Gag Transcripts Research Articles

Adult neurogenic deficits in HIV-1 Tg26 transgenic mice

BackgroundEven in the antiretroviral treatment (ART) era, HIV-1-infected patients suffer from milder forms of HIV-1-associated neurocognitive disorders (HAND). While the viral proteins Tat and gp120 have been shown to individually inhibit the proliferation and neural differentiation of neural stem cells (NSCs), no studies have characterized the effects of all the combined viral proteins on adult neurogenesis.MethodsThe HIV-1 Tg26 transgenic mouse model was used due to its clinical relevance to ART-controlled HIV-1-infected patients who lack active viral replication but suffer from continuous stress from the viral proteins. Quantitative RT-PCR analysis was performed to validate the expression of viral genes in the neurogenic zones. In vitro stemness and lineage differentiation assays were performed in cultured NSCs from HIV-1 Tg26 transgenic mice and their wild-type littermates. Hippocampal neurogenic lineage analysis was performed to determine potential changes in initial and late differentiation of NSCs in the subgranular zone (SGZ). Finally, fluorescent retroviral labeling of mature dentate granule neurons was performed to assess dendritic complexity and dendritic spine densities.ResultsVarying copy numbers of partial gag (p17), tat (unspliced and spliced variants), env (gp120), vpu, and nef transcripts were detected in the neurogenic zones of Tg26 mice. Significantly fewer primary neurospheres and a higher percentage of larger sized primary neurospheres were generated from Tg26 NSCs than from littermated wild-type mouse NSCs, implying that Tg26 mouse NSCs exhibit deficits in initial differentiation. In vitro differentiation assays revealed that Tg26 mouse NSCs have reduced neuronal differentiation and increased astrocytic differentiation. In the SGZs of Tg26 mice, significantly higher amounts of quiescent NSCs, as well as significantly lower levels of active NSCs, proliferating neural progenitor cells, and neuroblasts, were observed. Finally, newborn mature granule neurons in the dentate gyri of Tg26 mice had deficiencies in dendritic arborization, dendritic length, and dendritic spine density.ConclusionsBoth in vitro and in vivo studies demonstrate that HIV-1 Tg26 mice have early- and late-stage neurogenesis deficits, which could possibly contribute to the progression of HAND. Future therapies should be targeting this process to ameliorate, if not eliminate HAND-like symptoms in HIV-1-infected patients.

Ribosome profiling of the retrovirus murine leukemia virus

BackgroundThe retrovirus murine leukemia virus (MuLV) has an 8.3 kb RNA genome with a simple 5′-gag-pol-env-3′ architecture. Translation of the pol gene is dependent upon readthrough of the gag UAG stop codon; whereas the env gene is translated from spliced mRNA transcripts. Here, we report the first high resolution analysis of retrovirus gene expression through tandem ribosome profiling (RiboSeq) and RNA sequencing (RNASeq) of MuLV-infected cells.ResultsRibosome profiling of MuLV-infected cells was performed, using the translational inhibitors harringtonine and cycloheximide to distinguish initiating and elongating ribosomes, respectively. Meta-analyses of host cell gene expression demonstrated that the RiboSeq datasets specifically captured the footprints of translating ribosomes at high resolution. Direct measurement of ribosomal occupancy of the MuLV genomic RNA indicated that ~ 7% of ribosomes undergo gag stop codon readthrough to access the pol gene. Initiation of translation was found to occur at several additional sites within the 5′ leaders of the gag and env transcripts, upstream of their respective annotated start codons.ConclusionsThese experiments reveal the existence of a number of previously uncharacterised, ribosomally occupied open reading frames within the MuLV genome, with possible regulatory consequences. In addition, we provide the first direct measurements of stop codon readthrough efficiency during cellular infection.

D4 Expression of Human Endogenous Retrovirus Type-K (HML-2) is Activated by the Tat Protein of HIV-1

Human Endogenous Retroviruses (HERVs) make up 8% of the human genome. Expression of HERV-K (HML-2), our most recent genome entrant, is tightly regulated but becomes markedly increased following infection with HIV-1. To better understand the mechanisms involved in this activation, we explored the role of the HIV-1 proteins in inducing expression of these retroviral genes. Transfection experiments showed that, out of all HIV-1 proteins, Tat and Vif where the only ones inducing significant activation of HERV-K (HML-2) gene expression. Out of these two proteins, Tat alone was sufficient to cause significant HERV-K (HML-2) expression. Administration of recombinant HIV-1 Tat protein caused a 13-fold increase in HERV-K (HML-2) gag transcripts in Jurkat T cells and a 10-fold increase in primary lymphocytes, and expression of the HERV-K (HML-2) rec and np9 oncogenes was also markedly increased. Additionally, transfection of a plasmid encoding Tat caused a strong increase in HERV-K (HML-2) gag protein expression. Luciferase reporter gene assays demonstrated that the effect of Tat on HERV-K (HML-2) expression occurred at the level of the transcriptional promoter. The transcription factors NF-kB and NF-AT contribute to the Tat-induced activation of the promoter, as shown by mutational analysis of the HERV-K (HML-2) LTR, ChIP assays, and treatments with agents that inhibit NF-AT or NF-kB activation. These studies show that HIV-1 Tat plays an important role in activating expression of HERV-K (HML-2) in the setting of HIV-1 infection. By increasing expression of HERV-K oncogenes, Tat might potentiate HIV-related disease, but increased expression of HERV-K antigens might also contribute to immunological control of HIV infection.

Strategies to quantify unspliced and multiply spliced mRNA expression in HIV-2 infection

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.

Production of HIV Particles Is Regulated by Altering Sub-Cellular Localization and Dynamics of Rev Induced by Double-Strand RNA Binding Protein

Human immunodeficiency virus (HIV)-1 encoded Rev is essential for export from the nucleus to the cytoplasm, of unspliced and singly spliced transcripts coding for structural and nonstructural viral proteins. This process is spatially and temporally coordinated resulting from the interactions between cellular and viral proteins. Here we examined the effects of the sub-cellular localization and dynamics of Rev on the efficiency of nucleocytoplasmic transport of HIV-1 Gag transcripts and virus particle production. Using confocal microscopy and fluorescence recovery after bleaching (FRAP), we report that NF90ctv, a cellular protein involved in Rev function, alters both the sub-cellular localization and dynamics of Rev in vivo, which drastically affects the accumulation of the viral protein p24. The CRM1–dependent nuclear export of Gag mRNA linked to the Rev Response Element (RRE) is dependent on specific domains of the NF90ctv protein. Taken together, our results demonstrate that the appropriate intracellular localization and dynamics of Rev could regulate Gag assembly and HIV-1 replication.

Viral gene expression and provirus load of Orf-A defective FIV in lymphoid tissues and lymphocyte subpopulations of neonatal cats during acute and chronic infections

Neonatal cats were infected with a wild type (JSY3) or orf-A defective (JSY3ΔORF-A) feline immunodeficiency virus (FIV) to determine the provirus load and level of viral gene expression at the acute versus chronic stages of infection. FIV DNA in the thymus, lymph node, peripheral blood mononuclear cells (PBMCs) and lymphocyte subpopulations at week 8 post-infection was lower in animals infected with JSY3ΔORF-A as compared to that of JSY3. At week 16 we observed no significant difference in provirus load between the two groups except for B cells where it was higher in the JSY3 infection. In B cells proviral burden was found to be the same in animals infected with JSY3 for both time points. In the chronic stage, therefore, proviral burden dominates in B cells for JSY3, whereas the level of JSY3ΔORF-A was lower with comparable values for all lymphocytes at both weeks 8 and 16. Gene expression profiles as measured by real time PCR for gag and rev transcripts revealed decreased levels of JSY3ΔORF-A mRNAs as compared to that of JSY3. The JSY3 chronic phase infection showed viral gene expression to be higher in B cells relative to CD4+ and CD8+ cells. The presence of viral RNA in CD8 and B cells during the chronic infection implicates active virus replication. Hematological profiles revealed that there was a decline in the number of B cells in JSY3ΔORF-A-infected cats during the chronic stage of infection while no significant change was observed in animals infected with the wild type virus. Comparative analysis of cell numbers to provirus load and levels of viral transcripts in CD4+ and CD8+, however, did not correlate cell numbers to the levels of viral DNA and gene expression. It remains to be determined whether the relatively high virus burden in B cells as compared to CD4+ and CD8+ cells reflects a role for Orf-A in a shift to B cell virus load during the chronic stage of FIV infection.

Recombinant simian varicella viruses induce immune responses to simian immunodeficiency virus (SIV) antigens in immunized vervet monkeys

The varicella-zoster virus (VZV) Oka vaccine offers potential as a recombinant vaccine against other pathogens. In this study, recombinant simian varicella viruses (rSVV) expressing simian immunodeficiency virus (SIV) envelope (env, gp130) and gag antigens were constructed. Expression of the SIV env and gag transcripts and antigens in rSVV-infected Vero cells was confirmed. The rSVV-SIVenv and rSVV-SIVgag viruses replicated as efficiently as wild-type SVV in cell culture. The immunogenicity of rSVV-SIVenv and rSVV-SIVgag was investigated in immunized vervet monkeys. Humoral immune responses to the SIV gp130 and gag antigens were detected as early as 4 weeks after the initial immunization with higher antibody titers following a booster immunization. Cellular immune responses against the SIV gp130 antigen were detected by ELISPOT assay. The rSVV established latent infection in neural ganglia. A subsequent study will evaluate the ability of rSVV vaccines expressing SIV antigens to protect nonhuman primates against simian AIDS.

719. E1-Deleted Adenoviral Vectors Persist in Activated Lymphocytes and Cause Continued Transgene Expression and Stimulation of CD8+ Effector T Cells

Top of pageAbstract As initial attempts to develop a vaccine that induces broadly cross-reactive neutralizing antibodies to the human immunodeficiency virus (HIV)-1 have failed, current vaccine approaches are focused on induction of cell-mediated immune responses, especially CD8+ T cells, against more conserved internal proteins of HIV-1. The differentiation pathway of antigen-induced memory CD8+ T cells has raised doubts about the usefulness of vaccines that target their induction, a concern substantiated by an experiment of nature, which showed in African prostitutes a link between resistance to infection and constant exposure to antigen. This in turn predicts that HIV-1 vaccines that aim to induce memory CD8+ T cells may have limited efficacy in humans. We tested a new vaccine platform based on E1-deleted adenoviral vectors derived from chimpanzee serotypes expressing gag of HIV-1 for induction of gag-specific CD8+ T cells in mice. A single administration of the vaccines resulted in exceptionally transgene product-specific CD8+ T cell frequencies, which initially declined after 2–3 weeks and then resurged suggestive of an internal booster effect due to continued or renewed production of gag. Indeed gag transcripts could be detected in lymphatic tissues for over a year after a single administration of the replication-defective adenoviral vectors. Interestingly, transcripts were preferentially expressed by activated gag-specific T cells. These results put forward a novel vaccine concept based on constructs that achieve persistent transgene product expression and thus maintain high frequencies of specific CD8+ T cells which may be superior in providing an immediate line of defense against an invading pathogen compared to resting memory CD8+ T cells.

Human endogenous retrovirus (HERV)-K transcripts in gonadoblastomas and gonadoblastoma-derived germ cell tumours.

Gonadoblastomas are rare tumours of abnormal or dysgenetic gonads, often transforming to invasive seminomatous and nonseminomatous germ cell tumours (GCT). Because of the intimate association of noninvasive and invasive lesions, gonadoblastoma may provide clues as to the molecular pathogenesis of GCT. We studied the expression of the human endogenous retrovirus (HERV)-K gag gene in eight gonadoblastomas arising in phenotypically female patients, including two newborn girls. We also studied testicular biopsies with immature Sertoli cell nodules harbouring neoplastic germ cells, a lesion with morphological resemblance to gonadoblastoma. In five gonadoblastomas, invasive seminoma/dysgerminoma was noted, in two cases with formation of additional GCT components. HERV-K gag transcripts were found with moderate levels in gonocytes of all gonadoblastomas and in neoplastic germ cells in testicular Sertoli cell nodules. All invasive GCT except for teratomas displayed HERV-K transcripts. Thus, expression of HERV-K is induced during fetal or embryonal development and precedes invasive GCT formation. Although the specific role of HERV-K expression remains unknown, the findings place HERV-K expression in an appropriate time frame for it to have a role in the molecular pathogenesis of GCT and suggest a precursor-invasive tumour relationship for ovarian GCT equivalent to the more common carcinoma in situ of the testis and testicular GCT.

Inflammatory cytokine expression is correlated with the level of human immunodeficiency virus (HIV) transcripts in HIV-infected placental trophoblastic cells.

The inflammatory cytokines interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor alpha (TNF-alpha) have been associated with increased human immunodeficiency virus (HIV) expression and enhanced lymphocyte adhesion to trophoblastic cells in experimental systems. To determine if there is a correlation between the expression of these cytokines and the levels of HIV transcripts in trophoblasts of term placentas from HIV-infected women, we studied the placentae of 30 HIV-positive and 13 control gravidae. Twenty-three of the HIV-positive women received zidovudine (ZDV) as prophylaxis against HIV vertical transmission; only one of the seven women who did not receive ZDV was a transmitter, for an overall vertical transmission rate of 3.8%. Cytokine production was measured by enzyme-linked immunosorbent assay in the supernatants of trophoblastic cell cultures. Additionally, cytokine transcripts and HIV gag sequences were determined by a quantitative reverse transcription-PCR assay. In general, trophoblastic cells of HIV-positive placentas expressed significantly higher levels of IL-1beta, IL-6, and TNF-alpha than those of control placentas. All placentas from HIV-positive women expressed HIV gag transcripts at either a low (<156 copies per microg of total RNA) or a high (>156 copies per microg of total RNA) level. There was a statistically significant positive association between the basal level of TNF-alpha production and the level of HIV gag transcripts of HIV-positive placental trophoblastic cells. Nevertheless, these data, coupled with a low transmission rate, would indicate that some other factors, perhaps working in concert with cytokines, are necessary for vertical transmission of HIV from mother to infant.

Altered mouse mammary tumor virus transcript synthesis in T-cell lymphoma cells

Proviral copies of mouse mammary tumor virus (MMTV) are known to be amplified in certain T-cell lymphomas. Transcription of the amplified MMTV proviruses was studied in detail in two T-cell lymphoma lines and showed the production of deletions and premature termination of env mRNAs and the premature termination of gag transcripts. EL-4 cells produce three env mRNAs, and sequence analysis of cDNAs of the two smaller transcripts revealed large deletions encompassing the 3' half of the env gene. The deletion in at least one of the altered transcripts appeared to be produced by a splicing mechanism. T-cell lymphoma line ML of DBA/2 mice also synthesizes two smaller env transcripts, both of which result from premature termination of transcription. Both lines transcribe high levels of gag mRNAs of about 0.8 kilobases in length, terminating at the end of the region encoding MMTV phosphoprotein pp21. Restriction enzyme BamHI analysis of the amplified proviruses of EL-4 and ML cells as well as of additional non-mammary tumor cell types containing amplified MMTV proviruses suggested that the amplified proviruses were derived from exogenous viruses, or activated endogenous provirus MTV-1 in the case of DBA/2 strain tumor cells.

Ribozymes as Potential Anti-HIV-1 Therapeutic Agents

Certain RNA molecules, called ribozymes, possess enzymatic, self-cleaving activity. The cleavage reaction is catalytic and no energy source is required. Ribozymes of the "hammerhead" motif were identified in plant RNA pathogens. These ribozymes possess unique secondary (and possibly tertiary) structures critical for their cleavage ability. The present study shows precise cleavage of human immunodeficiency virus type 1 (HIV-1) sequences in a cell-free system by hammerhead ribozymes. In addition to the cell-free studies, human cells stably expressing a hammerhead ribozyme targeted to HIV-1 gag transcripts have been constructed. When these cells were challenged with HIV-1, a substantial reduction in the level of HIV-1 gag RNA relative to that in nonribozyme-expressing cells, was observed. The reduction in gag RNA was reflected in a reduction in antigen p24 levels. These results suggest the feasibility of developing ribozymes as therapeutic agents against human pathogens such as HIV-1.