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MPMV precursor polypeptide Pr78 Gag orchestrates assembly and packaging of genomic RNA (gRNA) into virus particles. Therefore, we have expressed recombinant full-length Pr78 Gag either with or without His 6 -tag in bacterial as well as eukaryotic cultures and purified the recombinant protein from soluble fractions of the bacterial cultures. The recombinant Pr78 Gag protein has the intrinsic ability to assemble in vitro to form virus like particles (VLPs). Consistent with this observation, the recombinant protein could form VLPs in both prokaryotes and eukaryotes. VLPs formed in eukaryotic cells by recombinant Pr78 Gag with or without His 6 -tag can encapsidate MPMV transfer vector RNA, suggesting that the inclusion of the His 6 -tag to the full-length Pr78 Gag did not interfere with its expression or biological function. This study demonstrates the expression and purification of a biologically active, recombinant Pr78 Gag , which should pave the way to study RNA-protein interactions involved in the MPMV gRNA packaging process.

Autonomous retrotransposons, in which replication and transcription are coupled, encode the essential gag and pol genes as a fusion or separate overlapping form(s) that are expressed in single transcripts regulated by a common upstream promoter. The element-specific expression strategies have driven development of relevant translational recoding mechanisms including ribosomal frameshifting to satisfy the protein stoichiometry critical for the assembly of infectious virus-like particles. Retrotransposons with different recoding strategies exhibit a mosaic distribution pattern across the diverse families of reverse transcribing elements, even though their respective distributions are substantially skewed towards certain family groups. However, only a few investigations to date have focused on the emergence of retrotransposons evolving novel expression strategy and causal genetic drivers of the structural variants. In this study, the bulk of genomic and transcribed sequences of a Ty3/gypsy-like CsRn1 retrotransposon in Clonorchis sinensis were analyzed for the comprehensive examination of its expression strategy. Our results demonstrated that structural variants with single open reading frame (ORF) have recurrently emerged from precedential CsRn1 copies encoding overlapping gag-pol ORFs by a single-nucleotide insertion in an upstream region of gag stop codon. In the parasite genome, some of the newly evolved variants appeared to undergo proliferative burst as active master lineages together with their ancestral copies. The genetic event was similarly observed in Opisthorchis viverrini, the closest neighbor of C. sinensis, whereas the resulting structural variants might have failed to overcome purifying selection and comprised minor remnant copies in the Opisthorchis genome., (Copyright © 2017 Elsevier B.V. All rights reserved.)

BACKGROUND Morphea, also known as localized scleroderma, is a rare autoimmune connective tissue disease characterized by skin fibrosis. UVA1 phototherapy is an important asset in the reduction of clinical manifestations in morphea. There are studies claiming that UV light modulates the expression of some human endogenous retroviral sequences. The aim of this study was to determine if the expression of HERV-K10 gag element is lowered by UVA1 phototherapy in morphea, a disease in which such irradiation has a soothing effect. MATERIAL AND METHODS The expression levels of the HERV-K10 gag were assessed by real-time PCR (polymerase chain reaction) in peripheral blood mononuclear cells (PBMC) and skin-punch biopsies of healthy volunteers and 9 morphea patients before and after phototherapy. Additionally, correlations between the HERV-K10 gag expression and age, disease duration, the Localized Scleroderma Skin Severity Index (LoSSI), and antinuclear antibody (ANA) titers were assessed. RESULTS In PBMC, HERV-K10 gag mRNA was significantly elevated after UVA1 phototherapy compared to healthy controls. Most of the patients responded with an increased expression level of this sequence. However, we found no statistical evidence at this point that phototherapy indeed has an effect on the HERV-K10 gag expression (there were no statistical differences in PBMC of morphea patients before and after phototherapy). Similarly, there was no statistically relevant effect of the UVA1 on the expression of HERV-K10 gag in skin. CONCLUSIONS At this point, the effect of UVA1 phototherapy on the expression of HERV-K10 gag cannot be statistically confirmed.

A novel form of copy number control (CNC) helps maintain a low number of Ty1 retrovirus-like transposons in the Saccharomyces genome. Ty1 produces an alternative transcript that encodes p22, a trans-dominant negative inhibitor of Ty1 retrotransposition whose sequence is identical to the C-terminal half of Gag. The level of p22 increases with copy number and inhibits normal Ty1 virus-like particle (VLP) assembly and maturation through interactions with full length Gag. A forward genetic screen for CNC-resistant (CNCR) mutations in Ty1 identified missense mutations in GAG that restore retrotransposition in the presence of p22. Some of these mutations map within a predicted UBN2 domain found throughout the Ty1/copia family of long terminal repeat retrotransposons, and others cluster within a central region of Gag that is referred to as the CNCR domain. We generated multiple alignments of yeast Ty1-like Gag proteins and found that some Gag proteins, including those of the related Ty2 elements, contain non-Ty1 residues at multiple CNCR sites. Interestingly, the Ty2-917 element is resistant to p22 and does not undergo a Ty1-like form of CNC. Substitutions conferring CNCR map within predicted helices in Ty1 Gag that overlap with conserved sequence in Ty1/copia, suggesting that p22 disturbs a central function of the capsid during VLP assembly. When hydrophobic residues within predicted helices in Gag are mutated, Gag level remains unaffected in most cases yet VLP assembly and maturation is abnormal. Gag CNCR mutations do not alter binding to p22 as determined by co-immunoprecipitation analyses, but instead, exclude p22 from Ty1 VLPs. These findings suggest that the CNCR alleles enhance retrotransposition in the presence of p22 by allowing productive Gag-Gag interactions during VLP assembly. Our work also expands the strategies used by retroviruses for developing resistance to Gag-like restriction factors to now include retrotransposons.

Ty1 Gag comprises the capsid of virus-like particles and provides nucleic acid chaperone (NAC) functions during retrotransposition in budding yeast. A subgenomic Ty1 mRNA encodes a truncated Gag protein (p22) that is cleaved by Ty1 protease to form p18. p22/p18 strongly inhibits transposition and can be considered an element-encoded restriction factor. Here, we show that only p22 and its short derivatives restrict Ty1 mobility whereas other regions of GAG inhibit mobility weakly if at all. Mutational analyses suggest that p22/p18 is synthesized from either of two closely spaced AUG codons. Interestingly, AUG1p18 and AUG2p18 proteins display different properties, even though both contain a region crucial for RNA binding and NAC activity. AUG1p18 shows highly reduced NAC activity but specific binding to Ty1 RNA, whereas AUG2p18 shows the converse behavior. p22/p18 affects RNA encapsidation and a mutant derivative defective for RNA binding inhibits the RNA chaperone activity of the C-terminal region (CTR) of Gag-p45. Moreover, affinity pulldowns show that p18 and the CTR interact. These results support the idea that one aspect of Ty1 restriction involves inhibition of Gag-p45 NAC functions by p22/p18-Gag interactions., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The well-established safety profile of the tuberculosis vaccine strain, Mycobacterium bovis bacille Calmette-Guérin (BCG), makes it an attractive vehicle for heterologous expression of antigens from clinically relevant pathogens. However, successful generation of recombinant BCG strains possessing consistent insert expression has encountered challenges in stability. Here, we describe a method for the development of large recombinant BCG accession lots which stably express the lentiviral antigens, human immunodeficiency virus (HIV) gp120 and simian immunodeficiency virus (SIV) Gag, using selectable leucine auxotrophic complementation. Successful establishment of vaccine stability stems from stringent quality control criteria which not only screen for highly stable complemented BCG ΔleuCD transformants but also thoroughly characterize postproduction quality. These parameters include consistent production of correctly sized antigen, retention of sequence-pure plasmid DNA, freeze-thaw recovery, enumeration of CFU, and assessment of cellular aggregates. Importantly, these quality assurance procedures were indicative of overall vaccine stability, were predictive for successful antigen expression in subsequent passaging both in vitro and in vivo, and correlated with induction of immune responses in murine models. This study has yielded a quality-controlled BCG ΔleuCD vaccine expressing HIV gp120 that retained stable full-length expression after 10(24)-fold amplification in vitro and following 60 days of growth in mice. A second vaccine lot expressed full-length SIV Gag for >10(68)-fold amplification in vitro and induced potent antigen-specific T cell populations in vaccinated mice. Production of large, well-defined recombinant BCG ΔleuCD lots can allow confidence that vaccine materials for immunogenicity and protection studies are not negatively affected by instability or differences between freshly grown production batches., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Recombinant adenoviral vectors (rAds) are lead vaccine candidates for protection against a variety of pathogens, including Ebola, HIV, tuberculosis, and malaria, due to their ability to potently induce T cell immunity in humans. However, the ability to induce protective cellular immunity varies among rAds. Here, we assessed the mechanisms that control the potency of CD8 T cell responses in murine models following vaccination with human-, chimpanzee-, and simian-derived rAds encoding SIV-Gag antigen (Ag). After rAd vaccination, we quantified Ag expression and performed expression profiling of innate immune response genes in the draining lymph node. Human-derived rAd5 and chimpanzee-derived chAd3 were the most potent rAds and induced high and persistent Ag expression with low innate gene activation, while less potent rAds induced less Ag expression and robustly induced innate immunity genes that were primarily associated with IFN signaling. Abrogation of type I IFN or stimulator of IFN genes (STING) signaling increased Ag expression and accelerated CD8 T cell response kinetics but did not alter memory responses or protection. These findings reveal that the magnitude of rAd-induced memory CD8 T cell immune responses correlates with Ag expression but is independent of IFN and STING and provide criteria for optimizing protective CD8 T cell immunity with rAd vaccines.

Advancement of new vaccines based on live viral vectors requires sensitive assays to analyze in vivo replication, gene expression and genetic stability. In this study, attenuated canine distemper virus (CDV) was used as a vaccine delivery vector and duplex 2-step quantitative real-time RT-PCR (RT-qPCR) assays specific for genomic RNA (gRNA) or mRNA have been developed that concurrently quantify coding sequences for the CDV nucleocapsid protein (N) and a foreign vaccine antigen (SIV Gag). These amplicons, which had detection limits of about 10 copies per PCR reaction, were used to show that abdominal cavity lymphoid tissues were a primary site of CDV vector replication in infected ferrets, and importantly, CDV gRNA or mRNA was undetectable in brain tissue. In addition, the gRNA duplex assay was adapted for monitoring foreign gene insert genetic stability during in vivo replication by analyzing the ratio of CDV N and SIV gag genomic RNA copies over the course of vector infection. This measurement was found to be a sensitive probe for assessing the in vivo genetic stability of the foreign gene insert., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Xenotransplantation represents one of alternative candidates for allotransplantation due to the chronic shortage of suitable human tissues; however, many obstacles remain. Expression and release of endogenous retroviral antigens by porcine cells after transplantation may evoke adverse immune responses in human subjects. Here, we examined whether human herpesvirus 1 (HHV-1) could induce the production of porcine endogenous retrovirus (PERV) antigens in porcine peripheral blood mononuclear cells (PBMCs)., Methods: Porcine PBMCs were infected with HHV-1 and examined for the production of PERV Gag protein and HHV-1 using antigen-capture ELISA and quantitative real-time polymerase chain reaction (PCR), respectively., Results: HHV-1 infection resulted in a 1.7- to 33.2-fold induction of PERV Gag relative to mock infection controls, compared to a 2.9- to 12.9-fold induction following treatment with PMA. Expression of PERV Gag was detected in porcine PBMCs and PK-15 cells after HHV-1 infection by double immunofluorescence staining of PERV Gag and HHV-1 antigen. The viability of HHV-1-infected porcine PBMCs was significantly lower than that of mock-infected cells. The HHV-1 level in the culture supernatant increased 5.2-fold relative to controls 24-h post-infection, indicative of active replication within these cells; decreased levels of HHV-1 were detected 72-h post-infection., Conclusions: These results suggest that HHV-1 may be capable of infecting transplanted porcine cells, resulting in strong direct induction of PERV antigen., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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., (© 2014 The Authors.)

CD4+ T cell-mediated immunity has increasingly received attention due to its contribution in the control of HIV viral replication; therefore, it is of great significance to improve CD4+ T cell responses to enhance the efficacy of HIV vaccines. Recent studies have suggested that macroautophagy plays a crucial role in modulating adaptive immune responses toward CD4+ T cells or CD8+ T cells. In the present study, a new strategy based on a macroautophagy degradation mechanism is investigated to enhance CD4+ T cell responses against the HIV/SIV gag antigen. Our results showed that when fused to the autophagosome-associated LC3b protein, SIVgag protein can be functionally targeted to autophagosomes, processed by autophagy-mediated degradation in autolysosomes/lysosomes, presented to MHC II compartments and elicit effective potential CD4 T cell responses in vitro. Importantly, compared with the SIVgag protein alone, SIVgag-LC3b fusion antigen can induce a stronger antigen-specific CD4+ T cell response in mice, which is characterized by an enhanced magnitude and polyfunctionality. This study provides insight for the immunological modulation between viral and mammalian cells via autophagy, and it also presents an alternative strategy for the design of new antigens in the development of effective HIV vaccines.

Purpose: Human endogenous retroviruses (HERV) encode 8% of the human genome. While HERVs may play a role in autoimmune and neoplastic disease, no mechanistic association has yet been established. We studied the expression and immunogenicity of a HERV-K GAG protein encoded on chromosome 22q11.23 in relation to the clinical course of prostate cancer., Experimental Design: In vitro expression of GAG-HERV-K was analyzed in panels of normal and malignant tissues, microarrays, and cell lines, and effects of demethylation and androgen stimulation were evaluated. Patient sera were analyzed for seroreactivity to GAG-HERV-K and other self-antigens by ELISA and seromics (protein array profiling)., Results: GAG-HERV-K expression was most frequent in prostate tissues and regulated both by demethylation of the promoter region and by androgen stimulation. Serum screening revealed that antibodies to GAG-HERV-K are found in a subset of patients with prostate cancer (33 of 483, 6.8%) but rarely in male healthy donors (1 of 55, 1.8%). Autoantibodies to GAG-HERV-K occurred more frequently in patients with advanced prostate cancer (29 of 191 in stage III-IV, 21.0%) than in early prostate cancer (4 of 292 in stages I-II, 1.4%). Presence of GAG-HERV-K serum antibody was correlated with worse survival of patients with prostate cancer, with a trend for faster biochemical recurrence in patients with antibodies to GAG-HERV-K., Conclusions: Preferential expression of GAG-HERV-K ch22q11.23 in prostate cancer tissue and increased frequency of autoantibodies observed in patients with advanced prostate cancer make this protein one of the first bona fide retroviral cancer antigens in humans, with potential as a biomarker for progression and biochemical recurrence rate of prostate cancer. Clin Cancer Res; 19(22); 6112-25. ©2013 AACR.

Caffeine and aspirin have been suggested to be involved in neurologic diseases, such as schizophrenia, and previous data have revealed that abnormal expression of HERV-W elements may be an important factor in the etiopathogenesis of those diseases. In this article, we reported that caffeine and aspirin contributed to the expression of HERV-W env and gag in Human SH-SY5Y neuroblastoma cells. Semi-quantitative RT-PCR and quantitative Real-time PCR were used to detect the mRNA of HERV-W env and gag in cells exposed to caffeine or aspirin. Western blotting was used to detect the protein of HERV-W env. Luciferase activity assay was employed to detect the activity of HERV-W env promoter. It was found that both caffeine and aspirin could increase the expression of HERV-W env and gag in human SH-SY5Y neuroblastoma cells. Caffeine could activate the HERV-W env promoter, while aspirin could not. With previous studies we can conjecture that HERVs might play a bridging role between environmental factors, such as drugs and neurologic diseases.

We compared the effect of the very strong pSFJ1-10 and moderately strong p7.5 promoters on the immunogenicity and pathogenicity of the replication-competent vaccinia virus (VV) LC16m8Δ (m8Δ) vector harboring the SIV gag gene in a vaccination regimen consisting of a recombinant BCG-SIVGag (rBCG-SIVGag) prime followed by a recombinant vaccinia boost. m8Δ/pSFJ/SIVGag synthesized more Gag protein than m8Δ/p7.5/SIVGag but replicated less efficiently in vitro. In addition, m8Δ/pSFJ/SIVGag was less pathogenic and elicited Gag-specific IFN-γ(+), CD107a(+), CD8(+) cells more efficiently than m8Δ/p7.5/SIVGag. Vaccination by this regimen elicited long-lasting Gag-specific CD8(+) T cells, the majority of which showed a CCR7(-) phenotype at over 8 weeks post-boost. Tetramer staining analyses revealed maintenance of Gag specific tetramer(+), CD62L(-), CD8(+) T cells for long time in vaccinated mice. However, Gag expression increased the neurotoxicity of the vaccinia vector, indicating the necessity of safety testing for each recombinant VV. We propose that this recombinant BCG prime-m8Δ/pSFJ/HIVGag boost regimen would be a promising vaccination procedure for preventing HIV infection., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Programmed -1 ribosomal frameshifting is widely used in the expression of RNA virus replicases and represents a potential target for antiviral intervention. There is interest in determining the extent to which frameshifting efficiency can be modulated before virus replication is compromised, and we have addressed this question using the alpharetrovirus Rous sarcoma virus (RSV) as a model system. In RSV, frameshifting is essential in the production of the Gag-Pol polyprotein from the overlapping gag and pol coding sequences. The frameshift signal is composed of two elements, a heptanucleotide slippery sequence and, just downstream, a stimulatory RNA structure that has been proposed to be an RNA pseudoknot. Point mutations were introduced into the frameshift signal of an infectious RSV clone, and virus replication was monitored following transfection and subsequent infection of susceptible cells. The introduced mutations were designed to generate a range of frameshifting efficiencies, yet with minimal impact on encoded amino acids. Our results reveal that point mutations leading to a 3-fold decrease in frameshifting efficiency noticeably reduce virus replication and that further reduction is severely inhibitory. In contrast, a 3-fold stimulation of frameshifting is well tolerated. These observations suggest that small-molecule inhibitors of frameshifting are likely to have potential as agents for antiviral intervention. During the course of this work, we were able to confirm, for the first time in vivo, that the RSV stimulatory RNA is indeed an RNA pseudoknot but that the pseudoknot per se is not absolutely required for virus viability.

Efficient monitoring of HIV-1-specific T-cells is crucial for the development of HIV-1 vaccines and immunotherapies. Currently, mainly peptides and vaccinia vectors are used for detection of HIV-1-specific cytotoxic T-lymphocytes (CTL), however, as HIV-1 is a variable virus, it is unknown to what extent the T-cell response against the autologous virus is under- or overestimated by using antigens from heterologous viral strains. Therefore, we established a new method for immunomonitoring of CTL using electroporation of peripheral blood mononuclear cells (PBMC) with mRNA derived from autologous viral strains. From six HIV-1-infected patients virus derived mRNA was produced after PCR-based cloning of autologous gag (n=5) and/or nef genes (n=3) from plasma and electroporated into PBMC from patients and healthy donors. Electroporation of PBMC with mRNA resulted in efficient protein expression with good induction of γ-interferon (γ-IFN) release by specific T-cells comparable to peptide pools and better than recombinant vaccinia viruses. Three mRNA encoded autologous Gag proteins and one autologous mRNA encoded Nef protein were better recognized by autologous PBMC in comparison to heterologous mRNA encoded Gag or Nef proteins (SF2 or HXB2). However, in one case each, mRNA encoded autologous Gag or Nef, respectively, was recognized less efficiently due to the presence of CTL escape mutations. In summary, electroporation of PBMC with mRNA is a very efficient, easy and rapid method for immunomonitoring of HIV-1-specific T-cell responses against autologous viral strains. Our data demonstrate that patients' CTL responses against autologous viral strains may be under- or overestimated by using antigens from heterologous viral strains., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Retroviral Gag proteins are encoded in introns and, because of this localization, they are subject to the default pathways of pre-mRNA splicing. Retroviruses regulate splicing and translation through a variety of intertwined mechanisms, including 5'- post-transcriptional control elements, 3'- constitutive transport elements, and viral protein RNA interactions that couple unspliced and singly spliced mRNAs to transport machinery. Sequences within the gag gene termed inhibitory or instability sequences also appear to affect viral mRNA stability and translation, and the action of these sequences can be countered by silent mutation or the presence of RNA interaction proteins like HIV-1 Rev. Here, we explored the requirements for mouse mammary tumor virus (MMTV) Gag expression using a combination of in vivo and in vitro expression systems., Results: We show that MMTV gag alleles are inhibited for translation despite possessing a functional open reading frame (ORF). The block to expression was post-transcriptional and targeted the mRNA but was not a function of mRNA transport or stability. Using bicistronic reporters, we show that inhibition of gag expression imparted a block to both cap-dependent and cap-independent translation onto the mRNA. Direct introduction of in vitro synthesized gag mRNA resulted in translation, implying a nuclear role in inhibition of expression. The inhibition of expression was overcome by intact proviral expression or by flanking gag with splice sites combined with a functional Rem-Rem response element (RmRE) interaction., Conclusions: Expression of MMTV Gag requires nuclear interactions involving the viral Rem protein, its cognate binding target the RmRE, and surprisingly, both a splice donor and acceptor sequence to achieve appropriate signals for translation of the mRNA in the cytoplasm.

HIV-1 candidate vaccines expressing an artificial polyprotein comprising Gag, Pol and Nef (GPN) and a secreted envelope protein (Env) were shown in recent Phase I/II clinical trials to induce high levels of polyfunctional T cell responses; however, Env-specific responses clearly exceeded those against Gag. Here, we assess the impact of the GPN immunogen design and variations in the formulation and vaccination regimen of a combined GPN/Env DNA vaccine on the T cell responses against the various HIV proteins. Subtle modifications were introduced into the GPN gene to increase Gag expression, modify the expression ratio of Gag to PolNef and support budding of virus-like particles. I.m. administration of the various DNA constructs into BALB/c mice resulted in an up to 10-fold increase in Gag- and Pol-specific IFNγ(+) CD8(+) T cells compared to GPN. Co-administering Env with Gag or GPN derivatives largely abrogated Gag-specific responses. Alterations in the molar ratio of the DNA vaccines and spatially or temporally separated administration induced more balanced T cell responses. Whereas forced co-expression of Gag and Env from one plasmid induced predominantly Env-specific T cells responses, deletion of the only H-2(d) T cell epitope in Env allowed increased levels of Gag-specific T cells, suggesting competition at an epitope level. Our data demonstrate that the biochemical properties of an artificial polyprotein clearly influence the levels of antigen-specific T cells, and variations in formulation and schedule can overcome competition for the induction of these responses. These results are guiding the design of ongoing pre-clinical and clinical trials.

Background: High mutation rates of human immunodeficiency virus (HIV) allows escape from T cell recognition preventing development of effective T cell vaccines. Vaccines that induce diverse T cell immune responses would help overcome this problem. Using SIV gag as a model vaccine, we investigated two approaches to increase the breadth of the CD8 T cell response. Namely, fusion of vaccine genes to ubiquitin to target the proteasome and increase levels of MHC class I peptide complexes and gene fragmentation to overcome competition between epitopes for presentation and recognition., Methodology/principal Findings: three vaccines were compared: full-length unmodified SIV-mac239 gag, full-length gag fused at the N-terminus to ubiquitin and 7 gag fragments of equal size spanning the whole of gag with ubiquitin-fused to the N-terminus of each fragment. Genes were cloned into a replication defective adenovirus vector and immunogenicity assessed in an in vitro human priming system. The breadth of the CD8 T cell response, defined by the number of distinct epitopes, was assessed by IFN-γ-ELISPOT and memory phenotype and cytokine production evaluated by flow cytometry. We observed an increase of two- to six-fold in the number of epitopes recognised in the ubiquitin-fused fragments compared to the ubiquitin-fused full-length gag. In contrast, although proteasomal targeting was achieved, there was a marked reduction in the number of epitopes recognised in the ubiquitin-fused full-length gag compared to the full-length unmodified gene, but there were no differences in the number of epitope responses induced by non-ubiquitinated full-length gag and the ubiquitin-fused mini genes. Fragmentation and ubiquitination did not affect T cell memory differentiation and polyfunctionality, though most responses were directed against the Ad5 vector., Conclusion/significance: Fragmentation but not fusion with ubiquitin increases the breadth of the CD8 T vaccine response against SIV-mac239 gag. Thus gene fragmentation of HIV vaccines may maximise responses.

HIV-2 infection is associated with a slower rate of disease progression with limited impact on the survival of the majority of infected adults, and much lower plasma viral load than HIV-1. In spite of the major differences in viremia, the quantitative assessment of HIV-2 proviral load documented levels similar to those observed in HIV-1 infected individuals, suggesting an equivalent number of circulating infected cells in both infections. It remains unclear whether this apparent paradox results from a contribution of latent/quiescent viruses or from transcriptional and/or post-transcriptional control of HIV-2 replication. In order to investigate these possibilities, a one-step and two-step reverse transcription quantitative real-time PCR based methods (RT-qPCR) for gag and tat mRNA HIV-2 transcripts were developed. These methods were validated and compared to assess the expression of HIV-2 gag and tat transcripts in parallel with proviral DNA and viral production. The results suggest that the two-step approach may allow a better detection of low level gag and tat mRNA HIV-2 transcripts., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Human T-lymphotropic virus I (HTLV-1) is associated with the T-cell malignancy known as adult T-cell leukemia/ lymphoma (ATLL) and with a disorder called HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Currently, the treatment of these diseases is based on symptom relief. RNA interference (RNAi) technology has been described as an efficient mechanism for development of new therapeutic methods. Thus, the aim of this study was to evaluate the inhibition of HTLV-1 structural proteins using short hairpin RNAs (shRNAs) expressed by non-viral vectors., Materials and Methods: Reporter plasmids that express enhanced green fluorescent protein-Gag (EGFP-Gag) and EGFP-Env fusion proteins and vectors that express shRNAs corresponding to the HTLV-1 gag and env genes were constructed. shRNA vectors and reporter plasmids were simultaneously transfected into HEK 293 cells., Results: Fluorescence microscopy, flow cytometry and real-time PCR showed that shRNAs were effective in inhibiting the fusion proteins., Conclusion: These shRNAs are effective against the expression of structural genes and may provide an approach to the development of new therapeutic agents.

Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus linked to prostate carcinoma and chronic fatigue syndrome. Here we report that NF-κB activation can markedly increase XMRV production. The inflammatory cytokine tumor necrosis factor alpha (TNF-α), which activates NF-κB, significantly augmented viral Gag protein production in XMRV-infected cells. Reporter assays showed that TNF-α and Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), an intrinsic NF-κB activator, increased long terminal repeat (LTR)-dependent XMRV transcription. We identified two NF-κB binding sites (designated κB-1 and κB-2) in the LTR U3 region of XMRV and demonstrated that both sites bind to the NF-κB component p65/RelA. Mutation of the κB-1 site, but not the κB-2 site, impaired responsiveness to TNF-α and LMP1 in reporter assays. A mutant XMRV with a mutation at the κB-1 site replicated significantly less efficiently than the wild-type XMRV in the prostate carcinoma LNCaP, DU145, and PC-3 cell lines, HEK293 cells, the EBV-immortalized cell line IB4, and the Burkitt's lymphoma cell line BJAB. These results demonstrate that TNF-α and EBV LMP1 enhance XMRV replication in prostate carcinoma and B-lineage cells through the κB-1 site in the XMRV LTR, suggesting that inflammation, EBV infection, and other conditions leading to NF-κB activation may promote XMRV spread in humans.

RNA-based compounds are promising agents to inactivate viruses. New specific hepatitis delta virus (HDV)-derived ribozymes are natural molecules that can be engineered to specifically target a viral RNA. We have designed specific on-off adaptor (SOFA)-HDV ribozymes targeting the tat and rev sequences of the human immunodeficiency virus type 1 (HIV-1) RNA. We show that the SOFA-HDV ribozymes cleave their RNA target in vitro. They inhibit the Tat-mediated transactivation of HIV-1 from 62% to 86% in different assays. In vivo, the amount of HIV RNA was decreased by 60 and 86% with two distinct ribozymes, which indicates that the inhibition of HIV production is directly correlated to the decline in spliced and unspliced viral RNAs. These SOFAHDV- ribozymes inhibited the expression and the viral production of four HIV-1 strains, indicating an extended potential to act on multiple HIV variants. In HEK 293T and HeLa cells transfected with pNL4-3 and the SOFA-HDV-ribozymes, the reduced RNA levels consequently decreased the Gag protein expression in the cell and virus production in the supernatant. When transfected before HIV-1 infection, the ribozymes prevented the incoming virus from being expressed. The ribozymes inhibited HIV production up to 90% when transfected in combination with the HIV protease inhibitor Atazanavir. Our results strongly suggest that SOFA-HDV ribozymes have a great potential to target HIV-1 and to be used as therapeutic agents in combination therapy.

Human endogenous retroviruses (HERVs) are remnants of ancient retroviral infections within the human genome. These molecular fossils draw parallels with present-day exogenous retroviruses and have been linked previously with immunopathology within rheumatoid arthritis (RA). Mechanisms of pathogenesis for HERV-K in RA such as molecular mimicry were investigated. To clarify a role for HERVs in RA, potential autoantigens implicated in autoimmunity were scanned for sequence identity with retroviral epitopes. Short retroviral peptides modelling shared epitopes were synthesized, to survey anti-serum of RA patients and disease controls. A novel real-time polymerase chain reaction (PCR) assay was also developed to quantify accurately levels of HERV-K (HML-2) gag expression, relative to normalized housekeeping gene expression. Both serological and molecular assays showed significant increases in HERV-K (HML-2) gag activity in RA patients, compared to disease controls. The real-time PCR assay identified significant up-regulation in HERV-K mRNA levels in RA patients compared to inflammatory and healthy controls. Exogenous viral protein expression and proinflammatory cytokines were also shown to exert modulatory effects over HERV-K (HML-2) transcription. From our data, it can be concluded that RA patients exhibited significantly elevated levels of HERV-K (HML-2) gag activity compared to controls. Additional factors influencing HERV activity within the synovium were also identified. The significant variation in RA patients, both serologically and transcriptionally, may be an indication that RA is an umbrella term for a number of separate disease entities, of which particular HERV polymorphisms may play a role in development.

Retroviral particles assemble a few thousand units of the Gag polyproteins. Proteolytic cleavage mediated by the retroviral protease forms the bioactive retroviral protein subunits before cell entry. We hypothesized that this process could be exploited for targeted, transient, and dose-controlled transduction of nonretroviral proteins into cultured cells. We demonstrate that gammaretroviral particles tolerate the incorporation of foreign protein at several positions of their Gag or Gag-Pol precursors. Receptor-mediated and thus potentially cell-specific uptake of engineered particles occurred within minutes after cell contact. Dose and kinetics of nonretroviral protein delivery were dependent upon the location within the polyprotein precursor. Proteins containing nuclear localization signals were incorporated into retroviral particles, and the proteins of interest were released from the precursor by the retroviral protease, recognizing engineered target sites. In contrast to integration-defective lentiviral vectors, protein transduction by retroviral polyprotein precursors was completely transient, as protein transducing retrovirus-like particles could be produced that did not transduce genes into target cells. Alternatively, bifunctional protein-delivering particle preparations were generated that maintained their ability to serve as vectors for retroviral transgenes. We show the potential of this approach for targeted genome engineering of induced pluripotent stem cells by delivering the site-specific DNA recombinase, Flp. Protein transduction of Flp after proteolytic release from the matrix position of Gag allowed excision of a lentivirally transduced cassette that concomitantly expresses the canonical reprogramming transcription factors (Oct4, Klf4, Sox2, c-Myc) and a fluorescent marker gene, thus generating induced pluripotent stem cells that are free of lentivirally transduced reprogramming genes.

In this study, mesenchymal stem cells (MSCs) embedded in biodegradable and water-swollen, elastic block copolymer scaffolds were assessed for MSC chondrogenesis. To determine the optimal conditions for chondrogenesis of the embedded rMSCs, transforming growth factor-beta 3 (TGF-beta 3) was physically conjugated with chondroitin sulfate (CS) and mixed into scaffolds, which were subsequently evaluated for the differentiation of transplanted rMSCs. In determination of CS-bound growth factors for chondrogenesis, scaffold mixed with rMSCs and TGF-beta 3 was then tested by growth factor release profiles, confocal laser microscopy, RT-PCR analysis, real time-QPCR, and histology. The results of several different analyses of the transplanted rMSCs embedded in the scaffolds showed that rMSCs coupled with a CS-bound TGF-beta 3 encapsulated scaffold evidenced superior cartilage tissue formation as measured by an assay of specific gene and protein expression. Moreover, the scaffold exhibited more rapid and more distinct morphology of differentiated rMSCs than was observed with other scaffolds, as determined by histology and immunochemical histology analysis. These results indicate that the elastic block copolymer scaffolds combined with a CS-bound TGF-beta 3 should prove very suitable matrix for cell-based cartilage tissue engineering., ((c) 2009 Wiley Periodicals, Inc.)

The CD8+ T-cell is a key mediator of antiviral immunity, potentially contributing to control of pathogenic lentiviral infection through both innate and adaptive mechanisms. We studied viral dynamics during antiretroviral treatment of simian immunodeficiency virus (SIV) infected rhesus macaques following CD8+ T-cell depletion to test the importance of adaptive cytotoxic effects in clearance of cells productively infected with SIV. As previously described, plasma viral load (VL) increased following CD8+ T-cell depletion and was proportional to the magnitude of CD8+ T-cell depletion in the GALT, confirming a direct relationship between CD8+ T-cell loss and viral replication. Surprisingly, first phase plasma virus decay following administration of antiretroviral drugs was not slower in CD8+ T-cell depleted animals compared with controls indicating that the short lifespan of the average productively infected cell is not a reflection of cytotoxic T-lymphocyte (CTL) killing. Our findings support a dominant role for non-cytotoxic effects of CD8+ T-cells on control of pathogenic lentiviral infection and suggest that cytotoxic effects, if present, are limited to early, pre-productive stages of the viral life cycle. These observations have important implications for future strategies to augment immune control of HIV.

Retroviruses express Gag and Pol proteins by translation of unspliced genome-length viral RNA. For some retroviruses, transport of unspliced viral RNA to the cytoplasm is mediated by small regulatory proteins such as human immunodeficiency virus Rev, while other retroviruses contain constitutive transport elements in their RNAs that allow transport without splicing. In this study, we found that the betaretrovirus Jaagsiekte sheep retrovirus (JSRV) encodes within the env gene a trans-acting factor (Rej) necessary for the synthesis of Gag protein from unspliced viral RNA. Deletion of env sequences from a JSRV proviral expression plasmid (pTN3) abolished its ability to produce Gag polyprotein in transfected 293T cells, and Gag synthesis could be restored by cotransfection of an env expression plasmid (DeltaGP). Deletion analysis localized the complementing activity (Rej) to the putative Env signal peptide, and a signal peptide expression construct showed Rej activity. Two other betaretroviruses, mouse mammary tumor virus (MMTV) and human endogenous retrovirus type K, encode analogous factors (Rem and Rec, respectively) that are encoded from doubly spliced env mRNAs. Reverse transcriptase-PCR cloning and sequencing identified alternate internal splicing events in the 5' end of JSRV env that could signify analogous doubly spliced Rej mRNAs, and cDNA clones expressing two of them also showed Rej activity. The predicted Rej proteins contain motifs similar to those found in MMTV Rem and other analogous retroviral regulatory proteins. Interestingly, in most cell lines, JSRV expression plasmids with Rej deleted showed normal transport of unspliced JSRV RNA to the cytoplasm; however, in 293T cells Rej modestly enhanced export of unspliced viral RNA (2.8-fold). Metabolic labeling experiments with [(35)S]methionine indicated that JSRV Rej is required for the synthesis of viral Gag polyprotein. Thus, in most cell lines, the predominant function of Rej is to facilitate translation of unspliced viral mRNA.

Jaagsiekte sheep retrovirus (JSRV) is a simple betaretrovirus causing a contagious lung cancer of sheep. JSRV encodes unspliced and spliced viral RNAs, among which unspliced RNA encodes Gag and Pol proteins and a singly spliced mRNA encodes Env protein. In another study we found that JSRV encodes a regulatory protein, Rej, that is responsible for synthesis of Gag polyprotein from unspliced viral RNA. Rej is encoded in the 5' end of env, and it enhances nuclear export or accumulation of cytoplasmic unspliced viral RNA in 293T cells but not in most other cell lines (A. Hofacre, T. Nitta, and H. Fan, J. Virol. 83:12483-12498, 2009). In this study, we found that mutations in the 3' end of env in the context of a cytomegalovirus-driven full-length JSRV expression construct abolished Gag protein synthesis and released viruses in 293T cells. These mutants also showed deficits in accumulation of unspliced viral RNA in the cytoplasm. These mutants defined a Rej-responsive element (RejRE). Inhibition of CRM1 but not Tap function prevented nuclear export/accumulation of cytoplasmic unspliced RNA in 293T cells, similarly to other complex retroviruses that express analogous regulator proteins (e.g., human immunodeficiency virus Rev). Structural modeling of the RejRE with Zuker M-fold indicated a region with a predicted stable secondary structure. Mutational analysis in this region indicated the importance of both secondary structures and primary nucleotide sequences in a central stem-bulge-stem structure. In contrast to 293T cells, mutations in the RejRE did not affect the levels of cytoplasmic unspliced RNA in 293 cells, although the unspliced RNA showed partial degradation, perhaps due to lack of translation. RejRE-containing RNA relocalized Rej protein from the nucleus to the cytoplasm in 293 and rat 208F cells, suggesting binding of Rej to the RejRE.

The prototype foamy virus (PFV) is a nonpathogenic retrovirus that shows promise as a vector for gene transfer. The PFV (pre)genomic RNA starts with a long complex leader that can be folded into an elongated hairpin, suggesting an alternative strategy to cap-dependent linear scanning for translation initiation of the downstream GAG open reading frame (ORF). We found that the PFV leader carries several short ORFs (sORFs), with the three 5'-proximal sORFs located upstream of a structural element. Scanning-inhibitory hairpin insertion analysis suggested a ribosomal shunt mechanism, whereby ribosomes start scanning at the leader 5'-end and initiate at the downstream ORF via bypass of the central leader regions, which are inhibitory for scanning. We show that the efficiency of shunting depends strongly on the stability of the structural element located downstream of either sORFs A/A' or sORF B, and on the translation event at the corresponding 5'-proximal sORF. The PFV shunting strategy mirrors that of Cauliflower mosaic virus in plants; however, in mammals shunting can operate in the presence of a less stable structural element, although it is greatly improved by increasing the number of base pairings. At least one shunt configuration was found in primate FV (pre)genomic RNAs.

The RNA silencing pathway is an intracellular innate response to virus infections and retro-transposons. Many plant viruses counter this host restriction by RNA silencing suppressor (RSS) activity of a double-stranded RNA-binding protein, e.g., tomato bushy stunt virus P19. Here, we demonstrate P19 and HIV-1 Tat function across the plant and animal kingdoms and suppress a common step in RNA silencing that is downstream of small RNA maturation. Our experiments reveal that RNA silencing in HIV-1 infected human cells severely attenuates the translational output of the unspliced HIV-1 gag mRNA, and possibly all HIV-1 transcripts. The attenuation in gag mRNA translation is exacerbated by K51A substitution in the Tat double-stranded RNA-binding domain. Tat, plant virus RSS, or Dicer downregulation rescues robust gag translation and bolsters HIV-1 virion production. The reversal of HIV-1 translation repression by plant RSS supports the recent finding in Arabidopsis that plant miRNAs operate by translational inhibition. Our results identify common features between RNA silencing suppression of plant and animal viruses. We suggest that RNA silencing-mediated translation repression plays a strategic role in determining the viral set-point in a newly HIV-1-infected patient.

A trial to evaluate the safety and immunogenicity of recombinant modified vaccinia Ankara (MVA) and fowlpox (FP) vectors expressing multiple HIV-1 proteins was conducted in twenty HIV-1 infected youth with suppressed viral replication on HAART. The MVA and FP-based multigene HIV-1 vaccines were safe and well tolerated. Increased frequencies of HIV-1 specific CD4+ proliferative responses and cytokine secreting cells were detected following immunization. Increased frequencies and breadth of HIV-1 specific CD8 T-cell responses were also detected. Plasma HIV-1-specific antibody levels and neutralizing activity were unchanged following vaccination. Poxvirus-based vaccines may merit further study in therapeutic vaccine protocols.

A vaccine against HIV-1 infection absolutely needs the ability to effectively elicit virus-specific immunity over a long term; nevertheless, there have been few studies indicating that the immunoinductivity of such a candidate vaccine has been researched for several years running. In a previous report, we demonstrated that recombinant BCG (rBCG) expressing the full-length gag gene of simian immunodeficiency virus (SIV) (rBCG-SIVgag) induced Gag-specific delayed-type hypersensitivity, T cell proliferation, gamma interferon (IFN gamma), and serum IgG responses in guinea pigs immunized intradermally (i.d.) with 0.1 mg for the 1-year period of study. Especially, the production of long-lasting Gag-specific serum IgG in the vaccinated animals perhaps reflects the persistent antigenic stimulation by rBCG-SIVgag. How long, we questioned, will such immune responses to Gag engendered by the rBCG-SIVgag vaccination persist without booster immunizations? To learn this, we examined Gag-specific IgG production in sera and Gag-specific IFN gamma mRNA expression in peripheral blood mononuclear cells (PBMC) in guinea pigs vaccinated with rBCG-SIVgag i.d. (0.1 mg) or orally (80 mg x 2) during a 3-year period. As a result, Gag-specific serum IgG was highly generated for 3 years at similar levels between the i.d. and the orally immunized guinea pigs (IgG2>IgG1). The enhancement of IFN gamma mRNA expression by in vitro restimulation with Gag antigen was also detected in PBMC from the two immunization groups throughout the 3-year observation period. In guinea pigs immunized i.d. with rBCG-SIVgag, a high level of Gag-specific IFN gamma response was observed at 1 year after vaccination, whereas the response has waned gradually. The current study indicates that i.d. and oral inoculations of rBCG-SIVgag elicit stable, strong, Gag-specific serum IgG production while exhibiting the different kinetics of Gag-specific IFN gamma responses between i.d. and oral vaccination routes. This suggests that the rBCG vector system expressing an appropriate size of foreign antigen gene should be suited for the induction of the antigen-specific humoral immunity concomitant with an IFN gamma response.

To study the immune responses elicited by multiple vectors and develop vaccines strategies against prevalent HIV-1 strains in China, we have examined the potency of vaccine regimens of plasmid DNA, adenovirus, and Sendai virus vectors expressing HIV-1 gag consensus sequence of HIV-1 isolates from China for inducing specific immune responses. In BALB/c mice, combination of these vectors induced higher Gag-specific cellular immune response than any regimen using single vector alone. The prime-boost-boost regimen consisting of the triple heterologous vectors induced Gag-specific T-cell responses the most efficiently. In rhesus macaques, the prime-boost-boost regimen induced potent Gag-specific cellular immune responses as well as long lasting humoral immune response, and each booster resulted in rapid and efficient expansion of Gag-specific T cells. These results indicate that this prime-boost-boost regimen using triple heterologous vectors is a promising AIDS vaccine candidate for efficiently inducing HIV-1-specific cellular and humoral immune responses. Its further studies as a promising scheme for therapeutic and/or prophylactic HIV-1 vaccines should be grounded.

Bovine leukemia virus (BLV) is a member of the family Retroviridae, genus Deltaretrovirus that has three important gene including gag, pol and env. This virus causes B-cell lymphocytosis and lymphosarcoma in cows. In the first step PCR product of gag gene of BLV isolated in different regions of Iran and BLV-FLK strain were cloned in to a pTZ57R/T vector, then insert were digested by BglII and XhoI restriction enzymes and cloned in to pET-28(a) as an expression vector. For the expression of p24 protein, the pET-28(a) recombinant vector was transformed in BL21(DE3) strain of E. coli competent cell and after induction of the protein having been expressed by IPTG, the presence of gag expressed protein was shown in immunoblotting and SDS-PAGE system. With respect to the remarkable frequency of infection to BLV in Iran and the necessity of controlling it through vaccination with recombinant vaccines of gp51, manufacturing and applying the recombinant p24 protein are vital goals in recognition and distinction between infection and responses caused by vaccine.

The brains of individuals with lentiviral-associated encephalitis contain an abundance of infected and activated macrophages. It has been hypothesized that encephalitis develops when increased numbers of infected monocytes traffic into the central nervous system (CNS) during the end stages of immunosuppression. The relationships between the infection of brain and systemic macrophages and circulating monocytes and the development of lentiviral encephalitis are unknown. We longitudinally examined the extent of monocyte/macrophage infection in blood and lymph nodes of pigtailed macaques that did or did not develop simian immunodeficiency virus encephalitis (SIVE). Compared to levels in macaques that did not develop SIVE, more ex vivo virus production was detected from monocyte-derived macrophages and nonadherent peripheral blood mononuclear cells (PBMCs) from macaques that did develop SIVE. Prior to death, there was an increase in the number of circulating PBMCs following a rise in cerebrospinal fluid viral load in macaques that did develop SIVE but not in nonencephalitic macaques. At necropsy, macaques with SIVE had more infected macrophages in peripheral organs, with the exception of lymph nodes. T cells and NK cells with cytotoxic potential were more abundant in brains with encephalitis; however, T-cell and NK-cell infiltration in SIVE and human immunodeficiency virus encephalitis was more modest than that observed in classical acute herpes simplex virus encephalitis. These findings support the hypothesis that inherent differences in host systemic and CNS monocyte/macrophage viral production are associated with the development of encephalitis.

Mouse cells are non-permissive to human immunodeficiency virus type 1 (HIV) in that there is a pronounced post-integration block to viral replication. We have recently demonstrated that mouse-human somatic cell hybrids that contain human chromosome 2 increase both HIV Capsid (CA) production and infectious virus release. Here we report on the isolation of three mouse-human microcell hybrids (MCHs) that behave similarly, starting from a pool of 500 MCH clones. Release of virus was specific to HIV and cell revertants that no longer contained any human chromosome fragments did not release CA or infectious virus. Two of the three cell clones were identical as judged by PCR STS content and fluorescence in situ hybridization (FISH) and contained a single 2-12 human chromosome chimera. The third cell clone only contained human chromosome 12, as determined by PCR, FISH, and microarray analyses. There were no consistent differences in Gag protein and spliced/unspliced viral RNA levels between mouse cell lines. CMV promoter-driven, codon-optimized gag-pol had no effect on infectious HIV release from these mouse cells, despite allowing Gag targeting and increasing CA production. These permissive mouse-human MCHs and their corresponding non-permissive revertants may prove useful for mechanistic studies and also for identifying the responsible gene(s) or factor(s) involved in the production of HIV.

Cross-presentation of normal self and candidate tumor Ags by bone marrow (BM)-derived APCs that have not been activated has been demonstrated as a major mechanism contributing to acquisition of tolerance by mature T cells that first encounter an Ag in the periphery (cross-tolerance). Following adoptive transfer of naive TCR-transgenic CD8(+) T cells into a host expressing a transgenic Ag that is a potentially targetable tumor Ag in normal hepatocytes as a self-Ag, we found that the majority of Ag-specific CD8(+) T cells were deleted, with the remaining cells rendered anergic. Studies in BM chimeric mice and with purified cell populations demonstrated that these events were not dependent on cross-presentation by BM-derived APCs including Kupffer cells or liver sinusoidal endothelial cells, and apparently can occur entirely as a consequence of direct recognition of Ag endogenously processed and presented by hepatocytes. Direct recognition of Ag-expressing hepatocytes in vivo induced a proliferative response and up-regulation of activation markers in responding CD8(+) T cells, but proliferating cells did not accumulate, with most cells rapidly eliminated, and the persisting T cells lost the capacity to proliferate in response to repeated Ag stimulation. The results suggest that parenchymal tissues may retain the capacity to directly regulate in vivo responses to self-Ags processed and presented in the context of class I MHC molecules.

To increase the safety and efficacy of human immunodeficiency virus vaccines, several groups have conducted studies using the macaque model with single-cycle replicating simian immunodeficiency viruses (SIVs). However, these constructs had poor or diminished efficacy compared to live attenuated vaccines. We previously showed that immunization of macaques with live attenuated SIV with a deletion in the nef gene and expressing gamma interferon (IFN-gamma) results in significantly enhanced safety and efficacy. To further enhance safety, we constructed and characterized single-cycle SIVs, pseudotyped with the glycoprotein of vesicular stomatitis virus, expressing different levels of macaque IFN-gamma. Expression of IFN-gamma did not alter the infectivity or antigenicity of pseudotyped SIV. The transduction of dendritic cells (DCs) by IFN-gamma-expressing particles resulted in the up-regulation of costimulatory and major histocompatibility complex molecules. Furthermore, T cells primed with DCs transduced by SIV particles expressing high levels of IFN-gamma and then stimulated with SIV induced significantly higher numbers of spot-forming cells in an enzyme-linked immunospot assay than did T cells primed with DCs transduced with SIV particles lacking the cytokine. In conclusion, we demonstrated that the transduction of DCs in vitro with pseudotyped single-cycle SIVs expressing IFN-gamma increased DC activation and augmented T-cell priming activity.

Oral immunization is a safe and easily applicable route to induce mucosal immunity to HIV infection. We examined the ability of oral attenuated Salmonella typhimurium (ST) vaccine expressing Gag for the efficiency of generating Gag-specific mucosal IgA and CD8+ T cell responses in intestinal lymphoid tissues. By optimizing the codon of HIV-1 gag to the preferred codon bias of Salmonella, the expression of Gag in Salmonella was dramatically improved. The oral ST-Gag vaccine by itself was not so powerful and induces little Gag-specific CD8+ T cell responses in the intestine. Nevertheless, we found that it potentiates otherwise weak intestinal CD8+ T cell responses in nasally primed mice with Gag p24 and cholera toxin adjuvant. Thus, the oral delivery of Salmonella expressing Gag would be utilized in combination with other parenteral vaccine to direct and strengthen intestinal HIV-specific CTL responses.

The 5' untranslated region (UTR) of retroviruses contain structured replication motifs that impose barriers to efficient ribosome scanning. Two RNA structural motifs that facilitate efficient translation initiation despite a complex 5' UTR are internal ribosome entry site (IRES) and 5' proximal post-transcriptional control element (PCE). Here, stringent RNA and protein analyses determined the 5' UTR of spleen necrosis virus (SNV), reticuloendotheliosis virus A (REV-A) and human T-cell leukemia virus type 1 (HTLV-1) exhibit PCE activity, but not IRES activity. Assessment of SNV translation initiation in the natural context of the provirus determined that SNV is reliant on a cap-dependent initiation mechanism. Experiments with siRNAs identified that REV-A and HTLV-1 PCE modulate post-transcriptional gene expression through interaction with host RNA helicase A (RHA). Analysis of hybrid SNV/HTLV-1 proviruses determined SNV PCE facilitates Rex/Rex responsive element-independent Gag production and interaction with RHA is necessary. Ribosomal profile analyses determined that RHA is necessary for polysome association of HTLV-1 gag and provide direct evidence that RHA is necessary for efficient HTLV-1 replication. We conclude that PCE/RHA is an important translation regulatory axis of multiple lymphotropic retroviruses. We speculate divergent retroviruses have evolved a convergent RNA-protein interaction to modulate translation of their highly structured mRNA.

Live-vector human immunodeficiency virus (HIV) vaccines are an integral part of a number of HIV vaccine regimens currently under evaluation that have yielded promising results in pre-clinical testing. In this report, a non-pathogenic protozoan parasitic vector, Leishmania tarentolae, which shares common target cells with HIV-1, was used to express full-length HIV-1 Gag protein. Immunization of BALB/c mice with recombinant L. tarentolae led to the expansion of HIV-1 Gag-specific T cells and stimulated CD8(+) T cells to produce gamma interferon in response to specific viral Gag epitopes. A booster immunization with recombinant L. tarentolae elicited effector memory HIV-1 Gag-specific CD4(+) T lymphocytes and increased antibody titres against HIV-1 Gag. Most importantly, immunization of human tonsillar tissue cultured ex vivo with Gag-expressing L. tarentolae vaccine vector elicited a 75% decrease in virus replication following exposure of the immunized tonsils to HIV-1 infection. These results demonstrated that recombinant L. tarentolae is capable of eliciting effective immune responses in mice and human systems, respectively, and suggest that this novel non-pathogenic recombinant vaccine vector shows excellent promise as a vaccination strategy against HIV-1.

The full-length viral RNA of human immunodeficiency virus type 1 (HIV-1) functions both as the mRNA for the viral structural proteins Gag and Gag/Pol and as the genomic RNA packaged within viral particles. The packaging signal which Gag recognizes to initiate genome encapsidation is in the 5' untranslated region (UTR) of the HIV-1 RNA, which is also the location of translation initiation complex formation. Hence, it is likely that there is competition between the translation and packaging processes. We studied the ability of Gag to regulate translation of its own mRNA. Gag had a bimodal effect on translation from the HIV-1 5' UTR, stimulating translation at low concentrations and inhibiting translation at high concentrations in vitro and in vivo. The inhibition was dependent upon the ability of Gag to bind the packaging signal through its nucleocapsid domain. The stimulatory activity was shown to depend on the matrix domain of Gag. These results suggest that Gag controls the equilibrium between translation and packaging, ensuring production of enough molecules of Gag to make viral particles before encapsidating its genome.

Few details are known about how the human immunodeficiency virus type 1 (HIV-1) genomic RNA is trafficked in the cytoplasm. Part of this process is controlled by the activity of heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2). The role of hnRNP A2 during the expression of a bona fide provirus in HeLa cells is investigated in this study. Using immunofluorescence and fluorescence in situ hybridization techniques, we show that knockdown of hnRNP A2 expression in HIV-1-expressing cells results in the rapid accumulation of HIV-1 genomic RNA in a distinct, cytoplasmic space that corresponds to the microtubule-organizing center (MTOC). The RNA exits in the nucleus and accumulates at the MTOC region as a result of hnRNP A2 knockdown even during the expression of a provirus harboring mutations in the hnRNP A2-response element (A2RE), the expression of which results in nuclear retention of genomic RNA. We also demonstrate that hnRNP A2 expression is required for downstream trafficking of genomic RNA from the MTOC in the cytoplasm. Genomic RNA localization at the MTOC that was both the result of hnRNP A2 knockdown and the overexpression of Rab7-interacting lysosomal protein had little effect on pr55Gag synthesis but negatively influenced virus production and infectivity. These data indicate that altered HIV-1 genomic RNA localization modulates viral assembly and that the MTOC serves as a central site to which HIV-1 genomic RNA converges following its exit from the nucleus, with the host protein, hnRNP A2, playing a central role in taking it to and from this site in the cell.

Natural transmission of human immunodeficiency virus (HIV) occurs at mucosal surfaces. During acute infection, intestinal and other mucosae are preferential sites of virus replication and rapidly become depleted of CD4(+) T cells. Therefore, mucosal immunity may be critical to control both initial infection and the massive early spread of virus. An attenuated D-alanine-requiring strain of the oral intracellular microorganism Listeria monocytogenes expressing HIV type 1 gag was shown to induce protective cell-mediated immunity in mice against viruses that express HIV gag when immunization occurs in the presence of a transient supply of D-alanine. In this study, we examined the efficacy of new attenuated strains that are able to synthesize d-alanine from a heterologous dal gene tightly regulated by an actA-promoted resolvase recombination system. In the absence of d-alanine, Gag-specific cytotoxic T lymphocytes (CTLs) were induced systemically after intravenous immunization, and one strain, Lmdd-gag/pARS, induced strong dose-dependent Gag-specific CTLs after oral immunization. A significant level of Gag-specific CD8(+) T cells was induced in the mucosal-associated lymphoid tissues (MALTs). Upon intravaginal challenge of these orally immunized mice with recombinant vaccinia virus (rVV) expressing HIV gag, gamma interferon- and tumor necrosis factor alpha-secreting Gag-specific CD8(+) T cells were dramatically increased in the spleen and MALTs. Oral immunization with Lmdd-gag/pARS led to complete protection against vaginal challenge by a homologous clade B gag-expressing rVV. In addition, strong cross-clade protection was seen against clades A and C and partial protection against clade G gag-expressing rVV. These results suggest that Lmdd-gag/pARS may be considered as a novel vaccine candidate for use against HIV/AIDS.

For some time there has been evidence suggesting an interaction between human cytomegalovirus (HCMV) and Human immunodeficiency virus (HIV) in the pathogenesis of AIDS. Here, the interaction of HCMV and HIV-1 was examined in monocyte/macrophage cells, two cell types known to be targets for both viruses in vivo. Infection experiments demonstrated that prior infection with HCMV impeded subsequent superinfection with HIV-1. In contrast, uninfected bystander cells within the population were still permissive for HIV-1 infection and were also found to express increased levels of Gag after HIV-1 superinfection. Analysis of CCR5, a co-receptor for HIV-1, on HCMV-infected and bystander cells showed a substantial loss of surface CCR5 expression on infected cells due to HCMV-induced reduction of total cellular CCR5. In contrast, uninfected bystander cells displayed increased surface CCR5 expression. Furthermore, the data suggested that soluble factor(s) secreted from HCMV-infected cells were responsible for the observed upregulation of CCR5 on uninfected bystander cells. Taken together, these results suggest that, whilst HCMV-infected monocytes/macrophages are refractory to infection with HIV-1, HCMV-uninfected bystander cells within a population are more susceptible to HIV-1 infection. On this basis, HCMV infection may contribute to the pathogenesis of HIV-1.

Approximately one million people in the world are dually infected with both HIV-1 and HIV-2. To identify potential interactions between these two human pathogens, we examined whether HIV-1 and HIV-2 Gag proteins can coassemble and functionally complement each other. We generated HIV-1- and HIV-2-based vectors with mutations in Gag; compared with wild-type vectors, these mutants had drastically decreased viral titers. Coexpression of the mutant HIV-1 and HIV-2 Gag could generate infectious viruses; furthermore, heterologous complementation in certain combinations showed efficiency similar to homologous complementation. Additionally, we used bimolecular fluorescence complementation analysis to directly demonstrate that HIV-1 and HIV-2 Gag can interact and coassemble. Taken together, our results indicate that HIV-1 and HIV-2 Gag polyproteins can coassemble and functionally complement each other during virus replication; to our knowledge, this is the first demonstration of its kind. These studies have important implications for AIDS treatment and the evolution of primate lentiviruses.

In complex retroviruses including simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1), the major structural proteins are encoded by the gag gene and translated as a precursor polyprotein, Pr55(Gag). An internal ribosome entry site (IRES) within the coding region of HIV-1 and HIV type 2 (HIV-2) gag RNA mediates expression of N-terminally truncated isoforms of the precursor polyprotein. In this study, we identify an N-terminally truncated SIV Pr55(Gag) isoform expressed from the SIV gag gene SIV p43. We demonstrate that translation of p43 occurs independently of Pr55(Gag) translation and initiates at an in-frame AUG within the gag transcript. We test several mechanisms that could mediate translation of p43 and report that translation of SIV p43 is driven by an IRES located entirely within the coding region of gag mRNA. Additionally, we present data that suggest SIV p43 affects viral replication in cell culture.

In cells, the expression of Gag protein, one of the major structural proteins of retroviruses, is sufficient for budding virus-like particles (VLPs) from the cell surface. We have previously reported that spheroplasts of Saccharomyces cerevisiae expressing HIV-1 Gag proteins from an episomal plasmid constitutively released a large amount of VLPs into culture media; however, commercially available ELISA kits which detect mature capsid of HIV-1 could not detect uncleaved 55-kDa Gag proteins released from budding yeast. We therefore developed a method to quantitate VLP levels released from budding yeast by using fusion protein from HIV-1 Gag and Firefly Luciferase. This system is useful for screening cellular factor(s) involved in retrovirus budding from S. cerevisiae.

Objective: To examine whether human endogenous retrovirus K10 is associated with autoimmune rheumatic disease., Design: A novel multiplex reverse transcription polymerase chain reaction (RT-PCR) system was developed to investigate HERV-K10 mRNA expression in patients with rheumatoid arthritis., Methods: 40 patients with rheumatoid arthritis, 17 with osteoarthritis, and 27 healthy individuals were recruited and total RNA was extracted from peripheral blood mononuclear cells (PBMCs) and analysed using multiplex RT-PCR for the level of HERV-K10 gag mRNA expression. Southern blot and DNA sequencing confirmed the authenticity of the PCR products., Results: Using the histidyl tRNA synthetase (HtRNAS) gene as a housekeeping gene in the optimised multiplex RT-PCR, a significantly higher level of HERV-K10 gag mRNA expression was found in rheumatoid arthritis than in osteoarthritis (p = 0.01) or in the healthy controls (p = 0.02)., Conclusion: There is enhanced mRNA expression of the HERV-K10 gag region in rheumatoid arthritis compared with osteoarthritis or healthy controls. This could contribute to the pathogenesis of rheumatoid arthritis.