The assembly-defective phenotype of an FIV Gag polyprotein containing the SIV nucleocapsid zinc finger motifs is reversed by including the SIV SP2 domain in the chimeric protein.

To gain insight into the functional relationship between the nucleocapsid (NC) domains of the Gag polyproteins of feline and simian immunodeficiency viruses, FIV and SIV, respectively, we generated two FIV Gag chimeric proteins containing different SIV NC and gag sequences. A chimeric FIV Gag protein (NC1) containing the SIV two zinc fingers motifs was incapable of assembling into virus-like particles. By contrast, another Gag chimera (NC2) differing from NC1 by the replacement of the C-terminal region of the FIV NC with SIV SP2 produced particles as efficiently as wild-type FIV Gag.

The impact of mutations affecting highly conserved amino acids in the simian immunodeficiency virus nucleocapsid protein on virion assembly, genomic RNA packaging and viral infectivity.

The nucleocapsid (NC) domain of the retroviral Gag polyproteins mediates the incorporation of the viral genomic RNA into virions. Although SIV is widely used as a model for human immunodeficiency virus type 1 (HIV-1) infections, the SIV NC has been the subject of few studies which have provided discrepant data on the relative contribution of the two NC zinc finger motifs to genomic RNA encapsidation. Here, we demonstrate that mutations affecting the first cysteine in the distal zinc finger motif (C33S) or the N-terminal NC basic domain (R7A/K8A) drastically impair virion assembly and viral RNA binding.

In vitro inhibitory effect of maraviroc on the association of the simian immunodeficiency virus envelope glycoprotein with CCR5.

Asian macaques infected with simian immunodeficiency viruses (SIVs) isolated from African non-human primates develop a disease similar to human AIDS. SIV enters its target cells by binding to CD4 and a coreceptor, typically CCR5. Maraviroc is an entry inhibitor of human immunodeficiency virus type 1 (HIV-1) that prevents the interaction between CCR5 and the surface subunit gp120 of the viral envelope glycoprotein (Env).

The Conserved Tyr176/Leu177 Motif in the α-Helix 9 of the Feline Immunodeficiency Virus Capsid Protein Is Critical for Gag Particle Assembly.

The capsid domain (CA) of the lentiviral Gag polyproteins has two distinct roles during virion morphogenesis. As a domain of Gag, it mediates the Gag-Gag interactions that drive immature particle assembly, whereas as a mature protein, it self-assembles into the conical core of the mature virion. Lentiviral CA proteins are composed of an N-terminal region with seven α-helices and a C-terminal domain (CA-CTD) formed by four α-helices.

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.

Analysis of the functional compatibility of SIV capsid sequences in the context of the FIV gag precursor.

The formation of immature lentiviral particles is dependent on the multimerization of the Gag polyprotein at the plasma membrane of the infected cells. One key player in the virus assembly process is the capsid (CA) domain of Gag, which establishes the protein-protein interactions that give rise to the hexagonal lattice of Gag molecules in the immature virion. To gain a better understanding of the functional equivalence between the CA proteins of simian and feline immunodeficiency viruses (SIV and FIV, respectively), we generated a series of chimeric FIV Gag proteins in which the CA-coding region was partially or totally replaced by its SIV counterpart.

Processing, fusogenicity, virion incorporation and CXCR4-binding activity of a feline immunodeficiency virus envelope glycoprotein lacking the two conserved N-glycosylation sites at the C-terminus of the V3 domain.

The process of feline immunodeficiency virus (FIV) entry into its target cells is initiated by the association of the surface (SU) subunit of the viral envelope glycoprotein (Env) with the cellular receptors CD134 and CXCR4. This event is followed by the fusion of the viral and cellular membranes, which is mediated by the transmembrane (TM) subunit of Env. We and others have previously demonstrated that the V3 domain of the SU subunit of Env is essential for CXCR4 binding.

Lentiviral Gag assembly analyzed through the functional characterization of chimeric simian immunodeficiency viruses expressing different domains of the feline immunodeficiency virus capsid protein.

To gain insight into the functional relationship between the capsid (CA) domains of the Gag polyproteins of simian and feline immunodeficiency viruses (SIV and FIV, respectively), we constructed chimeric SIVs in which the CA-coding region was partially or totally replaced by the equivalent region of the FIV CA. The phenotypic characterization of the chimeras allowed us to group them into three categories: the chimeric viruses that, while being assembly-competent, exhibit a virion-associated unstable FIV CA; a second group represented only by the chimeric SIV carrying the N-terminal domain (NTD) of the FIV CA which proved to be assembly-defective; and a third group constituted by the chimeric viruses that produce virions exhibiting a mature and stable FIV CA protein, and which incorporate the envelope glycoprotein and contain wild-type levels of viral genome RNA and reverse transcriptase. Further analysis of the latter group of chimeric SIVs demonstrated that they are non-infectious due to a post-entry impairment, such as uncoating of the viral core, reverse transcription or nuclear import of the preintegration complex.

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).

Understanding the process of envelope glycoprotein incorporation into virions in simian and feline immunodeficiency viruses.

The lentiviral envelope glycoproteins (Env) mediate virus entry by interacting with specific receptors present at the cell surface, thereby determining viral tropism and pathogenesis. Therefore, Env incorporation into the virions formed by assembly of the viral Gag polyprotein at the plasma membrane of the infected cells is a key step in the replication cycle of lentiviruses. Besides being useful models of human immunodeficiency virus (HIV) infections in humans and valuable tools for developing AIDS therapies and vaccines, simian and feline immunodeficiency viruses (SIV and FIV, respectively) are relevant animal retroviruses; the study of which provides important information on how lentiviral replication strategies have evolved.

Replacement of the V3 domain in the surface subunit of the feline immunodeficiency virus envelope glycoprotein with the equivalent region of a T cell-tropic human immunodeficiency virus type 1 results in a chimeric surface protein that efficiently binds to CXCR4.

Feline immunodeficiency virus (FIV) and the T cell-tropic strains of human immunodeficiency virus type 1 (HIV-1) share the use of the chemokine receptor CXCR4 for cell entry. To study this process further we developed a cell surface binding assay based on the expression of a soluble version of the FIV SU C-terminally tagged with the influenza virus hemagglutinin epitope (HA). The specificity of the assay was demonstrated by the following evidence: (1) the SU-HA protein bound to HeLa cells that express CXCR4 but not to MDCK cells that lack this chemokine receptor; and (2) binding of the SU-HA to HeLa cells was blocked by incubation with the CXCR4 antagonist AMD3100 as well as with the anti-CXCR4 monoclonal antibody (MAb) 12G5.

Palmitoylation of the feline immunodeficiency virus envelope glycoprotein and its effect on fusion activity and envelope incorporation into virions.

The feline immunodeficiency virus (FIV) envelope glycoprotein (Env) possesses a short cytoplasmic domain of 53 amino acids containing four highly conserved cysteines at Env positions 804, 811, 815 and 848. Since palmitoylation of transmembrane proteins occurs at or near the membrane anchor, we investigated whether cysteines 804, 811 and 815 are acylated and analyzed the relevance of these residues for Env functions. Replacement of cysteines 804, 811 and 815 individually or in combination by serine residues resulted in Env glycoproteins that were efficiently expressed and processed.

Mapping of the self-interaction domains in the simian immunodeficiency virus Gag polyprotein.

To gain a better understanding of the assembly process in simian immunodeficiency virus (SIV), we first established the conditions under which recombinant SIV Gag lacking the C-terminal p6 domain (SIV GagΔp6) assembled in vitro into spherical particles. Based on the full multimerization capacity of SIV GagΔp6, and to identify the Gag sequences involved in homotypic interactions, we next developed a pull-down assay in which a panel of histidine-tagged SIV Gag truncation mutants was tested for its ability to associate in vitro with GST-SIVGagΔp6. Removal of the nucleocapsid (NC) domain from Gag impaired its ability to interact with GST-SIVGagΔp6.

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.

In vitro binding of simian immunodeficiency virus matrix protein to the cytoplasmic domain of the envelope glycoprotein.

Incorporation of the envelope (Env) glycoprotein into budding virions is a key step in the replication cycle of lentiviruses. Previously, we provided genetic and biochemical evidence indicating that Env packaging into simian immunodeficiency virus (SIV) particles is mediated by the association of the Env cytoplasmic domain (CD) with the matrix (MA) domain of Gag. In this study, we developed an in vitro binding assay that, based on recombinant proteins expressed in bacteria, allowed us to demonstrate the physical interaction between the SIV Env CD and the MA in the absence of other viral or cellular proteins.

Importance of the short cytoplasmic domain of the feline immunodeficiency virus transmembrane glycoprotein for fusion activity and envelope glycoprotein incorporation into virions.

The mature form of the envelope (Env) glycoprotein of lentiviruses is a heterodimer composed of the surface (SU) and transmembrane (TM) subunits. Feline immunodeficiency virus (FIV) possesses a TM glycoprotein with a cytoplasmic tail of approximately 53 amino acids which is unusually short compared with that of the other lentiviral glycoproteins (more than 100 residues). To investigate the relevance of the FIV TM cytoplasmic domain to Env-mediated viral functions, we characterized the biological properties of a series of Env glycoproteins progressively shortened from the carboxyl terminus.

Mutations at the C-terminus of the simian immunodeficiency virus envelope glycoprotein affect gp120-gp41 stability on virions.

The transmembrane (TM) subunit of the envelope (Env) glycoprotein of the simian immunodeficiency virus (SIV) contains an unusually long cytoplasmic domain of 164 amino acids. Previously, we identified domains in the SIV TM cytoplasmic tail that are necessary for Env incorporation into virions and viral infectivity. In this study, we investigated the relevance to Env function of the highly conserved sequence comprising the immediate C-terminal 19 residues of TM.

Functional relationship between the matrix proteins of feline and simian immunodeficiency viruses.

To investigate the functional relationship between the matrix (MA) proteins of feline and simian immunodeficiency viruses (FIV and SIV, respectively), we generated chimeric proviruses in which the MA-coding region of an SIV infectious molecular clone was partially or fully replaced by its FIV counterpart. Chimeric SIV proviruses containing the amino-terminal 36 residues or the central and carboxy-terminal regions of the FIV MA assembled into virions as efficiently as wild-type SIV. However, the resulting virions were noninfectious in single-cycle infectivity assays.

Functional domains in the feline immunodeficiency virus nucleocapsid protein.

Retroviral nucleocapsid (NC) proteins are small Gag-derived products containing one or two zinc finger motifs that mediate genomic RNA packaging into virions. In this study, we addressed the role of the feline immunodeficiency virus (FIV) NC protein in the late stages of virus replication by analyzing the assembly phenotype of FIV NC mutant viruses and the RNA binding activity of a panel of recombinant FIV NC mutant proteins. Substitution of serine for the first cysteine residue in the NC proximal zinc finger was sufficient to impair both virion assembly and genomic RNA binding.

Second-site revertants of a simian immunodeficiency virus gp41 mutant defective in envelope glycoprotein incorporation.

We previously characterized a series of small in-frame deletions within the C-terminal third of the simian immunodeficiency virus (SIV) gp41 cytoplasmic domain that significantly impair the incorporation of the envelope (Env) glycoprotein into particles and Env-mediated virus entry. Among these mutations, removal of Env residues 832-837 caused the most drastic defective phenotype. In the present study, we introduced the Delta832-837 deletion into the PBj1.

Positive and negative modulation of virus infectivity and envelope glycoprotein incorporation into virions by amino acid substitutions at the N terminus of the simian immunodeficiency virus matrix protein.

The matrix (MA) protein of the simian immunodeficiency viruses (SIVs) is encoded by the amino-terminal region of the Gag precursor and is the component of the viral capsid that lines the inner surface of the virus envelope. Previously, we identified domains in the SIV MA that are involved in the transport of Gag to the plasma membrane and in particle assembly. In this study, we characterized the role in the SIV life cycle of highly conserved residues within the SIV MA region spanning the two N-terminal alpha-helices H1 and H2.

Differential degradation of amyloid beta genetic variants associated with hereditary dementia or stroke by insulin-degrading enzyme.

Inherited amino acid substitutions at position 21, 22, or 23 of amyloid beta (Abeta) lead to presenile dementia or stroke. Insulin-degrading enzyme (IDE) can hydrolyze Abeta wild type, yet whether IDE is capable of degrading Abeta bearing pathogenic substitutions is not known. We studied the degradation of all of the published Abeta genetic variants by recombinant rat IDE (rIDE).

Presenilin 1 overexpressions in Chinese hamster ovary (CHO) cells decreases the phosphorylation of retinoblastoma protein: relevance for neurodegeneration.

Mutations in the presenilin 1 (PS1) gene have been associated to familial Alzheimer disease although the exact pathogenic mechanism is unclear. We report that stable overexpression of wild type PS1 led to a decrease in cyclin-dependent kinase 4 (CDK 4) activity and retinoblastoma tumor suppressor protein (pRb) phosphorylation that correlated with decreased levels of beta-catenin and cyclin D1. PS1 mutant D385A also precipitated a similar effect suggesting that gamma-secretase cleavage is not essential for PS1-mediated CDK 4 inhibition.