Feline Immunodeficiency Virus Gag Research Articles

Detection and genetic characterization of feline retroviruses in domestic cats with different clinical signs and hematological alterations.

Feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) are globally distributed retroviruses that infect domestic cats and cause various syndromes that can lead to death. The aim of this study was to detect and genotype feline retroviruses in Mexican domestic cats. We used PCR assays to identify proviral DNA and viral RNA in 50 domestic cats with different clinical signs and hematological alterations. Endogenous FeLV (enFeLV) was identified in the genomic DNA of all cats in the study, and we detected transcripts of the LTR region of enFeLV in 48 individuals. Exogenous FeLV (exFeLV) was found in 13 cats. Furthermore, we detected FIV proviral DNA in 10 cats. The enFeLV sequences were shown to be the most variable, while the exFeLV sequences were highly conserved and related to previously reported subgroup A sequences. Sequencing of the FIV gag gene revealed the presence of subtype B in the infected cats.

Review and Perspectives on the Structure-Function Relationships of the Gag Subunits of Feline Immunodeficiency Virus.

The Gag polyprotein is implied in the budding as well as the establishment of the supramolecular architecture of infectious retroviral particles. It is also involved in the early phases of the replication of retroviruses by protecting and transporting the viral genome towards the nucleus of the infected cell until its integration in the host genome. Therefore, understanding the structure–function relationships of the Gag subunits is crucial as each of them can represent a therapeutic target. Though the field has been explored for some time in the area of Human Immunodeficiency Virus (HIV), it is only in the last decade that structural data on Feline Immunodeficiency Virus (FIV) Gag subunits have emerged. As FIV is an important veterinary issue, both in domestic cats and endangered feline species, such data are of prime importance for the development of anti-FIV molecules targeting Gag. This review will focus on the recent advances and perspectives on the structure–function relationships of each subunit of the FIV Gag polyprotein.

The molecular and serological investigation of Feline immunodeficiency virus and Feline leukemia virus in stray cats of Western Turkey

This study aimed to investigate the Feline immunodeficiency virus (FIV) / Feline leukemia virus (FeLV) infection prevalence among looking healthy stray cats in Western Turkey by serologic and molecular-based tests. A total of 1008 blood samples from the stray cats were used in this study. All samples were tested for FIV antibodies / proviral DNA and FeLV antibodies / antigens / proviral DNA. The genetic characterization and phylogenetic analysis of FeLV and FIV were carried out in this study. These cats also tested for Leishmaniasis and Toxoplasmosis previously. FIV Ab and proviral DNA detected in 25.2 % and 25.5 % of samples, respectively. FeLV Ab, Ag, proviral DNA positivity was in 45.2 %, in 3.3 %, in 69.7 %, respectively. The molecular detection and phylogenetic analysis of the current FeLV pol gene and FIV gag gene performed. The molecular characterization for the pol gene of FeLV (enFeLV and exFeLV) among Turkey's cat population was reported for the first time. The exFeLV pol sequences closer to the FeLV-A genotype, and the enFeLV pol sequences overlapped with other enFeLV. The current FIV gag sequences were clustered within the subtypes A, B, and C. The findings revealed FeLV subtype A and FIV subtype-A, subtype-B, subtype-C circulate among Turkish stray cats. Single and multiple co-infection positivity was found higher compared to previous reports.

Serum Samples from Co-Infected and Domestic Cat Field Isolates Nonspecifically Bind FIV and Other Antigens in Enzyme-Linked Immunosorbent Assays.

We evaluated enzyme-linked immunosorbent assay (ELISA) specificity for measuring seroantibody responses to two types of retroviral infections in domestic cats: feline immunodeficiency virus (FIV) and feline foamy virus (FFV). We compared the seroreactivity of specific pathogen-free (SPF) cat sera, sera from SPF cats inoculated with either FIV or FFV, and field isolates (e.g., shelter or privately owned cats). Sera from SPF cats experimentally infected with the cognate virus had significantly lower background in both FIV and FFV ELISAs compared to sera from negative field isolates. ELISA values for SPF cats exposed to either FIV or FFV tended to have higher OD values on the opposite ELISA antigen plate. FIV nonspecific background absorbance was greater than that of FFV, and 10 of 15 sera samples from FIV seronegative field samples were measured in the indeterminant range. These findings highlight that exposure to off-target pathogens elicit antibodies that may nonspecifically bind to antigens used in binding assays; therefore, validation using sera from SPF animals exposed during controlled infection results in the setting of a cutoff value that may be inappropriately low when applied to field samples. Our work also suggests that infection of domestic cats with pathogens other than FIV results in antibodies that cross-react with the FIV Gag antigen.

Properties and Functions of Feline Immunodeficiency Virus Gag Domains in Virion Assembly and Budding.

Feline immunodeficiency virus (FIV) is an important cat pathogen worldwide whose biological and pathophysiological properties resemble those of human immunodeficiency virus type 1 (HIV-1). Therefore, the study of FIV not only benefits its natural host but is also useful for the development of antiviral strategies directed against HIV-1 infections in humans. FIV assembly results from the multimerization of a single but complex viral polypeptide, the Gag precursor. In this review, we will first give an overview of the current knowledge of the proteins encoded by the FIV pol, env, rev, vif, and orf-A genes, and then we will describe and discuss in detail the critical roles that each of the FIV Gag domains plays in virion morphogenesis. Since retroviral assembly is an attractive target for therapeutic interventions, gaining a better understanding of this process is highly desirable.

Formation of RNA Granule-Derived Capsid Assembly Intermediates Appears To Be Conserved between Human Immunodeficiency Virus Type 1 and the Nonprimate Lentivirus Feline Immunodeficiency Virus.

During immature capsid assembly in cells, human immunodeficiency virus type 1 (HIV-1) Gag co-opts a host RNA granule, forming a pathway of intracellular assembly intermediates containing host components, including two cellular facilitators of assembly, ABCE1 and DDX6. A similar assembly pathway has been observed for other primate lentiviruses. Here we asked whether feline immunodeficiency virus (FIV), a nonprimate lentivirus, also forms RNA granule-derived capsid assembly intermediates. First, we showed that the released FIV immature capsid and a large FIV Gag-containing intracellular complex are unstable during analysis, unlike for HIV-1. We identified harvest conditions, including in situ cross-linking, that overcame this problem, revealing a series of FIV Gag-containing complexes corresponding in size to HIV-1 assembly intermediates. Previously, we showed that assembly-defective HIV-1 Gag mutants are arrested at specific assembly intermediates; here we identified four assembly-defective FIV Gag mutants, including three not previously studied, and demonstrated that they appear to be arrested at the same intermediate as the cognate HIV-1 mutants. Further evidence that these FIV Gag-containing complexes correspond to assembly intermediates came from coimmunoprecipitations demonstrating that endogenous ABCE1 and the RNA granule protein DDX6 are associated with FIV Gag, as shown previously for HIV-1 Gag, but are not associated with a ribosomal protein, at steady state. Additionally, we showed that FIV Gag associates with another RNA granule protein, DCP2. Finally, we validated the FIV Gag-ABCE1 and FIV Gag-DCP2 interactions with proximity ligation assays demonstrating colocalization in situ Together, these data support a model in which primate and nonprimate lentiviruses form intracellular capsid assembly intermediates derived from nontranslating host RNA granules.IMPORTANCE Like HIV-1 Gag, FIV Gag assembles into immature capsids; however, it is not known whether FIV Gag progresses through a pathway of immature capsid assembly intermediates derived from host RNA granules, as shown for HIV-1 Gag. Here we showed that FIV Gag forms complexes that resemble HIV-1 capsid assembly intermediates in size and in their association with ABCE1 and DDX6, two host facilitators of HIV-1 immature capsid assembly that are found in HIV-1 assembly intermediates. Our studies also showed that known and novel assembly-defective FIV Gag mutants fail to progress past putative intermediates in a pattern resembling that observed for HIV-1 Gag mutants. Finally, we used imaging to demonstrate colocalization of FIV Gag with ABCE1 and with the RNA granule protein DCP2. Thus, we conclude that formation of assembly intermediates derived from host RNA granules is likely conserved between primate and nonprimate lentiviruses and could provide targets for future antiviral strategies.

Sequence Instability in the Proviral Long Terminal Repeat and gag Regions from Peripheral Blood and Tissue-Derived Leukocytes of FIV-Infected Cats during the Late Asymptomatic Phase.

Feline immunodeficiency virus (FIV) infection results in viral persistence, a prolonged asymptomatic phase, and progressive immunopathology. During the asymptomatic phase, a cohort of experimentally FIV-infected cats exhibits features of viral latency in blood suggestive of inactive viral replication. We sought to investigate viral replication activity and genomic stability of the FIV proviral long terminal repeat (LTR) and the 5′ aspect of gag over time. FIV-infected cats during the asymptomatic phase demonstrated undetectable plasma FIV gag RNA transcripts and intermittent to undetectable blood-derived cell-associated FIV gag RNA. The LTR sequence demonstrated instability in blood-derived cells over time, in spite of low to undetectable viral replication. Sequence variation in the LTR was identified in CD4+ and CD21+ leukocytes from blood and surgically removed lymph nodes. Three single nucleotide polymorphisms (SNPs) in the LTR were commonly identified. Promoter functionality of a common LTR SNP and rare U3 mutation were examined by reporter gene assays and demonstrated either no change or increased basal FIV promoter function, respectively. In conclusion, this cohort of asymptomatic FIV-infected cats demonstrated instability of the LTR and 5’ gag sequences during the study period, in spite of undetectable plasma and rare to undetectable viral gag RNA, which suggests that blood may not accurately represent viral activity in asymptomatic FIV-infected cats.

Viral Reservoirs in Lymph Nodes of FIV-Infected Progressor and Long-Term Non-Progressor Cats during the Asymptomatic Phase.

BackgroundExamination of a cohort of cats experimentally infected with feline immunodeficiency virus (FIV) for 5.75 years revealed detectable proviral DNA in peripheral blood mononuclear cells (PBMCs) harvested during the asymptomatic phase, undetectable plasma viral RNA (FIV gag), and rarely detectable cell-associated viral RNA. Despite apparent viral latency in peripheral CD4+ T cells, circulating CD4+ T cell numbers progressively declined in progressor animals. The aim of this study was to explore this dichotomy of peripheral blood viral latency in the face of progressive immunopathology. The viral replication status, cellular immunophenotypes, and histopathologic features were compared between popliteal lymph nodes (PLNs) and peripheral blood. Also, we identified and further characterized one of the FIV-infected cats identified as a long-term non-progressor (LTNP).ResultsPLN-derived leukocytes from FIV-infected cats during the chronic asymptomatic phase demonstrated active viral gag transcription and FIV protein translation as determined by real-time RT-PCR, Western blot and in situ immunohistochemistry, whereas viral RNA in blood leukocytes was either undetectable or intermittently detectable and viral protein was not detected. Active transcription of viral RNA was detectable in PLN-derived CD4+ and CD21+ leukocytes. Replication competent provirus was reactivated ex vivo from PLN-derived leukocytes from three of four FIV-infected cats. Progressor cats showed a persistent and dramatically decreased proportion and absolute count of CD4+ T cells in blood, and a decreased proportion of CD4+ T cells in PLNs. A single long-term non-progressor (LTNP) cat persistently demonstrated an absolute peripheral blood CD4+ T cell count indistinguishable from uninfected animals, a lower proviral load in unfractionated blood and PLN leukocytes, and very low amounts of viral RNA in the PLN.ConclusionCollectively our data indicates that PLNs harbor important reservoirs of ongoing viral replication during the asymptomatic phase of infection, in spite of undetectable viral activity in peripheral blood. A thorough understanding of tissue-based lentiviral reservoirs is fundamental to medical interventions to eliminate virus or prolong the asymptomatic phase of FIV infection.

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.

Aberrant placental immune parameters in the feline immunodeficiency virus (FIV)-infected cat suggest virus-induced changes in T cell function

BackgroundImmune activity during pregnancy must be tightly regulated to ensure successful pregnancy. This regulation includes the suppression of inflammatory activity that could target the semi-allogeneic fetus. Tregs are immunosuppressive; Th17 cells are pro-inflammatory. A precise balance in the two cell populations is critical to pregnancy maintenance, while dysregulation in this balance accompanies compromised pregnancy in humans and mice. FIV is known to target Tregs preferentially in the infected cat. Therefore, it may be hypothesized that FIV infection alters the placental Treg/Th17 cell balance resulting in aberrant immunomodulator expression by these cells and consequent pregnancy perturbation.MethodsRNA was purified from random sections of whole placental tissues collected from both uninfected and FIV-infected queens at early pregnancy, including tissues from viable and nonviable fetuses. Real time qPCR was performed to quantify expression of intranuclear markers of Tregs (FoxP3) and Th17 cells (RORγ); cytokine products of Tregs (IL-10 and TGF-β), Th17 cells (IL-2, IL-6, and IL-17a), and macrophages (IL-1β); and the FIV gag gene. Pairwise comparisons were made to evaluate coexpression patterns between the cytokines and between the cytokines and the virus.ResultsBoth FoxP3 and RORγ were reduced in placentas of infected animals. Neither infection status nor fetal viability affected placental expression of IL-1β. However, fetal nonviability was associated with reduced levels of all other cytokines. Infection and fetal nonviability impacted coexpression of various cytokine pairs. No obvious bias toward Treg or Th17 cells was observed.ConclusionsFIV infection coupled with fetal nonviability alters expression patterns of T cell cytokines. These data suggest that functionally altered placental T cell leukocyte populations may occur in the infected queen and possibly contribute to fetal nonviability.

Feline Immunodeficiency Virus Gag Is a Nuclear Shuttling Protein

Lentiviral genomic RNAs are encapsidated by the viral Gag protein during virion assembly. The intracellular location of the initial Gag-RNA interaction is unknown. We previously observed feline immunodeficiency virus (FIV) Gag accumulating at the nuclear envelope during live-cell imaging, which suggested that trafficking of human immunodeficiency virus type 1 (HIV-1) and FIV Gag may differ. Here we analyzed the nucleocytoplasmic transport properties of both Gag proteins. We discovered that inhibition of the CRM1 nuclear export pathway with leptomycin B causes FIV Gag but not HIV-1 Gag to accumulate in the nucleus. Virtually all FIV Gag rapidly became intranuclear when the CRM1 export pathway was blocked, implying that most if not all FIV Gag normally undergoes nuclear cycling. In FIV-infected feline cells, some intranuclear Gag was detected in the steady state without leptomycin B treatment. When expressed individually, the FIV matrix (MA), capsid (CA), and nucleocapsid-p2 (NC-p2) domains were not capable of mediating leptomycin B-sensitive nuclear export of a fluorescent protein. In contrast, CA-NC-p2 did mediate nuclear export, with MA being dispensable. We conclude that HIV-1 and FIV Gag differ strikingly in a key intracellular trafficking property. FIV Gag is a nuclear shuttling protein that utilizes the CRM1 nuclear export pathway, while HIV-1 Gag is excluded from the nucleus. These findings expand the spectrum of lentiviral Gag behaviors and raise the possibility that FIV genome encapsidation may initiate in the nucleus.

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

BackgroundFeline immunodeficiency virus (FIV) is a lentivirus of cats that establishes a lifelong persistent infection with immunologic impairment.ResultsIn 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.ConclusionsCollectively, 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.

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

BackgroundFIV 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-1β, 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.MethodsWe 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.ResultsWe detected IL-4, IL-5, IL-6, IL-1β, 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.ConclusionFeline 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.

Occurrence of Feline Immunodeficiency Virus and Feline Leukemia Virus Infection in Cats

Problem statement: The objective of the present study was to evaluate occurrence of the Feline Immunodeficiency Virus (FIV) and the Feline Leukemia Virus (FeLV) infection in asymptomatic domestic cats in the metropolitan region of Belo Horizonte, Minas Gerais, using the Polymerase Chain Reaction (PCR) for FIV detection and SNAP Combo Plus for FeLV and FIV detection. Approach: Blood samples were collected from the jugular vein of 78 healthy cats, mixed breeds and ages and both sexes. Specific primers were designed for PCR to amplify a 244 bp fragment of FIV gag gene. Results: Five animals (6.41%) were positive by PCR and three animals (3.85%) were positive by SNAP Combo Plus for FIV and 14 animals were positive for FeLV (17.95%). Conclusion: These results suggest that there is a significant occurrence of asymptomatic infected animals which may serve as potential transmitters of FIV and FeLV.

Expression of Regulatory T cell (Treg) Activation Markers in Endometrial Tissues from Early and Late Pregnancy in the Feline Immunodeficiency Virus (FIV)-Infected Cat

Regulatory T cells (Tregs) support pregnancy maintenance by suppressing placental inflammation, while diminished Treg function may accompany reproductive failure. Experimental FIV infection frequently results in vertical transmission and increased pregnancy failure in the cat. The mechanism of reproductive compromise is unknown. We hypothesized that FIV infection alters endometrial Treg population dynamics and function, potentiating vertical transmission and reproductive failure. RNA collected from early and late gestation reproductive tissue and fetuses from FIV infected and control cats was probed for expression of FIV gag and Treg markers CD25, FOXP3, and CTLA4, using real time reverse-transcriptase (RT)-PCR. Frequent placental and fetal infection and reproductive failure were detected at early and late pregnancy. Expression of FOXP3 and CTLA4 was higher in early gestation tissues from control cats. FIV infection significantly reduced expression of FOXP3 and CTLA4 at early, but not late pregnancy. At late pregnancy, CTLA4 was expressed to higher levels in infected tissues. The number of tissues with decreased co-expression of FOXP3 and CTLA4 was significant in infected cats at early pregnancy. No significant changes in CD25 expression occurred between FIV-infected and control animals at early or late pregnancy. Differences in Treg marker expression were not significant between viable and non-viable pregnancies in infected cats. The detection of Treg markers in these feline tissues provides the first evidence of feline endometrial Tregs and suggests that such cells diminish as pregnancy progresses. These cells may be depleted or rendered less functional by viral infection, but understanding their role in pregnancy requires further study.

In vitro assembly of the feline immunodeficiency virus Gag polyprotein

The retroviral Gag protein is the only viral product that is necessary for the assembly of virions in mammalian cells. We have established an in vitro assembly system to study the assembly properties of purified feline immunodeficiency virus (FIV) Gag protein expressed in bacteria. Under fully defined conditions, the FIV Gag protein assembles into spherical particles of 33 nm in diameter which are morphologically similar to authentic immature particles, albeit smaller than virions. The in vitro assembly of FIV Gag into particles was found to be resistant to the addition of Triton X-100 and required the presence of RNA. Notably, we found that an amino acid substitution in the nucleocapsid domain of Gag that impairs RNA binding and blocks virion production in vivo, also abrogates Gag assembly in vitro. The development of an in vitro assembly system for FIV Gag protein will facilitate the study of the mechanisms by which this protein assembles into immature particles.

Role of the feline immunodeficiency virus L‐domain in the presence or absence of Gag processing: Involvement of ubiquitin and Nedd4‐2s ligase in viral egress

RNA-enveloped viruses bud from infected cells by exploiting the multivesicular body (MVB) pathway. In this context, ubiquitination of structural viral proteins and their direct interaction with cellular factors involved in the MVB biogenesis through short proline rich regions, named late domains (L-domains), are crucial mechanisms. Here we report that, in contrast with the human immunodeficiency virus (HIV), the feline immunodeficiency virus (FIV), a non-primate lentivirus, is strictly dependent for its budding on a "PSAP"-type L-domain, mapping in the carboxy-terminal region of Gag, irrespective of a functional viral protease. Moreover, we provide evidence that FIV egress is related to Gag ubiquitination, that is, linked to the presence of an active L-domain. Finally, although FIV Gag does not contain a PPxY motif, we show that the Nedd4-2s ubiquitin ligase enhances FIV Gag ubiquitination and it is capable to rescue viral mutants lacking a functional L-domain. In conclusion, our data bring to light peculiar aspects of FIV egress, but we also demonstrate that a non-primate lentivirus shares with HIV-1 a novel mechanism of connection to the cellular budding machinery.

Molecular Characterization of Feline Immunodeficiency Virus Budding

Infection of domestic cats with feline immunodeficiency virus (FIV) is an important model system for studying human immunodeficiency virus type 1 (HIV-1) infection due to numerous similarities in pathogenesis induced by these two lentiviruses. However, many molecular aspects of FIV replication remain poorly understood. It is well established that retroviruses use short peptide motifs in Gag, known as late domains, to usurp cellular endosomal sorting machinery and promote virus release from infected cells. For example, the Pro-Thr/Ser-Ala-Pro [P(T/S)AP] motif of HIV-1 Gag interacts directly with Tsg101, a component of the endosomal sorting complex required for transport I (ESCRT-I). A Tyr-Pro-Asp-Leu (YPDL) motif in equine infectious anemia virus (EIAV), and a related sequence in HIV-1, bind the endosomal sorting factor Alix. In this study we sought to identify and characterize FIV late domain(s) and elucidate cellular machinery involved in FIV release. We determined that mutagenesis of a PSAP motif in FIV Gag, small interfering RNA-mediated knockdown of Tsg101 expression, and overexpression of a P(T/S)AP-binding fragment of Tsg101 (TSG-5') each inhibited FIV release. We also observed direct binding of FIV Gag peptides to Tsg101. In contrast, mutagenesis of a potential Alix-binding motif in FIV Gag did not affect FIV release. Similarly, expression of the HIV-1/EIAV Gag-binding domain of Alix (Alix-V) did not disrupt FIV budding, and FIV Gag peptides showed no affinity for Alix-V. Our data demonstrate that FIV relies predominantly on a Tsg101-binding PSAP motif in the C terminus of Gag to promote virus release in HeLa cells, and this budding mechanism is highly conserved in feline cells.

Evolution of feline immunodeficiency virus Gag proteins

We evaluated the predicted biochemical properties of Gag proteins from a diverse group of feline immunodeficiency viruses (FIV) to determine how different evolutionary histories of virus and host have changed or constrained these important structural proteins. Our data are based on FIV sequences derived from domestic cat (FIVfca), cougar (FIVpco), and lions (FIVple). Analyses consisted of determining the selective forces acting at each position in the protein and the comparing predictions for secondary structure, charge, hydrophobicity and flexibility for matrix, capsid and nucleocapsid, and the C-terminal peptide, which comprise the Gag proteins. We demonstrate that differences among the FIV Gag proteins have largely arisen by neutral evolution, although many neutrally evolving regions have maintained biochemical features. Regions with predicted differences in biochemical features appear to involve intramolecular interactions and structural elements that undergo conformational changes during particle maturation. In contrast, the majority of sites involved in intermolecular contacts on the protein surface are constrained by purifying selection. There is also conservation of sites that interact with host proteins associated with cellular trafficking and particle budding. NC is the only protein with evidence of positive selection, two of which occur in the N-terminal region responsible for RNA binding and interaction with host proteins.

Application of RNA interference for inhibiting the replication of feline immunodeficiency virus in chronically infected cell lines

RNA interference (RNAi) is a process in which double-stranded RNA induces the post-transcriptional sequence-specific degradation of homologous messenger RNA. The present study was carried out to apply the RNAi technology to inhibit the replication of feline immunodeficiency virus (FIV). Four small interfering RNAs (siRNAs) homologous to the FIV gag gene were synthesized and transfected into a feline fibroblastic cell line chronically infected with FIV (CRFK/FIV). These synthetic siRNAs efficiently inhibited the replication of FIV. Next, we examined the effect of retroviral vector-mediated transfer of FIV-specific short hairpin RNA (shRNA) on the replication of FIV in a feline T-cell line chronically infected with FIV (FL4). The retroviral vector-mediated transfer of FIV-specific shRNA was shown to markedly inhibit the replication of FIV in the FL4 cells. These results provide useful information for the development of RNAi-based gene therapy strategy to control FIV infection.