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Immunization of macaques with attenuated simian immunodeficiency virus (SIV) with deletions in nef (SIVΔnef) is shown to elicit protective immunity to infection by pathogenic SIV, yet the mechanisms that orchestrate protection and prevent pathogenesis remains unknown. We utilized whole-genome transcriptional profiling to reveal molecular signatures of protective immunity in circulating CD8+ T cells of rhesus macaques vaccinated with SIVmac239Δnef and challenged with pathogenic SIVmac251. Our findings suggest that protective immunity to pathogenic SIV infection induced by SIVmac239∆nef is associated with balanced induction of T cell activation and immunoregulatory mechanisms and dampened activation of interferon-induced signaling pathways and cytolytic enzyme production as compared with pathogenic SIVmac251 infection of unvaccinated controls. We provide evidence that protective immunity to SIVmac251 correlates with induction of biomarkers of T cell activation, differentiation, signaling, and adhesion that were down regulated in unvaccinated controls. The study highlights potential immunomodulatory networks associated with protective immunity against the virus., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Three Indian rhesus macaques, Ad-SIV primed/protein boosted and exposed twice to high-dose mucosal SIV(mac251) challenges, exhibited elite control of viremia over 6.5 years. They were negative for host factors associated with control of SIV infection. After a third intrarectal challenge with SIV(smE660), all controlled viremia, with one (macaque #5) maintaining undetectable viremia in blood. Acquisition was not blocked, but virus was contained in the jejunum and draining lymph nodes. Polyfunctional memory T cell responses and high-titered neutralizing and non-neutralizing serum and mucosal antibodies were present before and maintained post-challenge. The level of protection seen for animal #5 was predicted from analyses of gene transcription in jejunum 2 weeks post-challenge. Macaques #7 and #9, exhibiting lower pre-challenge cellular and humoral immunity, partially controlled the SIV(smE660) challenge. Initial vaccine-induced control by macaque #5 extended to the SIV(smE660) challenge due to multiple immune mechanisms that were boosted and augmented by cryptic SIV exposure., (Published by Elsevier Inc.)

Hepatitis C virus (HCV) Core protein is implicated in the development of hepatocellular carcinoma (HCC). We utilized a HepG2 human hepatocyte cell line with inducible expression of HCV Core protein (HCV-1b) to investigate the early effects of Core protein on hepatocyte gene expression and to identify molecular processes modulated by the Core protein. A significant change was observed in the expression of 407 genes, which included genes regulating apoptosis, immune response, and cell cycle. Some of these genes were previously known to be tumor markers. The decreased expression of chemo-attractants such as TNFSF10, CCL20, and osteopontin was observed, which suggested that HCV Core expression could lead to suppression of inflammatory response as well as trafficking of macrophages and neutrophils to the site of HCV infection. An increased expression of anti-apoptosis factors including PAK2, API5, BH1, Tax1BP1, DAXX, and TNFAIP3/A20 was observed. Some of these genes were also linked to the regulation of NFKB activation and that the alteration of their expression levels, by HCV Core, might lead to the suppression NFKB activation of inflammatory responses. Our data suggested that Core expression may contribute to the viral persistence by protecting infected hepatocytes from cell death by the suppressing apoptosis and inflammatory reaction to HCV viral infection.

The Nef protein of human immunodeficiency virus and simian immunodeficiency virus is expressed early in infection and plays an important role in disease progression in vivo. In addition, Nef has been shown to modulate cellular functions. To decipher Nef-mediated changes in gene expression, we utilized DNA microarray analysis to elucidate changes in gene expression in a Jurkat CD4+ T-cell line stably expressing SIV-Nef protein under the control of an inducible promoter. Our results showed that genes associated with antigen presentation including members of the T-cell receptor and major histocompatibility class 1 complex were consistently down-regulated at the transcript level in SIV-Nef-expressing cells. In addition, Nef induced a transcriptional profile of cell-cycle-related genes that support the survival of Nef-expressing cells. Furthermore, Nef enhanced the transcription of genes encoding enzymes and factors that catalyze the biosynthesis of membrane glycolipids and phospholipids. In conclusion, gene expression profiling showed that SIV-Nef induces a transcriptional profile in CD4+ T cells that promotes immune evasion and cell survival, thus facilitating viral persistence.

We utilized the simian immunodeficiency virus model of AIDS to examine evolution of nef gene in gut-associated lymphoid tissue (GALT) during primary and early asymptomatic stages of infection. Macaques were infected with a cloned virus, SIVmac239/nef-stop harboring a premature stop codon in the nef gene. Restoration of the nef open reading frame occurred in GALT early at 3 days post-infection. Analysis of nef sequences by phylogenetic tools showed that evolution of nef was neutral thereafter, as evidenced by the ratio of synonymous to nonsynonymous substitutions, a star pattern in unrooted trees and distribution of amino acid replacements fitting a simple Poisson process. Two regions encoding for a nuclear localization signal and a CTL epitope were conserved. Thus, GALT was a site for strong positive selection of functional nef during initial stages of infection. However, evolution of the nef gene thereafter was neutral during early asymptomatic stage of infection.

The total number of circulating CD4+ and CD8+ T-cells undergoing clonal expansions following SIV(mac251) infection was determined using a T-cell receptor Vbeta chain (TRBV) third complementarity-determining region (CDR3) DNA heteroduplex tracking assay (HTA). This assay measures the number of newly expanding T-cell clones but not their antigenic specificity. Fewer expanding CD4+ (3-23 per animal) than CD8+ (18-37 per animal) clonotypes were observed during the acute phase of SIV infection. CD8+ T-cell expansions peaked at 4 weeks postinfection (wpi) concomitant with early reductions in viremia. Expanding clone TRBV transcripts ranged in frequency from the limit of detection of 2% to 40% of their TRBV subfamily's transcripts. The number of expanding CD4+ or CD8+ clones correlated with neither peak, subsequent slope, nor steady-state viremia. CDR3 repertoires in CD8-expressing cells in different anatomical compartments were also analyzed. Repertoires were polyclonal in the thymus, oligoclonal in mesenteric lymph nodes, peripheral blood mononuclear cells (PBMC), and spleen, and extremely oligoclonal in intra-epithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL). The lack of correlation between the number of expanding T-cell clonotypes and viremia levels may reflect the highly variable selection pressure imposed on SIV by T-cell responses targeting different epitopes in outbred macaques.

During primary simian immunodeficiency virus (SIV) infection, CD4+ T cells are severely depleted in gut-associated lymphoid tissue (GALT), while CD8+ T-cell numbers dramatically increase. To gain an understanding of the molecular basis of this disruption in T-cell homeostasis, host gene expression was monitored in longitudinal jejunum tissue biopsies from SIV-infected rhesus macaques by DNA microarray analysis. Transcription of cyclin E1, CDC2, retinoblastoma, transforming growth factor (TGF), fibroblast growth factor (FGF), and interleukin-2 was repressed while cyclins B1 and D2 and transcription factor E2F were upregulated, indicating a complex dysregulation of growth and proliferation within the intestinal mucosa. Innate, cell-mediated, and humoral immune responses were markedly upregulated in animals that significantly reduced their viral loads and retained more intestinal CD4+ T cells. We conclude that the alterations in intestinal gene expression during primary SIV infection were characteristic of a broad-range immune response, and reflective of the efficacy of viral suppression.

Hepatitis C virus (HCV) Core protein is implicated in viral pathogenesis by the modulation of hepatocyte gene expression and function. To determine the effect of Core protein on the cell-cycle control of hepatocytes, a HepG2 cell line containing a Flag-tagged Core under the control of an inducible promoter was generated. Initial Core protein expression included the presence of unprocessed (191 aa) and processed (173 aa) forms of the Core proteins with the processed form becoming dominant later. Expression of the 191 aa form of Core protein corresponded to an increase in the expression of the p21, a decrease in cdk2-dependent kinase activity, and a decrease in the percentage of cells in S-phase along with an accumulation of cells in the G(0)/G(1) phase of the cell cycle. As the processed form accumulated, the p21 levels started to decline, suggesting that Core protein regulates p21 expression in a biphasic manner. These findings implicate Core protein in potentially modulating hepatocyte cell cycle differentially in the early stages of infection through biphasic regulation of p21 cdk kinase inhibitor.

RANTES, a beta-chemokine, can suppress human immunodeficiency virus (HIV) as well as simian immunodeficiency virus (SIV) infections in T-lymphocyte cultures in vitro. However, the association of RANTES levels in peripheral blood with viral loads and disease outcome in HIV infection has been inconclusive. SIV-infected rhesus macaques were evaluated to determine whether RANTES gene expression correlated with suppression of viral infection in intestinal lymphoid tissues. Intestinal tissues were obtained from rhesus macaques infected with either pathogenic or nonpathogenic SIVmac variants at various stages of infection (primary acute, asymptomatic, and terminal). We examined the level of SIV infection (in situ hybridization), RANTES expression (quantitative competitive RT-PCR), and T-cell counts (immunohistochemistry). The most pronounced increase in RANTES gene expression in intestinal tissues was observed in primary SIV infection, which correlated with the pathogenicity of the infecting virus and not the tissue viral loads. Our results demonstrated that in contrast to the occurrence of viral suppression by RANTES in vitro, there was no direct correlation between high RANTES gene expression and suppression of viral loads in intestinal lymphoid tissues. Thus RANTES expression in the gut lymphoid tissue may not be a correlate for viral suppression. However, RANTES gene expression in primary SIV infection may be part of early host immune response to viral infection., (Copyright 1999 Academic Press.)

HIV-1BR was isolated from the autopsied brain tissue of a 57-year-old man who died of progressive dementing illness. This virus was shown to be HIV-1 by hybridization to HIV-specific DNA probes. The expression of viral proteins as tested by radioimmunoprecipitation assay revealed the presence of HIV-1-specific proteins. HIV-1BR replicated in cultures of CD4+ T-lymphoid cells and induced cytopathic effects in these cells. HIV-1BR also replicated in monocytoid cell lines. The genetic nature of this isolate was determined by molecular cloning and sequencing of the 3'-half of the genome. DNA sequence information established that HIV-1BR is a unique HIV-1 isolate. A stretch of approximately 30 bases in the nef gene of HIV-1BR was found duplicated when compared with the other sequenced HIV-1 genomes. The functional significance of this duplication remains to be determined.

The RNA of a diabetogenic variant of encephalomyocarditis (EMC) virus (D variant, ifp- phenotype) and a nondiabetogenic variant of EMC virus (B variant, ifp+ phenotype) which were derived from the same virus stock (M strain) have been compared. The size of both genomes is estimated at 7.7 kb. The poly(C) tract of EMC-D is estimated at 144 bases, whereas that of EMC-B is 141 bases in length. The untranslated 5' terminal 103 nucleotides are identical for B and D with preservation of a stable terminal hairpin structure. The entire open reading frame of both variants has been cloned and the restriction maps of 12 different enzymes are identical. These maps were compared to a computer-generated restriction map of another strain of EMC virus which has been cloned and sequenced by Palmenberg et al. (1984, Nucleic Acids Res. 12, 2969-2985). Approximately 50% of the restriction sites of the B and D variants had similar locations in this strain of EMC. We conclude that significant genetic variation exists between the M strain (B and D variants) and the Palmenberg strain of EMC virus. However, the B and D variants are very similar at the molecular level and comparison of their nucleotide sequences will be necessary to reveal the basis for their different biological properties.