Murine leukemia virus glycosylated Gag blocks apolipoprotein B editing complex 3 and cytosolic sensor access to the reverse transcription complex.

Pathogenic retroviruses have evolved multiple means for evading host restriction factors such as apolipoprotein B editing complex (APOBEC3) proteins. Here, we show that murine leukemia virus (MLV) has a unique means of counteracting APOBEC3 and other cytosolic sensors of viral nucleic acid. Using virus isolated from infected WT and APOBEC3 KO mice, we demonstrate that the MLV glycosylated Gag protein (glyco-Gag) enhances viral core stability. Moreover, in vitro endogenous reverse transcription reactions of the glyco-Gag mutant virus were substantially inhibited compared with WT virus, but only in the presence of APOBEC3. Thus, glyco-Gag rendered the reverse transcription complex in the viral core resistant to APOBEC3. Glyco-Gag in the virion also rendered MLV resistant to other cytosolic sensors of viral reverse transcription products in newly infected cells. Strikingly, glyco-Gag mutant virus reverted to glyco-Gag-containing virus only in WT and not APOBEC3 KO mice, indicating that counteracting APOBEC3 is the major function of glyco-Gag. Thus, in contrast to the HIV viral infectivity factor protein, which prevents APOBEC3 packaging in the virion, the MLV glyco-Gag protein uses a unique mechanism to counteract the antiviral action of APOBEC3 in vivo--namely, protecting the reverse transcription complex in viral cores from APOBEC3. These data suggest that capsid integrity may play a critical role in virus resistance to intrinsic cellular antiviral resistance factors that act at the early stages of infection.

Comparative ability of IL-12 and IL-28B to regulate Treg populations and enhance adaptive cellular immunity.

Improving the potency of immune responses is paramount among issues concerning vaccines against deadly pathogens. IL-28B belongs to the newly described interferon lambda (IFNlambda) family of cytokines, and has not yet been assessed for its potential ability to influence adaptive immune responses or act as a vaccine adjuvant. We compared the ability of plasmid-encoded IL-28B to boost immune responses to a multiclade consensus HIV Gag plasmid during DNA vaccination with that of IL-12. We show here that IL-28B, like IL-12, is capable of robustly enhancing adaptive immunity. Moreover, we describe for the first time how IL-28B reduces regulatory T-cell populations during DNA vaccination, whereas IL-12 increases this cellular subset. We also show that IL-28B, unlike IL-12, is able to increase the percentage of splenic CD8(+) T cells in vaccinated animals, and that these cells are more granular and have higher antigen-specific cytolytic degranulation compared with cells taken from animals that received IL-12 as an adjuvant. Lastly, we report that IL-28B can induce 100% protection from mortality after a lethal influenza challenge. These data suggest that IL-28B is a strong candidate for further studies of vaccine or immunotherapy protocols.

Dominant negative inhibition of human immunodeficiency virus particle production by the nonmyristoylated form of gag.

Myristoylation of human immunodeficiency virus (HIV) Gag protein is essential for membrane targeting of Gag and production of viral particles. We show here that coexpression of wild-type and nonmyristoylated forms of HIV Gag resulted in severe inhibition of viral particle production, indicating that the nonmyristoylated counterpart had a dominant negative effect on particle release. When coexpressed, the nonmyristoylated Gag partially incorporated into membrane and lipid raft fractions, likely through coassembly with the wild-type Gag. The membrane and raft associations of the wild-type Gag appeared unaffected, and yet particle production was severely impaired. When viral particles produced from the coexpressing cells were analyzed, the wild-type Gag was more abundant than the nonmyristoylated Gag. Confocal microscopy showed that both forms of Gag were diffusely distributed in the cytoplasm of coexpressing cells but that a portion of the wild-type Gag population was accumulated in EEA1- and CD63-positive endosomes. The intracellular accumulation of Gag was more frequently observed at late time points. The Gag accumulation was also observed on the cell surface protrusion. Electron microscopy of the coexpressing cells revealed budding arrest phenotypes, including the occurrence of interconnected virions on the plasma membrane, and intracellular budding. We also show that the inhibition of particle production and the Gag accumulation to endosomes were suppressed when the nucleocapsid (NC) domain was deleted from the nonmyristoylated Gag, although the NC-deleted Gag was still capable of coassembly. Overall, our data indicate that coassembly with the nonmyristoylated Gag impairs HIV particle release, a phenomenon that may involve NC-mediated Gag-Gag interaction.

In vivo depletion of CD4+CD25+ regulatory T cells in cats.

To establish a characterized model of regulatory T cell (Treg) depletion in the cat we assessed the kinetics of depletion and rebound in peripheral and central lymphoid compartments after treatment with anti-CD25 antibody as determined by cell surface markers and FOXP3 mRNA expression. An 82% decrease in circulating CD4+CD25+ Tregs was observed by day 11 after treatment. CD4+CD25+ cells were also reduced in the thymus (69%), secondary lymphoid tissues (66%), and gut (67%). Although CD4+CD25+ cells rebound by day 35 post-treatment, FOXP3 levels remain depressed suggesting anti-CD25 antibody treatment has a sustainable diminutive effect on the Treg population. To determine whether CD25+ Treg depletion strategies also deplete activated CD25+ effector cells, cats were immunized with feline immunodeficiency virus (FIV) p24-GST recombinant protein, allowing them to develop a measurable memory response, prior to depletion with anti-CD25 antibody. Anti-FIV p24-GST effector cell activity in peripheral blood after depletion was sustained as determined by antigen-specific T cell proliferation and humoral responses against FIV p24-GST with an ELISA for antigen-specific feline IgG. Furthermore, development of an anti-mouse response in Treg-depleted cats was similar to control levels indicating the retained capacity to respond to a novel antigen. We conclude that despite alterations in CD25+ cell levels during depletion, the feline immune system remains functional. We demonstrate here a model for the study of disease pathogenesis in the context of reduced numbers of immunosuppressive CD4+CD25+ Tregs throughout the feline immune system.

Recombinant HIV-1 Pr55gag virus-like particles: potent stimulators of innate and acquired immune responses.

Several previous reports have clearly demonstrated the strong effectiveness of human immunodeficiency virus (HIV) Gag polyprotein-based virus-like particles (VLP) to stimulate humoral and cellular immune responses in complete absence of additional adjuvants. Yet, the mechanisms underlying the strong immunogenicity of these particulate antigens are still not very clear. However, current reports strongly indicate that these VLP act as "danger signals" to trigger the innate immune system and possess potent adjuvant activity to enhance the immunogenicity of per se only weakly immunogenic peptides and proteins. Here, we review the current understanding of how various particle-associated substances and other impurities may contribute to the observed immune-activating properties of these complex immunogens.

Analytical study of rat retrotransposon VL30 RNA dimerization in vitro and packaging in murine leukemia virus.

A sequence of the rat retrotransposon virus-like 30 S RNA (VL30) located next to the 5' end of the Harvey murine sarcoma virus (HaMSV) genome was recently found to form stable dimeric RNA in vitro and to direct the efficient packaging of VL30-derived recombinant RNAs into MuLV virions. To study the structure-function relationships of the rat VL30 dimerization-encapsidation signal (E/DLS), we have performed biochemical and genetic studies of rat VL30 RNA dimerization in vitro. The results show that temperature and specific cation/RNA interactions are important for VL30 dimerization in vitro. VL30 RNA dimerization is optimal at 55 degrees C and Li+ dramatically enhances the stability of VL30 dimeric RNA. In addition, a genetic analysis of VL30 RNA dimerization reveals that a 5' G-rich sequence is critical for dimer formation and that a UGUCUUGUC repeat contributes to VL30 dimer stability. Interestingly enough, substitution of an A for a G in the 5' G-rich sequence is sufficient to abolish VL30 RNA dimerization in vitro. Taken together, these biochemical and genetic data indicate that dimerization of VL30 RNA involves non-canonical base-pairings and possible purine-purine interactions. Nucleocapsid protein NCp10 of murine leukemia virus (MuLV), a gag-encoded protein that is tightly associated with genomic RNA in the virion core, has been shown to have nucleic acid binding and annealing activities. Here we report that the viral NCp10 protein is able to bind tightly to annealing activities. Here we report that the viral NCp10 protein is able to bind tightly to the retrotransposon VL30 RNA and to activate its dimerization. Moreover, mutations in the 5' G-rich sequence of the VL30 dimerization sequence impaired NCp10 binding to RNA. Recombinant MLV-VL30 vectors with mutations in the VL30 dimerization sequence were constructed. Results obtained in vivo clearly show that the mutations that had a deleterious effect on the packaging of MLV-VL30 retroviral vector in vivo were those that impaired VL30 RNA dimerization and interactions with NCp10 in vitro, even the single mutation in the 5' G-rich region. Therefore, these findings suggest that packaging of VL30 RNA into MuLV virions requires specific interactions between RNA dimerization sequences and viral NC protein molecules.

HIV p17 enhances lymphocyte proliferation and HIV-1 replication after binding to a human serum factor.

OBJECTIVE: To analyse the role of recombinant HIV-1 protein p17 in the modulation of cell activity. METHODS: Peripheral blood mononuclear cells (PBMC) obtained from healthy donors were cultured in the presence or absence of p17 with mitogens such as phytohaemagglutinin or interleukin-2 and their response assayed by cell proliferation. Cross-linking experiments were employed to investigate the presence of a binding between p17 and factor(s) present in human serum. An immunoenzymatic assay for p24 antigen detection was used to analyse the effect of the addition of exogenous p17 to cultures of PBMC infected with HIV-1 in vitro. RESULTS: Purified recombinant p17 protein at a concentration of 0.25 microg/ml significantly increased the proliferation of preactivated PBMC obtained from healthy donors. This effect was obtained by binding p17 to factor(s) present in human serum and observed on both CD4+ and CD8+ T cells. Recombinant p17 also induced an increased rate of HIV-1 replication, probably due to enhanced T-cell proliferation. The activity of p17 protein was inhibited by anti-p17 antibodies generated by injecting recombinant p17 in rabbits, but not by human antibodies generated during the natural course of HIV infection. CONCLUSION: Characterization of the human factor(s) and identification of the interacting p17 epitope(s) will improve our understanding of the mechanisms used by HIV to efficiently replicate in our organisms.

HIV-1 p17 and IFN-gamma both induce fructose 1,6-bisphosphatase.

The p17 matrix protein of the human immunodeficiency virus type 1 (HIV-1) plays a crucial role in AIDS pathogenesis. It orchestrates viral assembly and directs the preintegration complex to the nucleus of infected cells. Recently, the three-dimensional structure of p17 was shown to resemble that of interferon-gamma (IFN-gamma), suggesting that both proteins might share analogous functions. We demonstrate that in monocytes, p17 shares with IFN-gamma the ability to induce 1alpha-hydroxylase activity and to activate fructose 1,6-bisphosphatase gene expression in the presence of 25-hydroxyvitamin D3. However, p17 does not bind to the IFN-gamma cell membrane receptor and fails to increase expression of IFN-gamma-induced proteins, such as tryptophanyl-tRNA synthetase, Fc gammaRI, and HLA DR or B7/BB1 antigens. Altogether, our results raise the possibility that the structural resemblance between p17 and IFN-gamma causes the selective activation of a common pathway resulting in the production of 1,25-dihydroxyvitamin D3. We also found that unlike IFN-gamma, p17 increases the intracellular ATP content. Since transport of the HIV-1 preintegration complex through the nuclear membrane is an ATP-dependent process, our observation suggests that p17 plays a double role in this active transport, not only by acting as a chaperone molecule but also by recruiting the necessary energy for this process.

A polypeptide encoded within the murine AIDS defective virus stimulates primary proliferation of CD8+ T-cells.

The murine AIDS (MAIDS) is a retrovirus-induced disease that shows severe immunodeficiency with abnormal lymphoproliferation in susceptible strains of mice. To clarify the antigenicity of gag gene products of the LP-BM5 defective virus, which is known as the causative virus of MAIDS, we expressed and purified the gag p12 gene product (P12) by using a baculovirus expression vector system. The P12 protein strongly stimulated the proliferation of normal C57BL/6 (B6) lymph node T-cells in vitro. Furthermore, a 25-mer synthetic polypeptide within the P12 sequence gave rise to the similar or even higher activation of T-cells. The phenotype of responding T-cells was found to be CD8+ CD44low, indicating that naive CD8+ T-cells respond against a peptide encoded within a MAIDS defective virus gag p12 gene. Finally, the expression of T-cell receptor (TcR) V beta on the responding CD8+ T-cells was analyzed. Although CD8+ T-cells with the particular V beta chains were expanded in response to the 25-mer peptide, this polypeptide does not seem to be a superantigen, since this response is MHC class I-restricted and the V beta preference is not striking. The presentation pathway of this highly antigenic polypeptide will be discussed.

Blocking of human immunodeficiency virus type-1 virion autolysis by autologous p2(gag) peptide.

Our previous study suggested that the p2(gag) peptide, AEAMSQVTNTATIM, inhibits human immunodeficiency virus type 1 (HIV-1) protease (PR) activity in vitro. In this study, Ala substitutions (Met4Ala and Thr8Ala) and deletion of amino acid Asn9 within the nona p2(gag) peptide (AEAMSQVTN) were found to decrease the inhibitory effect on HIV-1 PR activity. Furthermore, treatment of PMA-activated latently infected T lymphocytes, ACH-2 cells, with the p2(gag) peptide (100 and 250 micro M) resulted in a decrease in the amount of p24(gag )in the resultant viral lysates derived from the cell-free supernatant. In addition, the HIV-1-Tat-p2(gag) fusion peptide was synthesized to effectively deliver the p2(gag) peptide into the cells. The fusion peptide was incorporated into chronically infected T lymphocytes, CEM/LAV-1 cells, as detected on indirect immunofluorescence analysis using anti-p2(gag) peptide monoclonal antibodies, which recognize the nona peptide (AEAMSQVTN) derived from the N-terminus of the p2(gag) peptide, and cleaved by HIV-1 PR in vitro. Treatment of CEM/LAV-1 cells with the fusion peptide also resulted in a decrease in the amount of p24(gag )in the resultant viral lysate derived from the cell-free supernatant. Taken together, these data suggest that the p2(gag) peptide consequently blocks the autolysis of HIV-1 virions for the conservation of viral species.

Intraventricular administration of a FIV-envelope protein induces sleep architecture changes in rats.

Fifteen adult male Sprague-Dawley rats were implanted with a set of electrodes for standard sleep recordings. A stainless steel cannula was also implanted into the lateral ventricle of these rats. Fifteen additional rats were implanted with a cannula alone. Rats with electrodes were habituated for 3 days or more to the recording environment, then placed into 3 groups (n = 5). One group received saline (i.c.v.), while the other two groups received either the feline immunodeficiency virus envelope glycoprotein (FIV SU-Env) or a fragment of the Epstein-Barr virus envelope glycoprotein (EB gp105). Rats were then recorded for electrographic sleep-wake cycle evaluation for the following 4 h. Core temperature was assessed through a thermistor probe inserted into the rectum, immediately before and 1, 2, 3 and 4 h after the i.c.v. treatment condition. Results demonstrated that compared to saline, FIV SU-Env increased wakefulness and decreased REM sleep throughout the 4 h of recording. Likewise, FIV SU-Env decreased SWS2 for 2 h. In addition, EB gp105 administration elicited minor modifications of the sleep-wake cycle, causing only a transient reduction of REM sleep in the first hour of recording. None of the treatments altered body temperature. These findings strongly support and extend studies in FIV-infected cats in which we have found similar sleep abnormalities. In addition, these results are consistent with the hypothesis that the FIV SU-Env proteins are responsible for these neurological disturbances.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoregulatory effect of a synthetic peptide corresponding to a region of protein p24 of HIV.

The effect of a synthetic peptide, corresponding to a sequence of HIV-1 p24 protein (amino acids 218-237), on in vitro immune responses was studied. The peptide inhibited in a dose-dependent manner the induction of an anti-SRC antibody response and of a PPD-specific proliferative response of human PBL. On the other hand, PHA-induced proliferation of human PBL and PPD-induced proliferation of a PPD-specific human T-cell line were not modified by comparable amounts of the peptide. These results suggest that structures from a protein (p24), present in the serum throughout the course of HIV infection, are able to interfere with the inductive stages of specific immune responses. These findings may help to unravel some of the pathogenic mechanisms of AIDS and may contribute to the development of vaccine strategies.

NCp7 activates HIV-1Lai RNA dimerization by converting a transient loop-loop complex into a stable dimer.

Nucleocapsid protein 7 (NCp7), the human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein, was shown to strongly potentiate the dimerization of the retroviral genomic RNA. This process involves the interaction of two retroviral RNA monomer subunits near their 5'-ends. A region located upstream from the splice donor site was recently identified as being responsible for the formation of dimeric HIV-1 RNA. This region appeared to be confined within a stem-loop structure, with an autocomplementary sequence in the loop. In an in vitro study of spontaneous dimer formation, we reported that the 77-402 RNA transcript forms two distinct dimers differing in their thermostability: D37 and D55. We identified D37 as a "kissing" complex structure, formed via a loop-loop interaction between the two monomers, and D55 as a double stranded structure involving all nucleotides of the stem-loop via canonical base pairing. In this report, we have characterized the role of NCp7 in the HIV-1Lai RNA dimerization process by using in vitro dimerization assays with RNA transcripts of different lengths and dimer thermal dissociation. Our results show that the nucleocapsid protein NCp7 activates RNA dimerization very likely through interaction with the kissing complex and converts it into a stable dimer. Furthermore, this NCp7-promoted conversion only occurs if the 240-280 stem-loop structure is present in HIV-1Lai RNA molecules and contains the autocomplementary G257CGCGC262 sequence. This study suggests that, under physiological conditions, an NCp7-mediated RNA conformational change is involved in the maturation of the HIV-1 RNA dimer.

Inhibition of protein kinase C by a peptide conjugate homologous to a domain of the retroviral protein p15E.

Retroviral infection is associated with immunosuppression, which has been shown to be due, in part, to the action of the envelope protein p15E. We studied a synthetic peptide (CKS-17) homologous to a highly conserved domain of the retroviral envelope protein p15E, which, when conjugated to BSA (CKS-17-BSA), can inhibit IL-1- and phorbol ester-mediated responses in cultured murine thymoma cells, and Ca2(+)- and phosphatidylserine-dependent protein kinase C (PKC) activity of cell homogenates. We characterized the mechanism of inhibition of PKC by the peptide. Using PKC purified from rat brain we found that CKS-17-BSA inhibited PKC-catalyzed Ca2(+)- and phosphatidylserine-dependent histone phosphorylation with an estimated ID50 of 4 microM. CKS-17-BSA did not inhibit the catalytic subunit of cAMP-dependent protein kinase. CKS-17-BSA also inhibited the Ca2(+)- and PS-independent activity of a catalytic fragment of PKC that was generated by limited trypsin treatment. However, CKS-17-BSA did not act as a competitive inhibitor of PKC with respect to ATP or phosphoacceptor substrate, despite the similarity between the CKS-17 sequence and substrates and pseudosubstrates of PKC. We conclude that this peptide homologue of a retroviral envelope protein has a novel mechanism of inhibition of PKC.

Reconstitution of cleavage of human immunodeficiency virus type-1 (HIV-1) RNAs.

A human immunodeficiency virus type 1 (HIV-1) particle contains approximately 1200 molecules of gag proteins and two copies of a 9.2-kb genomic RNA which has been reported to be dimerized and rapidly cleaved and to form a complex with a nucleocapsid protein, p7 (NCp7), during viral budding. These suggest that the cleavage can be reconstituted with gag proteins in vitro. Here we show that the p15(gag) coding region of viral RNA is fragmented in viral particles and that in vitro-synthesized RNA transcripts of HIV-1 undergo cleavage which is activated by NCp7 and other factors. Single-stranded oligoribonucleotides were cleaved between C and A or U and A, leaving 2',3'-cyclic phosphate and 5'-hydroxyl termini. These findings might explain the rapid degradation of genomic RNAs in HIV-1 particles.

Conformational change of dimerization initiation site of HIV-1 genomic RNA by NCp7 or heat treatment.

Dimerization initiation site (DIS) of the human immunodeficiency virus type 1 (HIV-1) genome has been identified as a primary sequence that can form a stem-loop structure with a self-complementary sequence in the loop and a bulge in the stem. A DIS RNA fragment spontaneously forms a kissing dimer and is converted into an extended-duplex dimer by supplement of nucleocapsid protein NCp7. This two-step dimerization reaction can be also executed by a heat treatment instead of the binding proteins. However, it has not identified whether mechanisms of the conformational conversion by two different treatments are identical or not. In the present study, we used a series of DIS RNA oligonucleotides and the conformations of two extended-duplex dimers produced by the two different treatments were compared by the analysis of NMR spectra in the imino proton region. It was found that the effects of the two kinds of treatments are quite similar and the conformations of the two extended-duplex dimers are identical. These findings suggest that the conversion mechanisms DIS RNA by NCp7 and heat treatments are similar.

Inhibition of infectious human immunodeficiency virus type 1 particle formation by Gag protein-derived peptides.

Sequential overlapping Gag protein-derived oligopeptides of human immunodeficiency virus type 1 (HIV-1) 22 to 24 amino acids long, were synthesized and tested in vitro for antiviral activity. Two synthetic peptides, one derived from the matrix protein p17 (NPGLLETSEGCRQ, amino acids 47 to 59) and one located in the capsid protein p24 (PAATLEEMMTA, amino acids 339 to 349) inhibited the production of infectious virus when added to HIV-1-infected cultures when used in the range of 20 to 200 micrograms/ml. As shown by thin section electron microscopy, peptide treatment resulted in the release of immature, deformed virus particles suggesting that the two peptides interfered with assembly and maturation. Other Gag protein-derived oligopeptides had little or no influence on virus production. To characterize further the functionally active regions we synthesized peptide derivatives with three consecutive amino acids substituted by alanine; they did not cause inhibition. Therefore the regions responsible for inhibition were located between amino acids 50 to 61 in p17, and 342 to 350 in p24. These observations might lead to the development of a new antiviral strategy affecting the late stage of virus replication.

MHC class I surface expression in embryo-derived cell lines inducible with peptide or interferon.

It has long been recognized that the absence of expression of products of the major histocompatibility complex (MHC) during early development might allow the fetus to escape recognition by maternal lymphocytes. In addition to the MHC class I heavy chain and beta 2-microglobulin, antigenic peptide is an essential structural component of the class I molecule. Indeed, there is evidence that MHC-linked genes encoding peptide transporter molecules and possibly components of a proteolytic complex are necessary for MHC class I assembly and stability at the cell surface. Here we demonstrate that embryonic cells in general show a defect in MHC class I assembly. Surface expression was rescued in the presence of an appropriate antigenic peptide, or by treatment with interferon. Consistent with this, HAM1 messenger RNA was not constitutively expressed, but was inducible by interferon, and during differentiation in vitro. Thus, tolerance of the fetal allograft may in part be controlled at the level of peptide-dependent MHC class I assembly.