Feline Immunodeficiency Virus Proteins Research Articles

Looking for Novel Capsid Protein Multimerization Inhibitors of Feline Immunodeficiency Virus.

Feline immunodeficiency virus (FIV) is a member of the retroviridae family of viruses. It causes acquired immunodeficiency syndrome (AIDS) in worldwide domestic and non-domestic cats and is a cause of an important veterinary issue. The genome organization of FIV and the clinical characteristics of the disease caused by FIV are similar to human immunodeficiency virus (HIV). Both viruses infect T lymphocytes, monocytes, and macrophages, with a similar replication cycle in infected cells. Thus, the infection of cats with FIV is also a useful tool for the study and development of novel drugs and vaccines against HIV. Anti-retroviral drugs studied extensively with regards to HIV infection have targeted different steps of the virus replication cycle: (1) disruption of the interaction with host cell surface receptors and co-receptors; (2) inhibition of fusion of the virus and cell membranes; (3) blocking of the reverse transcription of viral genomic RNA; (4) interruption of nuclear translocation and integration of viral DNA into host genomes; (5) prevention of viral transcript processing and nuclear export; and (6) inhibition of virion assembly and maturation. Despite the great success of anti-retroviral therapy in slowing HIV progression in humans, a similar therapy has not been thoroughly investigated for FIV infection in cats, mostly because of the little structural information available for FIV proteins. The FIV capsid protein (CA) drives the assembly of the viral particle, which is a critical step in the viral replication cycle. During this step, the CA protein oligomerizes to form a protective coat that surrounds the viral genome. In this work, we perform a large-scale screening of four hundred molecules from our in-house library using an in vitro assembly assay of p24, combined with microscale thermophoresis, to estimate binding affinity. This screening led to the discovery of around four novel hits that inhibited capsid assembly in vitro. These may provide new antiviral drugs against FIV.

Densitometric analysis of Western blot assays for feline immunodeficiency virus antibodies

Western blot (WB) strips for antibodies directed to feline immunodeficiency virus (FIV) were analysed using reflectance densitometry by a semiautomatic densitometer. This method was used to quantify the antibody responses to different FIV proteins in both vaccinated and naturally or experimentally-infected cats. In order to increase reproducibility, reagents and protocols were accurately standardised and internal controls were added. In a first format, an internal control band consisting of feline IgG was added to each blot to minimise the effect of band intensity variation. In a second format, antibody concentrations were calculated from the ratio of the densities produced by test sera and by positive and negative standard sera. The sera under scrutiny were also examined by standard enzyme-linked immunosorbent assay (ELISA) and the results obtained compared with those of the corresponding WB. A statistically significant positive correlation was found between the results obtained with the two methods, and this was especially evident when ELISA titres were compared to corrected WB values ( P=0.001). Densitometric analysis of WB assays allowed to quantify the antibodies against FIV proteins and might be useful to investigate possible humoral immune correlates of protection in FIV vaccination studies and antibody production in the early phase of infection. The quantitation of antibodies to Gag and Env FIV antigens might be used to obtain further informations on the course of FIV disease, as previously demonstrated in human immunodeficiency virus-1 (HIV-1) infections.

The Feline Immunodeficiency Virus vif Gene Is Required for Productive Infection of Feline Peripheral Blood Mononuclear Cells and Monocyte-Derived Macrophages

The role of the feline immunodeficiency virus (FIV) vif gene in establishing productive infection in feline peripheral blood mononuclear cells (PBMCs) and monocyte-derived macrophages (MDMs) was examined in cell culture systems. A 375-bp deletion was introduced into the vif gene of the wild-type FIV-pPPR infectious molecular clone to produce Vif deletion mutant FIV-pPPRΔvif. This mutant FIV proviral construct expressed FIV proteins p24gag and gp100env in transfected Crandell feline kidney cells as measured by immunoprecipitation and Western blot analysis as well as immunocytochemical analysis; these cultures produced very low levels of virus by cocultivation of transfected cells with PBMCs and K-258 cells, as measured by production of p24gag. Replication kinetics of wild-type and vif-deleted virus were compared in PBMCs and monocyte-derived macrophages (MDMs) by infection with cell-free virus preparations. Similar to findings with other lentiviruses, the vif gene was found to be essential for establishment of productive FIV infection in both PBMCs and MDMs. This study indicates that vif is essential for productive FIV infection of host target cells in vitro and that FIV-pPPRΔvif may be an excellent candidate viral mutant for attenuated virus vaccine studies.

CXCR4 is required by a nonprimate lentivirus: heterologous expression of feline immunodeficiency virus in human, rodent, and feline cells.

A heterologous feline immunodeficiency virus (FIV) expression system permitted high-level expression of FIV proteins and efficient production of infectious FIV in human cells. These results identify the FIV U3 element as the sole restriction to the productive phase of replication in nonfeline cells. Heterologous FIV expression in a variety of human cell lines resulted in profuse syncytial lysis that was FIV env specific, CD4 independent, and restricted to cells that express CXCR4, the coreceptor for T-cell-line-adapted strains of human immunodeficiency virus. Stable expression of human CXCR4 in CXCR4-negative human and rodent cell lines resulted in extensive FIV Env-mediated, CXCR4-dependent cell fusion and infection. In feline cells, stable overexpression of human CXCR4 resulted in increased FIV infectivity and marked syncytium formation during FIV replication or after infection with FIV Env-expressing vectors. The use of CXCR4 is a fundamental feature of lentivirus biology independent of CD4 and a shared cellular link to infection and cytopathicity for distantly related lentiviruses that cause AIDS. Their conserved use implicates chemokine receptors as primordial lentivirus receptors.

Efficient transduction of nondividing human cells by feline immunodeficiency virus lentiviral vectors.

The molecular bases for species barriers to lentiviral replication are not well understood, but are of interest for explaining lentiviral pathogenesis, devising therapeutic strategies, and adapting lentiviruses to gene therapy. HIV-1-based lentiviral vectors efficiently transduce nondividing cells, but present complex safety concerns. Nonprimate (ungulate or feline) lentiviruses might provide safer alternatives, but these viruses display highly restricted tropisms, and their potential for adaptation as replication-defective vectors capable of transducing human cells is unknown. Feline immunodeficiency virus (FIV) does not infect humans or other non-Felidae despite prevalent natural exposure. Although long terminal repeat (LTR)-directed FIV expression was found to be negligible in human cells, promoter substitution enabled an env-deleted, three-plasmid, human cell-FIV lentiviral vector system to express high levels of FIV proteins and FIV vectors in human cells, thus bypassing the hazards of feline vector producer cells. Pseudotyped FIV vectors efficiently transduced dividing, growth-arrested, and postmitotic human targets. The experiments delineate mechanisms involved in species-restricted replication of this lentivirus and show that human cells support both productive- and infective-phase mechanisms of the FIV life cycle needed for efficient lentiviral vector transduction. Nonprimate lentiviral vectors may offer safety advantages, and FIV vectors provide unique experimental opportunities.

Eight-year observation and comparative study of specific pathogen-free cats experimentally infected with feline immunodeficiency virus (FIV) subtypes A and B: terminal acquired immunodeficiency syndrome in a cat infected with FIV petaluma strain.

Three specific pathogen-free cats experimentally infected with feline immunodeficiency virus (FIV) strains Petaluma, TM1 and TM2, respectively were observed for over 8 years. Without showing any significant clinical signs of immunodeficiency syndrome (AIDS) for 8 years and 4 months of asymptomatic phase, the Petaluma-infected cat exhibited severe stomatitis/gingivitis, anorexia, emaciation, hematological and immunological disorders such as severe anemia, lymphopenia, thrombocytopenia, and decrease of CD4/CD8 ratio to 0.075, and finally died with hemoperitoneum at 8 years and 8 months post-infection. Histopathological studies revealed that the cat had systemic lymphoid atrophy and bone marrow disorders indicating acute myelocytic leukemia (aleukemic type). Plasma viral titer of the cat at AIDS phase was considerably high and anti-FIV antibody titer was slightly low as compared with the other FIV-infected cats. In addition, immunoblotting analysis using serially collected serum/plasma samples of these cats revealed that antibodies against FIV proteins were induced in all the infected cats, however in the Petaluma-infected cat anti-Gag antibodies disappeared during the asymptomatic period. These results suggested that plasma viral load and anti-FIV Gag antibody response correlated with disease progression, and supported FIV-infected cats as a suitable animal model of human AIDS.

Feline Immunodeficiency Virus (FIV)-Specific Cytotoxic T Lymphocytes from Chronically Infected Cats Are Induced in Vitro by Retroviral Vector-Transduced Feline T Cells Expressing the FIV Capsid Protein

We have previously reported the presence of feline immunodeficiency virus (FIV)-specific, major histocompatibility complex (MHC)-restricted cytolytic T lymphocytes (CTL) in experimentally FIV-infected cats. However, the fine specificity of the CTL and the role of individual FIV proteins in inducing FIV-specific CTL responses remain unknown. In this study, we examined the in vitro induction and activity of FIV p24 capsid-specific CTL obtained from cats that had been experimentally infected with FIV Petaluma for 30 to 56 months. An amphotropic murine retrovital vector was used to generate transgenic primary feline T lymphoblasts that expressed the FIV capsid protein. When the autologous capsid-transduced T cells were used in vitro to stimulate CTL responses from peripheral blood mononuclear cells of chronically infected cats, MHC-restricted lysis of virus-infected target cells was observed. The majority of the CTL expressed CD8, and depletion of this population, but not CD4 cells, effectively diminished the CTL activity. When the autologous capsid-transduced T cells were used as target cells, lysis by capsid-induced effectors was not observed. Analysis of capsid-transduced T cell clones revealed a variable and low level of capsid expression among the clones. This study demonstrates the potential for using retroviral vectors as a means of inducing CTL effector cells that will specifically kill lentivirus-infected cells during lentiviral infection.

Nucleotide sequence and genomic organization of feline immunodeficiency virus.

An infectious molecular clone of the Petaluma strain of feline immunodeficiency virus (FIV) was isolated from a recombinant bacteriophage library containing genomic DNA prepared from FIV-infected Crandall feline kidney (CRFK) cells. The integrated provirus has a total length of 9472 base pairs. Three long open reading frames corresponding to GAG, POL, and ENV gene coding frames are evident. In addition, an open reading frame overlaps the 3' end of POL, in the region that encodes viral infectivity factor in the primate viruses. Several short open reading frames are present in the intergenic region between POL and ENV and within ENV, which may serve as exons for production of TAT and REV equivalents in FIV. Alignment of the predicted amino acid sequences of the FIV proteins with those of other lentiviruses indicates that FIV did not arise recently from any other characterized lentivirus.