Elizabeth Fink

Feline immunodeficiency virus (FIV) as a model for study of lentivirus infections: parallels with HIV.

FIV is a significant pathogen in the cat and is, in addition, the smallest available natural model for the study of lentivirus infections. Although divergent at the amino acid level, the cat lentivirus has an abundance of structural and pathophysiological commonalities with HIV and thus serves well as a model for development of intervention strategies relevant to infection in both cats and man. The following review highlights both the strengths and shortcomings of the FIV/cat model, particular as regards development of antiviral drugs.

Fine Definition of the CXCR4-Binding Region on the V3 Loop of Feline Immunodeficiency Virus Surface Glycoprotein

The chemokine receptor CXCR4 is shared by primary and laboratory-adapted strains of feline immunodeficiency virus (FIV) for viral entry. Our previous studies implicated a contiguous nine-amino-acid region of the V3 loop of the FIV envelope surface as important in CXCR4 binding and virus entry. The binding is specific for CXCR4 since it can be inhibited by AMD3100, a selective CXCR4 inhibitor. Additional site-directed mutagenesis was used to further reveal the key residues. Binding studies indicated that basic residues R395, K397, R399 as well as N398 are critical for CXCR4 binding. The effect of other amino acid residues on receptor binding depends on the type of amino acid residue substituted. The binding study results were confirmed on human CXCR4-expressing SupT1 cells and correlated with entry efficiency using a virus entry assay. Amino acid residues critical for CXCR4 are not critical for interactions with the primary binding receptor CD134, which has an equivalent role as CD4 for HIV-1 binding. The ELISA results show that W394 and W400 are crucial for the recognition by neutralizing anti-V3 antibodies. Since certain strains of HIV-1 also use CXCR4 as the entry receptor, the findings make the feline model attractive for development of broad-based entry antagonists and for study of the molecular mechanism of receptor/virus interactions.

OrfA Downregulates Feline Immunodeficiency Virus Primary Receptor CD134 on the Host Cell Surface and Is Important in Viral Infection

ABSTRACT Feline immunodeficiency virus (FIV) OrfA is an accessory protein that is critical for productive viral replication and infection in T cells. Here, we show that OrfA acts to markedly reduce cell surface expression of the FIV primary binding receptor. Downregulation does not occur at the transcriptional or translational level in that the amounts of CD134 mRNA and protein in total cell lysates are not altered between parental 104-C1 T cells and the same cell line stably expressing OrfA (104-C1-OrfA). Analysis by confocal microscopy revealed significant accumulation of CD134 in the Golgi apparatus of 104-C1 cells expressing OrfA. OrfA does not cause a generalized disruption of membrane trafficking in that surface expression of CD9 is unaffected by OrfA overexpression. Consistent with the above observations, OrfA-negative FIV-34TF10 productively infects CrFK (CD134-negative) and 104-C1-OrfA (CD134 downregulated by OrfA) cells but fails to productively infect either 104-C1 (CD134-positive) cells or GFox (CrFK cells overexpressing CD134) cells. FIV-34TF10 in which the OrfA reading frame is open (OrfArep) productively infects CrFK, GFox, 104-C1, and 104-C1-OrfA cells. We hypothesize that reduced surface expression of the receptor, a hallmark of retrovirus infections, may facilitate an increase in virus release from the infected cell by minimizing receptor interactions with budding virus particles.

Improved health and survival of FIV-infected cats is associated with the presence of autoantibodies to the primary receptor, CD134

We analyzed antibody responses in sera from feline immunodeficiency virus (FIV)-infected and uninfected cats. A strong antiviral response to the viral surface glycoprotein (SU) was noted in both natural and experimental infections. In addition, 143 of 226 FIV-infected animals (63%) also expressed antibodies to the primary binding receptor, CD134, whereas cats infected with other feline RNA viruses, including calicivirus, coronavirus, herpesvirus, and feline leukemia virus, did not. Both affinity-purified anti-CD134 and anti-SU antibodies blocked FIV infection ex vivo. FACS analyses revealed that the anti-CD134 antibodies bound to a cryptic epitope on the receptor that was only exposed when SU bound to CD134. Anti-CD134 binding caused displacement of SU from the surface of the cell and inhibition of infection. The presence of antibodies to CD134 correlated with lower virus loads and a better overall health status in FIV+ cats, whereas anti-SU antibodies were present independent of health status. The findings are consistent with a role for antireceptor antibodies in protection from virus spread and disease progression.

Identification of Amino Acid Residues Important for Heparan Sulfate Proteoglycan Interaction within Variable Region 3 of the Feline Immunodeficiency Virus Surface Glycoprotein

ABSTRACT Heparan sulfate proteoglycans (HSPGs) act as binding receptors or attachment factors for the viral envelope of many viruses, including strains of HIV and feline immunodeficiency virus (FIV). The FIV gp95 glycoprotein (SU) from laboratory-adapted strains (tissue culture adapted [TCA]) such as FIV-34TF10 can bind to HSPG, whereas SU from field strains (FS) such as FIV-PPR cannot. Previous studies indicate that SU-HSPG interactions occur within the V3 loop. We utilized a series of nested V3 peptides to further map the HSPG binding sites and found that both sides of the predicted V3 loop stem were critical for the binding but not the CXCR4 binding domain near the predicted tip of the V3 loop. Neutralization assays for TCA strain entry using the same set of V3 peptides showed that peptides targeting CXCR4 or HSPG binding sites can block infection, supporting the V3 loop as a critical neutralization target. Site-directed mutagenesis identified two highly conserved arginines, R379 and R389, on the N-terminal side of the V3 stem as critical for the contact between SU and HSPG. Residues K407, K409, K410, and K412 on the C-terminal side of the V3 stem form a second nonconserved domain necessary for HSPG binding, consistent with the observed specificity distinctions with FS FIV. Our findings discriminate structural determinants important for HSPG and CXCR4 binding by FIV SU and thus further define the importance of the V3 loop for virus entry and infection.

Type 1 diabetes pathogenesis is modulated by spontaneous autoimmune responses to endogenous retrovirus antigens in NOD mice

Secreted microvesicles (MVs) are potent inflammatory triggers that stimulate autoreactive B and T cells, causing Type 1 Diabetes in non‐obese diabetic (NOD) mice. Proteomic analysis of purified MVs released from islet cells detected the presence of endogenous retrovirus (ERV) antigens, including Env and Gag sequences similar to the well‐characterized murine leukemia retroviruses. This raises the possibility that ERV antigens may be expressed in the pancreatic islets via MV secretion. Using virus‐like particles produced by co‐expressing ERV Env and Gag antigens, and a recombinant gp70 Env protein, we demonstrated that NOD but not diabetes‐resistant mice developed anti‐Env autoantibodies that increase in titer as disease progresses. A lentiviral‐based RNA interference knockdown of Gag revealed that Gag contributes to the MV‐induced T‐cell response, whose diabetogenic function can be demonstrated via cell‐transfer into immune‐deficient mice. Finally, we observed that Gag and Env are expressed in NOD islet‐derived primary mesenchymal stem cells (MSCs). However, MSCs derived from the islets of diabetes‐resistant mice do not express the antigens. Taken together, abnormal ERV activation and secretion of MVs may induce anti‐retroviral responses to trigger autoimmunity.

FIV vaccine with receptor epitopes results in neutralizing antibodies but does not confer resistance to challenge

Feline immunodeficiency virus (FIV) is the feline analogue to human immunodeficiency virus (HIV) and utilizes parallel modes of receptor-mediated entry. The FIV surface glycoprotein (SU) is an important target for induction of neutralizing antibodies, and autoantibodies to the FIV binding receptor (CD134) block infection ex vivo; thus highlighting the potential for immunotherapies which utilize anti-receptor antibodies to block viral infection. To determine whether vaccination with CD134-SU complexes could induce protection against FIV infection, cats (n = 5 per group) were immunized with soluble CD134, recombinant FIV-SU protein, and/or CD134+SU complexes. Two trials were performed with different antigen combinations and vaccination schedules. In vivo generation of anti-CD134 and anti-SU IgG antibodies was measured, and in vitro neutralization assays were conducted. Immunization induced production of anti-CD134 and anti-SU antibodies that significantly inhibited FIV infection in vitro. However, no vaccine combination protected cats from FIV infection, and neat serum from vaccinated cats enhanced FIV growth in vitro. CD134+SU vaccinated cats exhibited increased CD4:CD8 ratio immediately prior to challenge, and antibodies were much more efficiently generated against vaccine by-products versus target antigens. Results suggest vaccination against viral and cryptic receptor epitopes yields neutralizing antibodies that synergistically inhibit FIV infection in vitro. Factors contributing to vaccine failure may include: (1) Heat-labile serum factors that enhance viral replication, (2) changes in circulating target cell populations induced by vaccination, and (3) weak immunogenicity of neutralizing epitopes compared to off-target vaccine components. Results reinforce the need to monitor vaccine preparation components and avoid non-specific immune stimulation during vaccination.Feline immunodeficiency virus: In vivo protection remains elusiveA vaccine candidate for feline immunodeficiency virus elicits strong immunological reaction in vitro, but no protection to live cats. The feline analog to human immunodeficiency virus, FIV shares a similar infection paradigm and has only one partially effective vaccine. A US team, led by Colorado State University’s Susan VandeWoude, immunized cats using a complex of an FIV surface protein and a feline cell-surface protein known to facilitate FIV’s entry into immune cells. Tissue culture assays yielded promising results; however, this did not translate to live-animal protection. The researchers highlighted multiple factors that could explain the lack of success, including circulatory pro-infection factors, and immune responses generated against vaccine by-products rather than intended targets. While the vaccine candidate failed, the research provides invaluable guidance for future efforts into FIV vaccination with implications for HIV vaccine trials.