Terminal-repeat retrotransposons with GAG domain in plant genomes: a new testimony on the complex world of transposable elements

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MRKAd5 HIV-1 Gag/Pol/Nef Vaccine-Induced T-Cell Responses Inadequately Predict Distance of Breakthrough HIV-1 Sequences to the Vaccine or Viral Load

Background: The sieve analysis for the Step trial found evidence that breakthrough HIV-1 sequences for MRKAd5/HIV-1 Gag/Pol/Nef vaccine recipients were more divergent from the vaccine insert than placebo sequences in regions with predicted epitopes. We linked the viral sequence data with immune response and acute viral load data to explore mechanisms for and consequences of the observed sieve effect. Methods: Ninety-one male participants (37 placebo and 54 vaccine recipients) were included; viral sequences were obtained at the time of HIV-1 diagnosis. T-cell responses were measured 4 weeks post-second vaccination and at the first or second week post-diagnosis. Acute viral load was obtained at RNA-positive and antibody-negative visits. Findings: Vaccine recipients had a greater magnitude of post-infection CD8+ T cell response than placebo recipients (median 1.68% vs 1.18%; p = 0.04) and greater breadth of post-infection response (median 4.5 vs 2; p = 0.06). Viral sequences for vaccine recipients were marginally more divergent from the insert than placebo sequences in regions of Nef targeted by pre-infection immune responses (p = 0.04; Pol p = 0.13; Gag p = 0.89). Magnitude and breadth of pre-infection responses did not correlate with distance of the viral sequence to the insert (p. 0.50). Acute log viral load trended lower in vaccine versus placebo recipients (estimated mean 4.7 vs 5.1) but the difference was not significant (p = 0.27). Neither was acute viral load associated with distance of the viral sequence to the insert (p>0.30). Interpretation: Despite evidence of anamnestic responses, the sieve effect was not well explained by available measures of T-cell immunogenicity. Sequence divergence from the vaccine was not significantly associated with acute viral load. While point estimates suggested weak vaccine suppression of viral load, the result was not significant and more viral load data would be needed to detect suppression.

Human Endogenous Retrovirus K(HML-2) Gag and Env specific T-cell responses are not detected in HTLV-I-infected subjects using standard peptide screening methods

Abstract Background An estimated 10–20 million individuals are infected with the retrovirus human T-cell leukemia virus type 1 (HTLV-1). While the majority of these individuals remain asymptomatic, 0.3-4% develop a neurodegenerative inflammatory disease, termed HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HAM/TSP results in the progressive demyelination of the central nervous system and is a differential diagnosis of multiple sclerosis (MS). The etiology of HAM/TSP is unclear, but evidence points to a role for CNS-inflitrating T-cells in pathogenesis. Recently, the HTLV-1-Tax protein has been shown to induce transcription of the human endogenous retrovirus (HERV) families W, H and K. Intriguingly, numerous studies have implicated these same HERV families in MS, though this association remains controversial. Results Here, we explore the hypothesis that HTLV-1-infection results in the induction of HERV antigen expression and the elicitation of HERV-specific T-cells responses which, in turn, may be reactive against neurons and other tissues. PBMC from 15 HTLV-1-infected subjects, 5 of whom presented with HAM/TSP, were comprehensively screened for T-cell responses to overlapping peptides spanning HERV-K(HML-2) Gag and Env. In addition, we screened for responses to peptides derived from diverse HERV families, selected based on predicted binding to predicted optimal epitopes. We observed a lack of responses to each of these peptide sets. Conclusions Thus, although the limited scope of our screening prevents us from conclusively disproving our hypothesis, the current study does not provide data supporting a role for HERV-specific T-cell responses in HTLV-1 associated immunopathology.

The MHC-haplotype influences primary, but not memory, immune responses to an immunodominant peptide containing T- and B-cell epitopes of the caprine arthritis encephalitis virus Gag protein

In this report, we describe a short peptide, containing a T helper- and a B-cell epitope, located in the Gag protein of the caprine arthritis encephalitis virus (CAEV). This T-cell epitope is capable of inducing a robust T-cell proliferative response in vaccinated goats with different genetic backgrounds and to provide help for a strong antibody response to the B-cell epitope, indicating that it may function as a universal antigen-carrier for goat vaccines. The primary immune response of goats homozygous for MHC class I and II genes showed an MHC-dependent partitioning in rapid-high and slow-low responses, whereas the memory immune response was strong in both groups, demonstrating that a vaccine based on this immunodominant T helper epitope is capable to overcome genetic differences.

Phylogenetic analysis of the gag region encoding the matrix protein of small ruminant lentiviruses: comparative analysis and molecular epidemiological applications

Little sequence information exists on the matrix-protein (MA) encoding region of small ruminant lentiviruses (SRLV). Fifty-two novel sequences were established and permitted a first phylogenetic analysis of this region of the SRLV genome. The variability of the MA encoding region is higher compared to the gag region encoding the capsid protein and surprisingly close to that reported for the env gene. In contrast to primate lentiviruses, the deduced amino acid sequences of the N- and C-terminal domains of MA are variable. This permitted to pinpoint a basic domain in the N-terminal domain that is conserved in all lentiviruses and likely to play an important functional role. Additionally, a seven amino acid insertion was detected in all MVV strains, which may be used to differentiate CAEV and MVV isolates. A molecular epidemiology analysis based on these sequences indicates that the Italian lentivirus strains are closely related to each other and to the CAEV-CO strain, a prototypic strain isolated three decades ago in the US. This suggests a common origin of the SRLV circulating in the monitored flocks, possibly related to the introduction of infected goats in a negative population. Finally, this study shows that the MA region is suitable for phylogenetic studies and may be applied to monitor SRLV eradication programs.

Vaccination with a T-cell-priming Gag peptide of caprine arthritis encephalitis virus enhances virus replication transiently in vivo

CD4+ T cells are involved in several immune response pathways used to control viral infections. In this study, a group of genetically defined goats was immunized with a synthetic peptide known to encompass an immunodominant helper T-cell epitope of caprine arthritis encephalitis virus (CAEV). Fifty-five days after challenge with the molecularly cloned CAEV strain CO, the vaccinated animals had a higher proviral load than the controls. The measurement of gamma interferon and interleukin-4 gene expression showed that these cytokines were reliable markers of an ongoing immune response but their balance did not account for more or less efficient control of CAEV replication. In contrast, granulocyte-macrophage colony-stimulating factor appeared to be a key cytokine that might support virus replication in the early phase of infection. The observation of a potential T-cell-mediated enhancement of virus replication supports other recent findings showing that lentivirus-specific T cells can be detrimental to the host, suggesting caution in designing vaccine candidates.

A Gag peptide encompassing B- and T-cell epitopes of the caprine arthritis encephalitis virus functions as modular carrier peptide

Short synthetic peptides are important tools in biomedical research permitting to generate hapten specific polyclonal sera for analytical purposes or functional studies. In this paper we provide proof of principle that a peptide located in a highly conserved portion of the Gag protein of the caprine arthritis encephalitis virus and carrying an immunodominant T helper cell epitope functions as an efficient carrier peptide, mediating a strong antibody response to a peptidic hapten encompassing a well-characterized B cell epitope of Env. The carrier and hapten peptides were collinearly synthesized permutating their molecular arrangement. While the antibody response to the hapten was similar for both constructs, the antibody response to a B cell epitope overlapping the T helper cell epitope of the Gag carrier peptide was considerably different. This permits a modular use of the carrier peptide to generate antibody directed exclusively to the hapten peptide or a strong humoral response to both carrier- and hapten-peptide. Finally, we have mapped the epitopes involved in this polarized antibody response and discussed the potential immunological implications.

A cellular cleavage pathway for the Moloney murine leukemia virus precursor protein, Pr65$\rm\sp{gag}$: Identification of a new viral protein containing CAp30 and NCp10 sequences

The origin and structure of P55$\sp{\rm gag},$ a gag encoded polyprotein lacking the nucleocapsid protein, NCp10, have been explored. Evidence shows that P55$\sp{\rm gag}$ is formed by non-viral proteolytic cleavage of the Moloney murine leukemia virus (MoMuLV)gag precursor protein, Pr65$\sp{\rm gag}.$ P55$\sp{\rm gag}$ is produced in cells infected by a viral protease deletion mutant and by a recombinant murine sarcoma virus known to lack the protease gene, implying that a cellular protease is responsible for the cleavage. Structural and immunological studies show that the protein cleavage site is upstream of the CAp30-NCp10 viral proteolytic junction, implying that P55$\sp{\rm gag}$ lacks the carboxy-terminal residues of CAp30. During the course of studying P55$\sp{\rm gag},$ another protein was discovered, which I named nucleocapsid-related protein(NCRP). NCRP possesses the portion of CAp30 that is lacking in P55$\sp{\rm gag}.$ NCRP possesses antigenic epitopes present in CAp30 and NCp10. NCRP was observed in virus lysates and in nuclear lysates of MoMuLV infected cells; it was not detected in the cytoplasmic fractions of MoMuLV infected cells. Our results indicated that NCRP originates from Pr65$\sp{\rm gag},$ resulting from the same cellular proteolytic cleavage event that produces the viral cellular protein P55$\sp{\rm gag}.$ P55$\sp{\rm gag}$- and NCRP-like proteins also were observed in AKV murine leukemia virus (AKV MuLV) and feline leukemia virus (FeLV) infected cells and in their respective virus particles. The site of cleavage that yields P55$\sp{\rm gag}$ and NCRP is within the carboxy terminus of CAp30, likely within a motif highly conserved among mammalian type C retroviruses. This new motif, called the capsid conserved motif (CCM), overlaps a region containing both a possible bipartite nuclear targeting sequence and a region homologous with the U1 small nuclear ribonucleoprotein 70-kD protein. This domain, when intact, may act as a nuclear targeting sequence for the gag precursor proteins Pr65$\sp{\rm gag}$ and CAp30. Nuclei of cells infected with MoMuLV were examined for the presence of gag proteins. Both Pr65$\sp{\rm gag}$ and CAp30 were detected in the nuclear fraction of MoMuLV, AKV MuLV and FeLV infected cells. P55$\sp{\rm gag}$ was never detected in the nucleus of MoMuLV, AKV MuLV and FeLV infected cells or in their respective virus particles. I propose that NCRP may be involved in sequestering viral genomic RNA for the purposes of encapsidation and intracellular viral genomic RNA dimerization. ^

Generation of a recombinant Gag virus-like-particle panel for the evaluation of p24 antigen detection by diagnostic HIV tests.

BACKGROUND Detection of HIV-1 p24 antigen permits early identification of primary HIV infection and timely intervention to limit further spread of the infection. Principally, HIV screening should equally detect all viral variants, but reagents for a standardised test evaluation are limited. Therefore, we aimed to create an inexhaustible panel of diverse HIV-1 p24 antigens. METHODS We generated a panel of 43 recombinantly expressed virus-like particles (VLPs), containing the structural Gag proteins of HIV-1 subtypes A-H and circulating recombinant forms (CRF) CRF01_AE, CRF02_AG, CRF12_BF, CRF20_BG and group O. Eleven 4th generation antigen/antibody tests and five antigen-only tests were evaluated for their ability to detect VLPs diluted in human plasma to p24 concentrations equivalent to 50, 10 and 2 IU/ml of the WHO p24 standard. Three tests were also evaluated for their ability to detect p24 after heat-denaturation for immune-complex disruption, a pre-requisite for ultrasensitive p24 detection. RESULTS Our VLP panel exhibited an average intra-clade p24 diversity of 6.7%. Among the 4th generation tests, the Abbott Architect and Siemens Enzygnost Integral 4 had the highest sensitivity of 97.7% and 93%, respectively. Alere Determine Combo and BioRad Access were least sensitive with 10.1% and 40.3%, respectively. Antigen-only tests were slightly more sensitive than combination tests. Almost all tests detected the WHO HIV-1 p24 standard at a concentration of 2 IU/ml, but their ability to detect this input for different subtypes varied greatly. Heat-treatment lowered overall detectability of HIV-1 p24 in two of the three tests, but only few VLPs had a more than 3-fold loss in p24 detection. CONCLUSIONS The HIV-1 Gag subtype panel has a broad diversity and proved useful for a standardised evaluation of the detection limit and breadth of subtype detection of p24 antigen-detecting tests. Several tests exhibited problems, particularly with non-B subtypes.

Proteasome inhibition interferes with Gag polyprotein processing, release, and maturation of HIV-1 and HIV-2

Retrovirus assembly and maturation involve folding and transport of viral proteins to the virus assembly site followed by subsequent proteolytic cleavage of the Gag polyprotein within the nascent virion. We report that inhibiting proteasomes severely decreases the budding, maturation, and infectivity of HIV. Although processing of the Env glycoproteins is not changed, proteasome inhibitors inhibit processing of Gag polyprotein by the viral protease without affecting the activity of the HIV-1 viral protease itself, as demonstrated by in vitro processing of HIV-1 Gag polyprotein Pr55. Furthermore, this effect occurs independently of the virus release function of the HIV-1 accessory protein Vpu and is not limited to HIV-1, as proteasome inhibitors also reduce virus release and Gag processing of HIV-2. Electron microscopy analysis revealed ultrastructural changes in budding virions similar to mutants in the late assembly domain of p6gag, a C-terminal domain of Pr55 required for efficient virus maturation and release. Proteasome inhibition reduced the level of free ubiquitin in HIV-1-infected cells and prevented monoubiquitination of p6gag. Consistent with this, viruses with mutations in PR or p6gag were resistant to detrimental effects mediated by proteasome inhibitors. These results indicate the requirement for an active proteasome/ubiquitin system in release and maturation of infectious HIV particles and provide a potential pharmaceutical strategy for interfering with retrovirus replication.

Foamy virus reverse transcriptase is expressed independently from the Gag protein.

In the foamy virus (FV) subgroup of retroviruses the pol genes are located in the +1 reading frame relative to the gag genes and possess potential ATG initiation codons in their 5' regions. This genome organization suggests either a + 1 ribosomal frameshift to generate a Gag-Pol fusion protein, similar to all other retroviruses studied so far, or new initiation of Pol translation, as used by pararetroviruses, to express the Pol protein. By using a genetic approach we have ruled out the former possibility and provide evidence for the latter. Two down-mutations (M53 and M54) of the pol ATG codon were found to abolish replication and Pol protein expression of the human FV isolate. The introduction of a new ATG in mutation M55, 3' to the down-mutated ATG of mutation M53, restored replication competence, indicating that the pol ATG functions as a translational initiation codon. Two nonsense mutants (M56 and M57), which functionally separated gag and pol with respect to potential frame-shifting sites, were also replication-competent, providing further genetic evidence that FVs express the Pol protein independently from Gag. Our results show that during a particular step of the replication cycle, FVs differ fundamentally from all other retroviruses.

TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis, is a toroid-shaped molecule that binds transcripts containing GAG or UAG repeats separated by two nucleotides.

The trp RNA-binding attenuation protein of Bacillus subtilis, TRAP, regulates both transcription and translation by binding to specific transcript sequences. The optimal transcript sequences required for TRAP binding were determined by measuring complex formation between purified TRAP protein and synthetic RNAs. RNAs were tested that contained repeats of different trinucleotide sequences, with differing spacing between the repeats. A transcript containing GAG repeats separated by two-nucleotide spacers was bound most tightly. In addition, transmission electron microscopy was used to examine the structure of TRAP and the TRAP-transcript complex. TRAP was observed to be a toroid-shaped oligomer when free or when bound to either a natural or a synthetic RNA.

Analyse du trafic et de la distribution intracellulaire de la protéine Gag du VIH-1 dans les cellules HEK 293T : importance de l'efficacité de la relâche virale

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Étude du trafic intracellulaire de la protéine Gag du VIH-1 : rôle des facteurs de l'hôte

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.