The structure of FIV reverse transcriptase and its implications for non-nucleoside inhibitor resistance.

Reverse transcriptase (RT) is the target for the majority of anti-HIV-1 drugs. As with all anti-AIDS treatments, continued success of RT inhibitors is persistently disrupted by the occurrence of resistance mutations. To explore latent resistance mechanisms potentially accessible to therapeutically challenged HIV-1 viruses, we examined RT from the related feline immunodeficiency virus (FIV). FIV closely parallels HIV-1 in its replication and pathogenicity, however, is resistant to all non-nucleoside inhibitors (NNRTI). The intrinsic resistance of FIV RT is particularly interesting since FIV harbors the Y181 and Y188 sensitivity residues absent in both HIV-2 and SIV. Unlike RT from HIV-2 or SIV, previous efforts have failed to make FIV RT susceptible to NNRTIs concluding that the structure or flexibility of the feline enzyme must be profoundly different. We report the first crystal structure of FIV RT and, being the first structure of an RT from a non-primate lentivirus, enrich the structural and species repertoires available for RT. The structure demonstrates that while the NNRTI binding pocket is conserved, minor subtleties at the entryway can render the FIV RT pocket more restricted and unfavorable for effective NNRTI binding. Measuring NNRTI binding affinity to FIV RT shows that the "closed" pocket configuration inhibits NNRTI binding. Mutating the loop residues rimming the entryway of FIV RT pocket allows for NNRTI binding, however, it does not confer sensitivity to these inhibitors. This reveals a further layer of resistance caused by inherent FIV RT variances that could have enhanced the dissociation of bound inhibitors, or, perhaps, modulated protein plasticity to overcome inhibitory effects of bound NNRTIs. The more "closed" conformation of FIV RT pocket can provide a template for the development of innovative drugs that could unlock the constrained pocket, and the resilient mutant version of the enzyme can offer a fresh model for the study of NNRTI-resistance mechanisms overlooked in HIV-1.

Expression of APOBEC3 Lentiviral Restriction Factors in Cats.

Feline immunodeficiency virus (FIV) is a naturally occurring T-cell tropic lentiviral disease of felids with many similarities to HIV/AIDS in humans. Similar to primate lentiviral-host interactions, feline APOBEC3 (A3) has been shown to inhibit FIV infection in a host-specific manner and feline A3 degradation is mediated by FIV Vif. Further, infection of felids with non-native FIV strains results in restricted viral replication in both experimental and naturally occurring infections. However, the link between molecular A3-Vif interactions and A3 biological activity during FIV infection has not been well characterized. We thus examined expression of the feline A3 genes A3Z2, A3Z3 and A3Z2-Z3 during experimental infection of domestic cats with host-adapted domestic cat FIV (referred to as FIV) and non-adapted Puma concolor FIV (referred to as puma lentivirus, PLV). We determined A3 expression in different tissues and blood cells from uninfected, FIV-infected, PLV-infected and FIV/PLV co-infected cats; and in purified blood cell subpopulations from FIV-infected and uninfected cats. Additionally, we evaluated regulation of A3 expression by cytokines, mitogens, and FIV infection in cultured cells. In all feline cells and tissues studied, there was a striking difference in expression between the A3 genes which encode FIV inhibitors, with A3Z3 mRNA abundance exceeding that of A3Z2-Z3 by 300-fold or more. Interferon-alpha treatment of cat T cells resulted in upregulation of A3 expression, while treatment with interferon-gamma enhanced expression in cat cell lines. In cats, secondary lymphoid organs and peripheral blood mononuclear cells (PBMC) had the highest basal A3 expression levels and A3 genes were differentially expressed among blood T cells, B cells, and monocytes. Acute FIV and PLV infection of cats, and FIV infection of primary PBMC resulted in no detectable change in A3 expression with the exception of significantly elevated A3 expression in the thymus, the site of highest FIV replication. We conclude that cat A3 expression is regulated by cytokine treatment but, by and large, lentiviral infection did not appear to alter expression. Differences in A3 expression in different blood cell subsets did not appear to impact FIV viral replication kinetics within these cells. Furthermore, the relative abundance of A3Z3 mRNA compared to A3Z2-Z3 suggests that A3Z3 may be the major active anti-lentiviral APOBEC3 gene product in domestic cats.

Effects of an oral superoxide dismutase enzyme supplementation on indices of oxidative stress, proviral load, and CD4:CD8 ratios in asymptomatic FIV-infected cats.

This study was designed to test the effect of antioxidant supplementation on feline immunodeficiency virus (FIV)-infected felines. Six acutely FIV-infected cats (> or =16 weeks post-inoculation) were given a propriety oral superoxide dismutase (SOD) supplement (Oxstrin; Nutramax Laboratories) for 30 days. Following supplementation, the erythrocyte SOD enzyme concentration was significantly greater in the supplemented FIV-infected group than the uninfected control group or the unsupplemented FIV-infected group. The CD4+ to CD8+ ratio increased significantly (0.66-0.88) in the SOD supplemented FIV-infected cats but not in the unsupplemented FIV-infected cats. Proviral load and reduced glutathione (GSH) levels in leukocyte cell types did not change significantly following supplementation. Antioxidant supplementation resulted in an increase in SOD levels, confirming the oral bioavailability of the compound in FIV-infected cats. This result warrants further investigation with trials of antioxidant therapy in FIV-infected cats that are showing clinical manifestations of their disease, as well as in other feline patients where oxidative stress likely contributes to disease pathogenesis, such as diabetes mellitus and chronic renal failure.

Adenosine deaminase activity in cats infected with feline immunodeficiency virus.

Adenosine deaminase (ADA), an enzyme involved in purine metabolism, has been shown to be of clinical importance in several diseases in humans. To investigate whether ADA is of any clinical significance in cats, plasma adenosine deaminase (P-ADA) and T cell adenosine deaminase (T-ADA) activities were measured in feline immunodeficiency virus (FIV) negative and positive cats. The AIDS-related complex (ARC) group showed a significant elevation in P-ADA activity compared to the asymptomatic carrier (AC), and FIV-negative groups (P<0.005). T-ADA activity was significantly elevated in FIV-positive cats compared to the FIV-negative group (P<0.05) and this elevation was attributed to the increase in the ARC group (P<0.01). A correlation was found between P-ADA and T-ADA activities in the FIV-negative group. T-ADA activity and CD4(+)cell number showed a strong negative correlation in FIV-positive cats (P<0.0005). CD4(+) cell numbers were significantly reduced in the ARC group compared to the healthy controls (P<0.005). Our results showed that T-ADA is increased in FIV-positive cats during the ARC stage. These results also suggest that ADA may be an indicator of T cell activation in the ARC stage of FIV infection.

Quantitative measurement of antibody inhibiting reverse transcriptase activity in cats naturally infected with feline immunodeficiency virus.

Quantitative measurement of reverse transcriptase-inhibiting (RTI) antibodies in Japanese household cats naturally infected with feline immunodeficiency virus (FIV) was performed by poly A-linked colorimetric reverse transcriptase assay (PAC-RTA). Eight FIV-seropositive plasma samples were diluted twofold from 1:10 to 1:160 and incubated with FIV RT. Fifty percent RTI activity (RTI50) was calculated from a dose response PAC-RTA curve. The plasma of FIV-seropositive cats showed different RTI activities against two Japanese isolates and Petaluma strain. Six of eight plasma samples showed RTI activities against the Japanese isolates (subtype B), but only one showed RTI activity against Petaluma strain (subtype A). It is important to use the appropriate strain as a source of RT for detection of RTI antibody in cats.

Molecular and functional interactions of cat APOBEC3 and feline foamy and immunodeficiency virus proteins: different ways to counteract host-encoded restriction.

Defined host-encoded feline APOBEC3 (feA3) cytidine deaminases efficiently restrict the replication and spread of exogenous retroviruses like Feline Immunodeficiency Virus (FIV) and Feline Foamy Virus (FFV) which developed different feA3 counter-acting strategies. Here we characterize the molecular interaction of FFV proteins with the diverse feA3 proteins. The FFV accessory protein Bet is the virus-encoded defense factor which is shown here to bind all feA3 proteins independent of whether they restrict FFV, a feature shared with FIV Vif that induces degradation of all feA3s including those that do not inactivate FIV. In contrast, only some feA3 proteins bind to FFV Gag, a pattern that in part reflects the restriction pattern detected. Additionally, one-domain feA3 proteins can homo- and hetero-dimerize in vitro, but a trans-dominant phenotype of any of the low-activity feA3 forms on FFV restriction by one of the highly-active feA3Z2 proteins was not detectable.

Comparative neurovirulence in lentiviral infections: The roles of viral molecular diversity and select proteases.

All lentiviruses infect the brain, causing chronic neurological disease in their respective hosts. To examine the relationship(s) between lentivirus molecular diversity and the development of neurological disease, we examined in vitro and in vivo models of lentivirus neurovirulence using different recombinant viruses derived from human (HIV-1) and feline (FIV) immunodeficiency viruses. Both in vitro and in vivo studies of FIV neurovirulence showed that the FIV envelope derived from a neurovirulent strain was a principal determinant of neuropathogenesis, although systemic immunosuppression was also an integral feature of FIV neurovirulence. Studies of HIV-1 envelope sequences derived from brain or blood indicate that molecular diversity is greater in viruses from patients with HIV-associated dementia (HAD), compared to nondemented individuals. Moreover, the hypervariable V3 domain of HIVgp120, regardless of the HIV-1 clade from which it was derived, was an important region for mediating neurotoxicity in vitro but the level of viral replication did not influence neurotoxicity. For both the HIV-1 and FIV envelopes and HIV-1 Tat, induction of matrix metalloproteinase (MMP)-2 in macrophages was a consistent finding. Neurotoxicity caused by supernatants from HIV-infected or transfected macrophages, containing MMP-2, was greater than direct neurotoxicity levels caused by direct exposure of neurons to virus in assays of total neuronal death, but not in assays of neuronal apoptosis. Proteinase-activated receptor (PAR)-1 and its ligand thrombin were also induced during HIV infection, chiefly on astrocytes. PAR-1 activation resulted in gliosis and neurobehavioral changes in an animal model and resulted in N-methyl-D-aspartate (NMDA) receptor-mediated neuronal death. These findings suggest that the lentivirus envelope, which is a domain of extensive molecular diversity in brain-derived lentivirus isolates, directly influences neuropathogenesis through the activation of select proteases, underscoring the importance of concentrating on individual viral genes and proteases in the development of neuroprotective agents for HIV-related neurological disease.

Apoptosis and CD4+ lymphocyte depletion following feline immunodeficiency virus infection of a T-lymphocyte cell line.

An interleukin-2-dependent feline T-lymphocyte cell line (FCD4-D), of which 65% of cells express CD4, was inoculated with the NCSU-1 isolate of feline immunodeficiency virus (FIV(NCSU-1)) and subsequently monitored for percentage of viable cells, percentage of apoptotic cells, percentage of CD4-expressing cells, and virus production. A decrease in viability from 91% to 12% over an 11-day postinoculation period was associated with an increase in the percentage of cells with nuclear morphology suggestive of apoptosis from < 5% to 97% based on ethidium bromide and acridine orange fluorescence. These changes were associated with a 24% reduction in the percentage of viable CD4-expressing cells at 7 days postinoculation. The relative amount of low-molecular-weight nuclear DNA was greater in FIV-infected cultures than in uninfected cultures from day 7 to day 15 postinoculation. This DNA was characterized by cleavage into fragments differing in size by approximately 180 base pairs. Ultrastructurally, nuclear chromatin and cytoplasm were condensed into discrete electron-dense bodies, and cell membrane projections were lost. Syncytia were occasionally present in FIV-inoculated cultures. Cytologic changes were associated with a logarithmic rise in Mg+2-dependent reverse transcriptase levels in culture supernatants on days 4-7 postinoculation. Supplementation of FIV-inoculated culture medium with 1 mM ZnCl2 enhanced viability, decreased the percentage of cells undergoing apoptosis, and prevented the loss of CD4+ lymphocytes at 7 days postinoculation. These data suggest that feline CD4+ lymphocytes die by apoptosis following in vitro infection with FIV(NCSU-1). The feline/FIV model may be a suitable system to investigate the mechanisms of lentivirus-associated CD4+ lymphocyte depletion in vivo.

Ultrasensitive retrovirus detection by a reverse transcriptase assay based on product enhancement.

Reverse transcriptase (RT) is an indispensable component of infectious retroviruses. We have developed an ultrasensitive RT test in which RNA of bacteriophage MS2 serves as the template for RT-mediated cDNA synthesis. A fragment of the cDNA is selectively amplified by polymerase chain reaction and the amplification product is analyzed by Southern blot hybridization or enzyme immunoassay. The procedure was 10(6) to 10(7) times more sensitive than a conventional RT test and detected as little as 10(-9) unit of murine leukemia virus RT, which corresponded to 2.1 x 10(2) molecules, a number present in 3-11 virions. As a screening assay for filterable particle-associated RT, it was positive with supernatants from cell cultures producing human immunodeficiency virus (HIV) type 1 or human T-cell leukemia virus (HTLV) type 1 or 2, but was negative with nonproducer cultures. It was positive with plasma samples from all tested individuals infected with HIV-1, HIV-2, or HTLV-1 and sera from cats infected with feline leukemia virus or feline immunodeficiency virus. Control samples from blood donors or uninfected cats were negative. Density banding experiments with culture supernatants showed that the RT activity was associated with virus particles. The assay should detect all replication-competent retroviruses or similar agents. It may be used as a screening assay for such agents, for quantitation of the viral load, drug susceptibility testing of RT, and control of virus inactivation in biological products.

Analysis of the S3 and S3' subsite specificities of feline immunodeficiency virus (FIV) protease: development of a broad-based protease inhibitor efficacious against FIV, SIV, and HIV in vitro and ex vivo.

The S3 and S3' subsite binding specificities of HIV and feline immunodeficiency virus proteases (FIV) proteases (PRs) have been explored by using C2-symmetric competitive inhibitors. The inhibitors evaluated contained (1S, 2R, 3R, 4S)-1,4-diamino-1, 4-dibenzyl-2,3-diol as P1 and P1' units, Val as P2 and P2' residues, and a variety of amino acids at the P3 and P3' positions. All inhibitors showed very high potency against HIV PR in vitro, and their Ki values ranged between 1.1 and 2.6 nM. In contrast to the low restriction of P3 and P3' residues observed in HIV PR, FIV PR exhibited strong preference for small hydrophobic groups at the S3 and S3' subsites. Within this series, the most effective inhibitor against FIV PR contained Ala at P3 and P3'. Its Ki of 41 nM was 415- and 170-fold lower than those of the inhibitors without the P3 and P3' moieties or with the Phe at these positions, respectively. In addition, these compounds were tested against mutant FIV PRs, which contain amino acid substitutions corresponding to those in native HIV PR at homologous sites, and their efficacy of inhibition progressively increased up to 5-fold. The most potent FIV PR inhibitor was selected for examination of its effectiveness in tissue culture, and it was able to block nearly 100% of virus production in an acute infection at 1 microg/ml (1.1 microM) against HIV, FIV, and simian immunodeficiency virus. Furthermore, it was not toxic to cells, and even after 2 months of culture there was no sign of resistance development by virus. The findings suggest that inhibitors with small P3 residue may be efficacious against a broad range of HIV variants as well as interspecies PRs.