex or no HIV-related symptoms. All patients had serum antibodies reacting with HIV-2 in an indirect immunofluorescence assay. All serum tested contained antibodies reacting with the envelope glycoprotein of the virus in an immunoprecipitation assay. Cross-reactivity of serum antibodies with HIV-1 was detected in a minority of patients and varied according to the assay used. Retroviral isolates were obtained from the blood lymphocytes of 11 patients and were all identified as HIV-2 by nucleic acid hybridization; none hybridized with an HIV-1 probe. These findings indicate that some cases of AIDS in West Africa may be caused by HIV-2, but the extent of the spread of this virus and its clinical correlates will require careful epidemiologic investigation.

Evidence for Editing of Human Papillomavirus DNA by APOBEC3 in Benign and Precancerous Lesions

Cytidine deaminases of the APOBEC3 family all have specificity for single-stranded DNA, which may become exposed during replication or transcription of double-stranded DNA. Three human APOBEC3A (hA3A), hA3B, and hA3H genes are expressed in keratinocytes and skin, leading us to determine whether genetic editing of human papillomavirus (HPV) DNA occurred. In a study of HPV1a plantar warts and HPV16 precancerous cervical biopsies, hyperedited HPV1a and HPV16 genomes were found. Strictly analogous results were obtained from transfection experiments with HPV plasmid DNA and the three nuclear localized enzymes: hA3A, hA3C, and hA3H. Thus, stochastic or transient overexpression of APOBEC3 genes may expose the genome to a broad spectrum of mutations that could influence the development of tumors.

Massive APOBEC3 Editing of Hepatitis B Viral DNA in Cirrhosis

DNA viruses, retroviruses and hepadnaviruses, such as hepatitis B virus (HBV), are vulnerable to genetic editing of single stranded DNA by host cell APOBEC3 (A3) cytidine deaminases. At least three A3 genes are up regulated by interferon-α in human hepatocytes while ectopic expression of activation induced deaminase (AICDA), an A3 paralog, has been noted in a variety of chronic inflammatory syndromes including hepatitis C virus infection. Yet virtually all studies of HBV editing have confined themselves to analyses of virions from culture supernatants or serum where the frequency of edited genomes is generally low (≤10−2). We decided to look at the nature and frequency of HBV editing in cirrhotic samples taken during removal of a primary hepatocellular carcinoma. Forty-one cirrhotic tissue samples (10 alcoholic, 10 HBV+, 11 HBV+HCV+ and 10 HCV+) as well as 4 normal livers were studied. Compared to normal liver, 5/7 APOBEC3 genes were significantly up regulated in the order: HCV±HBV>HBV>alcoholic cirrhosis. A3C and A3D were up regulated for all groups while the interferon inducible A3G was over expressed in virus associated cirrhosis, as was AICDA in ∼50% of these HBV/HCV samples. While AICDA can indeed edit HBV DNA ex vivo, A3G is the dominant deaminase in vivo with up to 35% of HBV genomes being edited. Despite these highly deleterious mutant spectra, a small fraction of genomes survive and contribute to loss of HBeAg antigenemia and possibly HBsAg immune escape. In conclusion, the cytokine storm associated with chronic inflammatory responses to HBV and HCV clearly up regulates a number of A3 genes with A3G clearly being a major restriction factor for HBV. Although the mutant spectrum resulting from A3 editing is highly deleterious, a very small part, notably the lightly edited genomes, might help the virus evolve and even escape immune responses.

Spermatozoa as potential carriers of HIV

In order to investigate the role of germ cells in the sexual transmission of immunodeficiency virus (HIV), spermatozoa from healthy HIV-seronegative men were incubated in vitro with HIV1. After washing, they were cocultured with peripheral blood leukocytes from seronegative blood donors. Reverse transcriptase assays and p24 antigen tests were performed in culture supernatants. Electron microscopy examination of these HIV-incubated spermatozoa was carried out, as well as the search for CD4 molecules on their surface. Although virus bound to and seemed to enter spermatozoa despite the absence of detectable CD4 epitopes on their surface, no replication of HIV was apparent. However, HIV particles on the surface of spermatozoa were capable of infecting CD4 T lymphocytes. Present results would seem to preclude artificial insemination between an HIV-seropositive man and an HIV-seronegative woman.

Protective activity of tetracycline analogs against the cytopathic effect of the human immunodeficiency viruses in CEM cells

Tetracycline analogs were evaluated for anti-HIV activity in CEM cells; minocycline and doxycycline were the most active of these in inhibiting the virus-induced cytopathic effect between 7 and 14 days post-infection. The active concentrations (0.3-1.5 micrograms/ml) were devoid of toxicity in uninfected cultures. Virus production, however, was not inhibited, indicating a dissociation between protection against cell death and suppression of virus growth. These protected cells could be maintained in culture for 6-7 weeks, even in the absence of the compounds. After that period, virus production ceased and cells could then be cultivated for several months without loss of viability or reappearance of virus production. As HIV stocks produced in the presence of tetracycline analogs were unable to induce cell death, we suggest that the cytopathogenicity of HIV may be due in some cases to the presence of tetracycline-sensitive contaminating microorganisms.

In vitro sensitivity of human herpesvirus-6 to antiviral drugs

We studied the sensitivity of human herpesvirus-6 (HHV-6) to 4 antiviral drugs known to be effective in the treatment of infections with other human herpesviruses and human immunodeficiency virus. HHV-6 was grown in peripheral blood lymphocytes, and virus multiplication was quantified by evaluation of the cytopathic effect by molecular hybridization and indirect immunofluorescence assay. The 50% and 90% inhibitory concentrations (IC50 and IC90) were determined for each drug. The results obtained by the 3 different quantification techniques were found to correlate, and enabled us to conclude that HHV-6 replication was readily inhibited by foscarnet or ganciclovir. In contrast, inhibition of HHV-6 replication was observed only at high concentrations of acyclovir, and was not detected at the tested concentrations of zidovudine.

CONCOMITANT INFECTION BY HUMAN HERPESVIRUS 6, HTLV-I, AND HIV-2

SiR,-HIV-1 and HIV-2 are the causative agents of AIDS; HTLV-1, another human lymphotropic retrovirus, has been associated with adult T-cell leukaemia (ATL); and a new lymphotropic virus, human herpesvirus 6 (HHV-6, previously known as HBLV), has been isolated from patients with AIDS or lymphoproliferative disorders. 1-3 We have isolated HHV-6 from a patient with both HTLV-I and HIV-2 infections. A 19-year-old woman from the Ivory Coast was admitted to hospital with typical ATL and opportunistic infections. Despite

Murine APOBEC1 is a powerful mutator of retroviral and cellular RNA in vitro and in vivo.

Mammalian APOBEC molecules comprise a large family of cytidine deaminases with specificity for RNA and single-stranded DNA (ssDNA). APOBEC1s are invariably highly specific and edit a single residue in a cellular mRNA, while the cellular targets for APOBEC3s are not clearly established, although they may curtail the transposition of some retrotransposons. Two of the seven member human APOBEC3 enzymes strongly restrict human immunodeficiency virus type 1 in vitro and in vivo. We show here that ssDNA hyperediting of an infectious exogenous gammaretrovirus, the Friend-murine leukemia virus, by murine APOBEC1 and APOBEC3 deaminases occurs in vitro. Murine APOBEC1 was able to hyperdeaminate cytidine residues in murine leukemia virus genomic RNA as well. Analysis of the edited sites shows that the deamination in vivo was due to mouse APOBEC1 rather than APOBEC3. Furthermore, murine APOBEC1 is able to hyperedit its primary substrate in vivo, the apolipoprotein B mRNA, and a variety of heterologous RNAs. In short, murine APOBEC1 is a hypermutator of both RNA and ssDNA in vivo, which could exert occasional side effects upon overexpression.

Genetic Editing of HBV DNA by Monodomain Human APOBEC3 Cytidine Deaminases and the Recombinant Nature of APOBEC3G

Hepatitis B virus (HBV) DNA is vulnerable to editing by human cytidine deaminases of the APOBEC3 (A3A-H) family albeit to much lower levels than HIV cDNA. We have analyzed and compared HBV editing by all seven enzymes in a quail cell line that does not produce any endogenous DNA cytidine deaminase activity. Using 3DPCR it was possible to show that all but A3DE were able to deaminate HBV DNA at levels from 10−2 to 10−5 in vitro, with A3A proving to be the most efficient editor. The amino terminal domain of A3G alone was completely devoid of deaminase activity to within the sensitivity of 3DPCR (∼10−4 to 10−5). Detailed analysis of the dinucleotide editing context showed that only A3G and A3H have strong preferences, notably CpC and TpC. A phylogenic analysis of A3 exons revealed that A3G is in fact a chimera with the first two exons being derived from the A3F gene. This might allow co-expression of the two genes that are able to restrict HIV-1Δvif efficiently.