Synthesis and comparison of substituted 1,2,3-dithiazole and 1,2,3-thiaselenazole as inhibitors of the feline immunodeficiency virus (FIV) nucleocapsid protein as a model for HIV infection.

We report the first biological evaluation the 1,2,3-thiaselenazole class of compound and utilising a concise synthetic approach of sulfur extrusion, selenium insertion of the 1,2,3-dithiazoles. We created a small diverse library of compounds to contrast the two ring systems. This approach has highlighted new structure activity relationship insights and lead to the development of sub-micro molar anti-viral compounds with reduced toxicity. The 1,2,3-thiaselenazole represents a new class of potential compounds for the treatment of FIV and HIV.

Novel epidithiodiketopiperazines as anti-viral zinc ejectors of the Feline Immunodeficiency Virus (FIV) nucleocapsid protein as a model for HIV infection.

Focused libraries of multi-substituted epidithiodiketopiperazines (ETP) were prepared and evaluated for efficacy of inhibiting the nucleocapsid protein function of the Feline Immunodeficiency Virus (FIV) as a model for HIV. This activity was compared and contrasted to observed toxicity utilising an in-vitro cell culture approach. This resulted in the identification of several promising lead compounds with nanomolar potency in cells with low toxicity and a favorable therapeutic index.

Feline Immunodeficiency Virus Evolutionarily Acquires Two Proteins, Vif and Protease, Capable of Antagonizing Feline APOBEC3.

The interplay between viral and host proteins has been well studied to elucidate virus-host interactions and their relevance to virulence. Mammalian genes encode apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins, which act as intrinsic restriction factors against lentiviruses. To overcome APOBEC3-mediated antiviral actions, lentiviruses have evolutionarily acquired an accessory protein, viral infectivity factor (Vif), and Vif degrades host APOBEC3 proteins via a ubiquitin/proteasome-dependent pathway. Although the Vif-APOBEC3 interaction and its evolutionary significance, particularly those of primate lentiviruses (including HIV) and primates (including humans), have been well investigated, those of nonprimate lentiviruses and nonprimates are poorly understood. Moreover, the factors that determine lentiviral pathogenicity remain unclear. Here, we focus on feline immunodeficiency virus (FIV), a pathogenic lentivirus in domestic cats, and the interaction between FIV Vif and feline APOBEC3 in terms of viral virulence and evolution. We reveal the significantly reduced diversity of FIV subtype B compared to that of other subtypes, which may associate with the low pathogenicity of this subtype. We also demonstrate that FIV subtype B Vif is less active with regard to feline APOBEC3 degradation. More intriguingly, we further reveal that FIV protease cleaves feline APOBEC3 in released virions. Taken together, our findings provide evidence that a lentivirus encodes two types of anti-APOBEC3 factors, Vif and viral protease.IMPORTANCE During the history of mammalian evolution, mammals coevolved with retroviruses, including lentiviruses. All pathogenic lentiviruses, excluding equine infectious anemia virus, have acquired the vif gene via evolution to combat APOBEC3 proteins, which are intrinsic restriction factors against exogenous lentiviruses. Here we demonstrate that FIV, a pathogenic lentivirus in domestic cats, antagonizes feline APOBEC3 proteins by both Vif and a viral protease. Furthermore, the Vif proteins of an FIV subtype (subtype B) have attenuated their anti-APOBEC3 activity through evolution. Our findings can be a clue to elucidate the complicated evolutionary processes by which lentiviruses adapt to mammals.

Neurologic disease in feline immunodeficiency virus infection: disease mechanisms and therapeutic interventions for NeuroAIDS.

Feline immunodeficiency virus (FIV) is a lentivirus that causes immunosuppression through virus-mediated CD4+ T cell depletion in feline species. FIV infection is complicated by virus-induced disease in the nervous system. FIV enters the brain soon after primary infection and is detected as FIV-encoded RNA, DNA, and proteins in microglia, macrophages, and astrocytes. FIV infection activates neuroinflammatory pathways including cytokines, chemokines, proteases, and ROS with accompanying neuronal injury and loss. Neurobehavioral deficits during FIV infection are manifested as impaired motor and cognitive functions. Several treatment strategies have emerged from studies of FIV neuropathogenesis including the therapeutic benefits of antiretroviral therapies, other protease inhibitors, anti-inflammatory, and neurotrophic compounds. Recently, insulin's antiviral, anti-inflammatory, and neuroprotective effects were investigated in models of lentivirus brain infection. Insulin suppressed HIV-1 replication in human microglia as well as FIV replication of lymphocytes. Insulin treatment diminished cytokine and chemokine activation in HIV-infected microglia while also protecting neurons from HIV-1 Vpr protein-mediated neurotoxicity. Intranasal (IN) insulin delivery for 6 weeks suppressed FIV expression in the brains of treated cats. IN insulin also reduced neuroinflammation and protected neurons in the hippocampus, striatum, and neocortex of FIV-infected animals. These morphological and molecular effects of IN insulin were confirmed by neurobehavioral studies that showed IN insulin-treated FIV-infected animals displayed improved motor and cognitive performance compared to sham-treated FIV-infected animals. Thus, FIV infection of the nervous system provides a valuable comparative in vivo model for discovering and evaluating disease mechanisms as well as developing therapeutic strategies for NeuroAIDS in humans.

Viruses as indicators of contemporary host dispersal and phylogeography: an example of feline immunodeficiency virus (FIVPle ) in free-ranging African lion (Panthera leo).

Measuring contemporary dispersal in highly mobile terrestrial species is challenging, especially when species are characterized by low levels of population differentiation. Directly transmitted viruses can be used as a surrogate for traditional methods of tracking host movement. Feline immunodeficiency virus (FIV) is a species-specific lentivirus, which has an exceptionally high mutation rate and circulates naturally in wild felids. Using samples derived from 35 lion (Panthera leo) prides, we tested the prediction that FIV in lions (FIVPle ) can be used to track the dispersal of individuals between prides. As FIVPle subtypes are geographically structured throughout Africa, we predicted that this marker could be used to detect phylogeographic structure of lions at smaller spatial scales. Phylogenetic analyses of FIVPle pol-RT sequences showed that core pride members (females and subadults) shared evolutionary close viral lineages which differed from neighbouring core prides, whereas sequences from sexually mature males associated with the same pride were always the most divergent. In six instances, natal pride associations of divergent male lions could be inferred, on the assumption that FIVPle infections are acquired during early life stages. Congruence between the genetic pattern of FIV and pride structure suggests that vertical transmission plays an important role in lion FIV dynamics. At a fine spatial scale, significant viral geographic structuring was also detected between lions occurring north of the Olifants River within the Kruger National Park (KNP) and those occupying the southern and central regions. This pattern was further supported by phylogenetic analyses and the confinement of FIVPle subtype E to the northern region of KNP. The study provides new insights into the use of retroviral sequences to predict host dispersal and fine-scale contemporary geographic structure in a social felid species.

Error correction and statistical analyses for intra-host comparisons of feline immunodeficiency virus diversity from high-throughput sequencing data.

BACKGROUND: Infection with feline immunodeficiency virus (FIV) causes an immunosuppressive disease whose consequences are less severe if cats are co-infected with an attenuated FIV strain (PLV). We use virus diversity measurements, which reflect replication ability and the virus response to various conditions, to test whether diversity of virulent FIV in lymphoid tissues is altered in the presence of PLV. Our data consisted of the 3' half of the FIV genome from three tissues of animals infected with FIV alone, or with FIV and PLV, sequenced by 454 technology. RESULTS: Since rare variants dominate virus populations, we had to carefully distinguish sequence variation from errors due to experimental protocols and sequencing. We considered an exponential-normal convolution model used for background correction of microarray data, and modified it to formulate an error correction approach for minor allele frequencies derived from high-throughput sequencing. Similar to accounting for over-dispersion in counts, this accounts for error-inflated variability in frequencies - and quite effectively reproduces empirically observed distributions. After obtaining error-corrected minor allele frequencies, we applied ANalysis Of VAriance (ANOVA) based on a linear mixed model and found that conserved sites and transition frequencies in FIV genes differ among tissues of dual and single infected cats. Furthermore, analysis of minor allele frequencies at individual FIV genome sites revealed 242 sites significantly affected by infection status (dual vs. single) or infection status by tissue interaction. All together, our results demonstrated a decrease in FIV diversity in bone marrow in the presence of PLV. Importantly, these effects were weakened or undetectable when error correction was performed with other approaches (thresholding of minor allele frequencies; probabilistic clustering of reads). We also queried the data for cytidine deaminase activity on the viral genome, which causes an asymmetric increase in G to A substitutions, but found no evidence for this host defense strategy. CONCLUSIONS: Our error correction approach for minor allele frequencies (more sensitive and computationally efficient than other algorithms) and our statistical treatment of variation (ANOVA) were critical for effective use of high-throughput sequencing data in understanding viral diversity. We found that co-infection with PLV shifts FIV diversity from bone marrow to lymph node and spleen.

Mutations in the feline immunodeficiency virus envelope glycoprotein confer resistance to a dominant-negative fragment of Tsg101 by enhancing infectivity and cell-to-cell virus transmission.

The Pro-Ser-Ala-Pro (PSAP) motif in the p2 domain of feline immunodeficiency virus (FIV) Gag is required for efficient virus release, virus replication, and Gag binding to the ubiquitin-E2-variant (UEV) domain of Tsg101. As a result of this direct interaction, expression of an N-terminal fragment of Tsg101 containing the UEV domain (referred to as TSG-5') inhibits FIV release. In these respects, the FIV p2(Gag) PSAP motif is analogous to the PTAP motif of HIV-1 p6(Gag). To evaluate the feasibility of a late domain-targeted inhibition of virus replication, we created an enriched Crandell-Rees feline kidney (CRFK) cell line (T5'(hi)) that stably expresses high levels of TSG-5'. Here we show that mutations in either the V3 loop or the second heptad repeat (HR2) domain of the FIV envelope glycoprotein (Env) rescue FIV replication in T5'(hi) cells without increasing FIV release efficiency. TSG-5'-resistance mutations in Env enhance virion infectivity and the cell-cell spread of FIV when diffusion is limited using a semi-solid growth medium. These findings show that mutations in functional domains of Env confer TSG-5'-resistance, which we propose enhances specific infectivity and the cell-cell transmission of virus to counteract inefficient virus release. This article is part of a Special Issue entitled: Viral Membrane Proteins-Channels for Cellular Networking.

Structural elements in the Gag polyprotein of feline immunodeficiency virus involved in Gag self-association and assembly.

The Gag polyprotein of feline immunodeficiency virus (FIV) assembles at the plasma membrane of the infected cells. We aimed to identify the FIV Gag domains that interact and promote Gag multimerization. To do this we generated a series of Gag subdomains and tested their ability to associate with full-length Gag and be recruited into extracellular virus-like particles (VLPs). Removal of 37 residues from the C-terminus of FIV Gag and deletion of the N-terminal and central regions of the nucleocapsid (NC) domain attenuated but did not abrogate association with wild-type Gag, whereas a Gag mutant protein encompassing the matrix (MA) and capsid (CA) domains interacted poorly with full-length Gag. Association with wild-type Gag was abolished by deleting most of the NC together with the N-terminal 40 residues of the MA, which most likely reflects the inability of this Gag mutant to bind RNA. Notably, the CA-NC Gag subdomain both associated with wild-type Gag and was recruited into particles in a proportion close to 50 % of the total Gag-related protein mass of VLPs. Moreover, both a Gag protein lacking the C-terminal p2 peptide and a nonmyristoylated version of the polyprotein exhibited a transdominant-negative effect on the assembly of wild-type Gag. Analysis of Gag mutants carrying internal deletions within the CA revealed that the N-terminal and the C-terminal domains of the CA are necessary for Gag assembly. Our results demonstrate that the FIV CA-NC region constitutes the principal self-interaction domain of Gag and that the RNA-binding capacity of Gag is necessary for its multimerization.

Accessory genes confer a high replication rate to virulent feline immunodeficiency virus.

Feline immunodeficiency virus (FIV) is a lentivirus that causes AIDS in domestic cats, similar to human immunodeficiency virus (HIV)/AIDS in humans. The FIV accessory protein Vif abrogates the inhibition of infection by cat APOBEC3 restriction factors. FIV also encodes a multifunctional OrfA accessory protein that has characteristics similar to HIV Tat, Vpu, Vpr, and Nef. To examine the role of vif and orfA accessory genes in FIV replication and pathogenicity, we generated chimeras between two FIV molecular clones with divergent disease potentials: a highly pathogenic isolate that replicates rapidly in vitro and is associated with significant immunopathology in vivo, FIV-C36 (referred to here as high-virulence FIV [HV-FIV]), and a less-pathogenic strain, FIV-PPR (referred to here as low-virulence FIV [LV-FIV]). Using PCR-driven overlap extension, we produced viruses in which vif, orfA, or both genes from virulent HV-FIV replaced equivalent genes in LV-FIV. The generation of these chimeras is more straightforward in FIV than in primate lentiviruses, since FIV accessory gene open reading frames have very little overlap with other genes. All three chimeric viruses exhibited increased replication kinetics in vitro compared to the replication kinetics of LV-FIV. Chimeras containing HV-Vif or Vif/OrfA had replication rates equivalent to those of the virulent HV-FIV parental virus. Furthermore, small interfering RNA knockdown of feline APOBEC3 genes resulted in equalization of replication rates between LV-FIV and LV-FIV encoding HV-FIV Vif. These findings demonstrate that Vif-APOBEC interactions play a key role in controlling the replication and pathogenicity of this immunodeficiency-inducing virus in its native host species and that accessory genes act as mediators of lentiviral strain-specific virulence.

Neurosteroid-mediated regulation of brain innate immunity in HIV/AIDS: DHEA-S suppresses neurovirulence.

Neurosteroids are cholesterol-derived molecules synthesized within the brain, which exert trophic and protective actions. Infection by human and feline immunodeficiency viruses (HIV and FIV, respectively) causes neuroinflammation and neurodegeneration, leading to neurological deficits. Secretion of neuroinflammatory host and viral factors by glia and infiltrating leukocytes mediates the principal neuropathogenic mechanisms during lentivirus infections, although the effect of neurosteroids on these processes is unknown. We investigated the interactions between neurosteroid-mediated effects and lentivirus infection outcomes. Analyses of HIV-infected (HIV(+)) and uninfected human brains disclosed a reduction in neurosteroid synthesis enzyme expression. Human neurons exposed to supernatants from HIV(+) macrophages exhibited suppressed enzyme expression without reduced cellular viability. HIV(+) human macrophages treated with sulfated dehydroepiandrosterone (DHEA-S) showed suppression of inflammatory gene (IL-1ß, IL-6, TNF-α) expression. FIV-infected (FIV(+)) animals treated daily with 15 mg/kg body weight. DHEA-S treatment reduced inflammatory gene transcripts (IL-1ß, TNF-α, CD3ε, GFAP) in brain compared to vehicle-(ß-cyclodextrin)-treated FIV(+) animals similar to levels found in vehicle-treated FIV(-) animals. DHEA-S treatment also increased CD4(+) T-cell levels and prevented neurobehavioral deficits and neuronal loss among FIV(+) animals, compared to vehicle-treated FIV(+) animals. Reduced neuronal neurosteroid synthesis was evident in lentivirus infections, but treatment with DHEA-S limited neuroinflammation and prevented neurobehavioral deficits. Neurosteroid-derived therapies could be effective in the treatment of virus- or inflammation-mediated neurodegeneration.

Differential type 1 interferon-regulated gene expression in the brain during AIDS: interactions with viral diversity and neurovirulence.

The lentiviruses, human and feline immunodeficiency viruses (HIV-1 and FIV, respectively), infect the brain and cause neurovirulence, evident as neuronal injury, inflammation, and neurobehavioral abnormalities with diminished survival. Herein, different lentivirus infections in conjunction with neural cell viability were investigated, concentrating on type 1 interferon-regulated pathways. Transcriptomic network analyses showed a preponderance of genes involved in type 1 interferon signaling, which was verified by increased expression of the type 1 interferon-associated genes, Mx1 and CD317, in brains from HIV-infected persons (P<0.05). Leukocytes infected with different strains of FIV or HIV-1 showed differential Mx1 and CD317 expression (P<0.05). In vivo studies of animals infected with the FIV strains, FIV(ch) or FIV(ncsu), revealed that FIV(ch)-infected animals displayed deficits in memory and motor speed compared with the FIV(ncsu)- and mock-infected groups (P<0.05). TNF-α, IL-1ß, and CD40 expression was increased in the brains of FIV(ch)-infected animals; conversely, Mx1 and CD317 transcript levels were increased in the brains of FIV(ncsu)-infected animals, principally in microglia (P<0.05). Gliosis and neuronal loss were evident among FIV(ch)-infected animals compared with mock- and FIV(ncsu)-infected animals (P<0.05). Lentiviral infections induce type 1 interferon-regulated gene expression in microglia in a viral diversity-dependent manner, representing a mechanism by which immune responses might be exploited to limit neurovirulence.

FIV establishes a latent infection in feline peripheral blood CD4+ T lymphocytes in vivo during the asymptomatic phase of infection.

BACKGROUND: Feline immunodeficiency virus (FIV) is a lentivirus of cats that establishes a lifelong persistent infection with immunologic impairment. RESULTS: In an approximately 2 year-long experimental infection study, cats infected with a biological isolate of FIV clade C demonstrated undetectable plasma viral loads from 10 months post-infection onward. Viral DNA was detected in CD4+CD25+ and CD4+CD25- T cells isolated from infected cats whereas viral RNA was not detected at multiple time points during the early chronic phase of infection. Viral transcription could be reactivated in latently infected CD4+ T cells ex vivo as demonstrated by detectable FIV gag RNA and 2-long terminal repeat (LTR) circle junctions. Viral LTR and gag sequences amplified from peripheral blood mononuclear cells during early and chronic stages of infection demonstrated minimal to no viral sequence variation. CONCLUSIONS: Collectively, these findings are consistent with FIV latency in peripheral blood CD4+ T cells isolated from chronically infected cats. The ability to isolate latently FIV-infected CD4+ T lymphocytes from FIV-infected cats provides a platform for the study of in vivo mechanisms of lentiviral latency.

Feline tetherin efficiently restricts release of feline immunodeficiency virus but not spreading of infection.

Domestic cats endure infections by all three subfamilies of the retroviridae: lentiviruses (feline immunodeficiency virus [FIV]), gammaretroviruses (feline leukemia virus [FeLV]), and spumaretroviruses (feline foamy virus [FFV]). Thus, cats present an insight into the evolution of the host-retrovirus relationship and the development of intrinsic/innate immune mechanisms. Tetherin (BST-2) is an interferon-inducible transmembrane protein that inhibits the release of enveloped viruses from infected cells. Here, we characterize the feline homologue of tetherin and assess its effects on the replication of FIV. Tetherin was expressed in many feline cell lines, and expression was induced by interferons, including alpha interferon (IFN-α), IFN-ω, and IFN-γ. Like human tetherin, feline tetherin displayed potent inhibition of FIV and HIV-1 particle release; however, this activity resisted antagonism by either HIV-1 Vpu or the FIV Env and "OrfA" proteins. Further, as overexpression of complete FIV genomes in trans could not overcome feline tetherin, these data suggest that FIV lacks a functional tetherin antagonist. However, when expressed stably in feline cell lines, tetherin did not abrogate the replication of FIV; indeed, syncytium formation was significantly enhanced in tetherin-expressing cells infected with cell culture-adapted (CD134-independent) strains of FIV (FIV Fca-F14 and FIV Pco-CoLV). Thus, while tetherin may prevent the release of nascent viral particles, cell-to-cell spread remains efficient in the presence of abundant viral receptors and tetherin upregulation may enhance syncytium formation. Accordingly, tetherin expression in vivo may promote the selective expansion of viral variants capable of more efficient cell-to-cell spread.

Imbalance of placental regulatory T cell and Th17 cell population dynamics in the FIV-infected pregnant cat.

BACKGROUND: An appropriate balance in placental regulatory T cells (Tregs), an immunosuppressive cell population, and Th17 cells, a pro-inflammatory cell population, is essential in allowing tolerance of the semi-allogeneic fetus. TGF-ß and IL-6 are cytokines that promote differentiation of Tregs and Th17 cells from a common progenitor; aberrant expression of the cytokines may perturb the balance in the two cell populations. We previously reported a pro-inflammatory placental environment with decreased levels of FoxP3, a Treg marker, and increased levels of IL-6 in the placentas of FIV-infected cats at early pregnancy. Thus, we hypothesized that FIV infection in the pregnant cat causes altered placental Treg and Th17 cell populations, possibly resulting in placental inflammation. METHODS: We examined the effect of FIV infection on Treg and Th17 populations in placentas at early pregnancy using quantitative confocal microscopy to measure FoxP3 or RORγ, a Th17 marker, and qPCR to quantify expression of the key cytokines TGF-ß and IL-6. RESULTS: FoxP3 and RORγ were positively correlated in FIV-infected placentas at early pregnancy, but not placentas from normal cats, indicating virus-induced alteration in the balance of these cell populations. In control cats the expression of IL-6 and RORγ was positively correlated as predicted, but this relationship was disrupted in infected animals. TGF-ß was reduced in infected queens, an occurrence that could dysregulate both Treg and Th17 cell populations. Co-expression analyses revealed a highly significant positive correlation between IL-6 and TGF-ß expression in control animals that did not occur in infected animals. CONCLUSION: Collectively, these data point toward potential disruption in the balance of Treg and Th17 cell populations that may contribute to FIV-induced inflammation in the feline placenta.

Regulation of lentivirus neurovirulence by lipopolysaccharide conditioning: suppression of CXCL10 in the brain by IL-10.

Lentivirus infections including HIV and feline immunodeficiency virus (FIV) cause neurovirulence, which is largely mediated by innate immunity. To investigate the interactions between neurovirulence and repeated conditioning by innate immune activation, models of lentivirus infection were exposed to LPS. Gene expression in HIV-infected (HIV+) and control (HIV-) patient brains was compared by real time RT-PCR and immunocytochemistry. Supernatants from mock and HIV-infected monocyte-derived macrophages exposed to LPS were applied to human neurons. FIV-infected (FIV+) and control (FIV-) animals were exposed repeatedly to LPS postinfection together with concurrent neurobehavioral testing, viral load, and host gene analyses. Brains from HIV+ individuals exhibited induction of CD3epsilon, CXCL10, and granzyme A expression (p < 0.05). Supernatants from HIV+ monocyte-derived macrophages induced CXCL10 expression in neurons, which was diminished by IL-10 treatment (p < 0.05). LPS-exposed FIV+ animals demonstrated lower plasma and brain viral loads (p < 0.05). Neuronal CXCL10 expression was increased in FIV+ animals but was suppressed by LPS exposure, together with reduced brain CD3epsilon and granzyme A expression (p < 0.05). In conjunction with preserved NeuN-positive neuronal counts in parietal cortex (p < 0.05), FIV+ animals exposed to LPS also showed less severe neurobehavioral deficits (p < 0.05). Repeated LPS exposures suppressed CXCL10 in the brain and ensuing T cell infiltration with a concomitant reduction in neurovirulence. Thus, innate immune chronic conditioning exerted beneficial effects on neurovirulence through suppression of a specific chemotactic factor, CXCL10, mediated by IL-10, leading to reduced leukocyte infiltration and release of neurotoxic factors.

Immunomodulator expression in trophoblasts from the feline immunodeficiency virus (FIV)-infected cat.

BACKGROUND: FIV infection frequently compromises pregnancy under experimental conditions and is accompanied by aberrant expression of some placental cytokines. Trophoblasts produce numerous immunomodulators that play a role in placental development and pregnancy maintenance. We hypothesized that FIV infection may cause dysregulation of trophoblast immunomodulator expression, and aberrant expression of these molecules may potentiate inflammation and compromise pregnancy. The purpose of this project was to evaluate the expression of representative pro-(TNF-α, IFN-γ, IL-1ß, IL-2, IL-6, IL-12p35, IL-12p40, IL-18, and GM-CSF) and anti-inflammatory cytokines (IL-4, IL-5, and IL-10); CD134, a secondary co-stimulatory molecule expressed on activated T cells (FIV primary receptor); the chemokine receptor CXCR4 (FIV co-receptor); SDF-1α, the chemokine ligand to CXCR4; and FIV gag in trophoblasts from early-and late-term pregnancy. METHODS: We used an anti-cytokeratin antibody in immunohistochemistry to identify trophoblasts selectively, collected these cells using laser capture microdissection, and extracted total RNA from the captured cell populations. Real time, reverse transcription-PCR was used to quantify gene expression. RESULTS: We detected IL-4, IL-5, IL-6, IL-1ß, IL-12p35, IL-12p40, and CXCR4 in trophoblasts from early-and late-term pregnancy. Expression of cytokines increased from early to late pregnancy in normal tissues. A clear, pro-inflammatory microenvironment was not evident in trophoblasts from FIV-infected queens at either stage of pregnancy. Reproductive failure was accompanied by down-regulation of both pro-and anti-inflammatory cytokines. CD134 was not detected in trophoblasts, and FIV gag was detected in only one of ten trophoblast specimens collected from FIV-infected queens. CONCLUSION: Feline trophoblasts express an array of pro-and anti-inflammatory immunomodulators whose expression increases from early to late pregnancy in normal tissues. Non-viable pregnancies were associated with decreased expression of immunomodulators which regulate trophoblast invasion in other species. The detection of FIV RNA in trophoblasts was rare, suggesting that the high rate of reproductive failure in FIV-infected queens was not a direct result of viral replication in trophoblasts. The influence of placental immune cells on trophoblast function and pregnancy maintenance in the FIV-infected cat requires additional study.

Neurobehavioral performance in feline immunodeficiency virus infection: integrated analysis of viral burden, neuroinflammation, and neuronal injury in cortex.

Human immunodeficiency virus (HIV) infection causes motor and neurocognitive abnormalities affecting >50% of children and 20% of adults with HIV/AIDS (acquired immunodeficiency syndrome). The closely related lentivirus, feline immunodeficiency virus (FIV), also causes neurobehavioral deficits. Herein, we investigated the extent to which FIV infection affected specific motor and cognitive tasks in conjunction with viral burden and immune responses within the brain. Neonatal animals were infected with a neurovirulent FIV strain (FIV-Ch) and assessed in terms of systemic immune parameters, viral burden, neurobehavioral performance, and neuropathological features. FIV-infected animals displayed less weight gain and lower blood CD4(+) T-cell levels than mock-infected animals (p < 0.05). Gait analyses disclosed greater gait width with increased variation in FIV-infected animals (p < 0.05). Maze performance showed that FIV-infected animals were slower and made more navigational errors than mock-infected animals (p < 0.05). In the object memory test, the FIV-infected group exhibited fewer successful steps with more trajectory errors compared with the mock-infected group (p < 0.05). Performance on the gait, maze, and object memory tests was inversely correlated with F4/80 and CD3 epsilon expression (p < 0.05) and with viral burden in parietal cortex (p < 0.05). Amino acid analysis in cortex showed that D-serine levels were reduced in FIV-infected animals, which was accompanied by diminished kainate and AMPA receptor subunit expression (p < 0.05). The neurobehavioral findings in FIV-infected animals were associated with increased gliosis and reduced cortical neuronal counts (p < 0.05). The present studies indicated that specific motor and neurocognitive abilities were impaired in FIV infection and that these effects were closely coupled with viral burden, neuroinflammation, and neuronal loss.

Acute mucosal pathogenesis of feline immunodeficiency virus is independent of viral dose in vaginally infected cats.

BACKGROUND: The mucosal pathogenesis of HIV has been shown to be an important feature of infection and disease progression. HIV-1 infection causes depletion of intestinal lamina propria CD4+ T cells (LPL), therefore, intestinal CD4+ T cell preservation may be a useful correlate of protection in evaluating vaccine candidates. Vaccine studies employing the cat/FIV and macaque/SIV models frequently use high doses of parenterally administered challenge virus to ensure high plasma viremia in control animals. However, it is unclear if loss of mucosal T cells would occur regardless of initial viral inoculum dose. The objective of this study was to determine the acute effect of viral dose on mucosal leukocytes and associated innate and adaptive immune responses. RESULTS: Cats were vaginally inoculated with a high, middle or low dose of cell-associated and cell-free FIV. PBMC, serum and plasma were assessed every two weeks with tissues assessed eight weeks following infection. We found that irrespective of mucosally administered viral dose, FIV infection was induced in all cats. However, viremia was present in only half of the cats, and viral dose was unrelated to the development of viremia. Importantly, regardless of viral dose, all cats experienced significant losses of intestinal CD4+ LPL and CD8+ intraepithelial lymphocytes (IEL). Innate immune responses by CD56+CD3- NK cells correlated with aviremia and apparent occult infection but did not protect mucosal T cells. CD4+ and CD8+ T cells in viremic cats were more likely to produce cytokines in response to Gag stimulation, whereas aviremic cats T cells tended to produce cytokines in response to Env stimulation. However, while cell-mediated immune responses in aviremic cats may have helped reduce viral replication, they could not be correlated to the levels of viremia. Robust production of anti-FIV antibodies was positively correlated with the magnitude of viremia. CONCLUSIONS: Our results indicate that mucosal immune pathogenesis could be used as a rapid indicator of vaccine success or failure when combined with a physiologically relevant low dose mucosal challenge. We also show that innate immune responses may play an important role in controlling viral replication following acute mucosal infection, which has not been previously identified.

Lentivirus-associated MAPK/ERK2 phosphorylates EMD and regulates infectivity.

Infection of a cell by lentiviruses, such as human immunodeficiency virus type 1 or feline immunodeficiency virus, results in the formation of a reverse transcription complex, the pre-integration complex (PIC), where viral DNA is synthesized. In non-dividing cells, efficient nuclear translocation of the PIC requires the presence of the inner nuclear lamina protein emerin (EMD). Here, we demonstrate that EMD phosphorylation is induced early after infection in primary non-dividing cells. Furthermore, we demonstrate that EMD phosphorylation is dependent on virion-associated mitogen-activated protein kinase (MAPK). Specific inhibition of MAPK activity with kinase inhibitors markedly reduced EMD phosphorylation and resulted in decreased integration of the proviral DNA into chromatin. Similarly, when a MEK1 kinase-inactive mutant was expressed in virus-producer cells, virus-induced phosphorylation of EMD was impaired and viral integration reduced during the subsequent infection. Expression of constitutively active MEK1 kinase in producer cells did not result in modulation of EMD phosphorylation or viral integration during subsequent infection. These studies demonstrate that, in addition to phosphorylating components of the PICs at an early step of infection, virion-associated MAPK plays a role in facilitating cDNA integration after nuclear translocation through phosphorylation of target-cell EMD.