Location and function of retroviral and SV40 sequences that enhance biochemical transformation after microinjection of DNA.

Journal ArticleBiochemical transformation of thymidine-kinase-deficient (TK-) mouse L cells is enhanced 20 to 40 fold when microinjected plasmid DNA contains regions of the genomes of Rous sarcoma virus or simian virus 40 in addition to the complete herpes simplex virus tk gene, irrespective of the orientation and location of the enhancer. The enhancing sequence of RSV DNA has been localized to 143 bp that include 88 bp at the 5' end of the long terminal repeat; the enhancing segment of SV40 DNA lies 42-276 bp upstream from the initiation site for early transcription. The RSV enhancer does not provide a favored integration site within microinjected plasmids and does not affect the frequency of integration into host cell DNA. When microinjected cells were grown into cell lines in nonselective medium, few cells containing integrated HSV tk without RSV DNA could grow in selective medium. In contrast, over 80% of cells from lines containing the HSV tk gene linked to the RSV enhancer could grow in selective medium. Thus the RSV enhancer affects the expression rather than the integration of the tk gene

Transcriptional Regulation of Latent Feline Immunodeficiency Virus in Peripheral CD4+ T-lymphocytes

Feline immunodeficiency virus (FIV), the lentivirus of domestic cats responsible for feline AIDS, establishes a latent infection in peripheral blood CD4+ T-cells approximately eight months after experimental inoculation. In this study, cats experimentally infected with the FIV-C strain in the asymptomatic phase demonstrated an estimated viral load of 1 infected cell per approximately 103 CD4+ T-cells, with about 1 copy of viral DNA per cell. Approximately 1 in 10 proviral copies was capable of transcription in the asymptomatic phase. The latent FIV proviral promoter was associated with deacetylated, methylated histones, which is consistent with a condensed chromatin structure. In contrast, the transcriptionally active FIV promoter was associated with histone acetylation and demethylation. In addition, RNA polymerase II appeared to be paused on the latent viral promoter, and short promoter-proximal transcripts were detected. Our findings for the FIV promoter in infected cats are similar to results obtained in studies of human immunodeficiency virus (HIV)-1 latent proviruses in cell culture in vitro studies. Thus, the FIV/cat model may offer insights into in vivo mechanisms of HIV latency and provides a unique opportunity to test novel therapeutic interventions aimed at eradicating latent virus

Analysis of multiply spliced transcripts in lymphoid tissue reservoirs of rhesus macaques infected with RT-SHIV during HAART.

Highly active antiretroviral therapy (HAART) can reduce levels of human immunodeficiency virus type 1 (HIV-1) to undetectable levels in infected individuals, but the virus is not eradicated. The mechanisms of viral persistence during HAART are poorly defined, but some reservoirs have been identified, such as latently infected resting memory CD4⁺ T cells. During latency, in addition to blocks at the initiation and elongation steps of viral transcription, there is a block in the export of viral RNA (vRNA), leading to the accumulation of multiply-spliced transcripts in the nucleus. Two of the genes encoded by the multiply-spliced transcripts are Tat and Rev, which are essential early in the viral replication cycle and might indicate the state of infection in a given population of cells. Here, the levels of multiply-spliced transcripts were compared to the levels of gag-containing RNA in tissue samples from RT-SHIV-infected rhesus macaques treated with HAART. Splice site sequence variation was identified during development of a TaqMan PCR assay. Multiply-spliced transcripts were detected in gastrointestinal and lymphatic tissues, but not the thymus. Levels of multiply-spliced transcripts were lower than levels of gag RNA, and both correlated with plasma virus loads. The ratio of multiply-spliced to gag RNA was greatest in the gastrointestinal samples from macaques with plasma virus loads <50 vRNA copies per mL at necropsy. Levels of gag RNA and multiply-spliced mRNA in tissues from RT-SHIV-infected macaques correlate with plasma virus load

Species Tropism of Chimeric SHIV Clones Containing HIV-1 Subtype-A and Subtype-E Envelope Genes

AbstractTo analyze HIV-1 genes in a nonhuman primate model for lentivirus infection and AIDS, recombinant SIV/HIV-1 (SHIV) clones were constructed from two HIV-1 subtype-A isolates (HIV-1SF170 and HIV-1Q23–17 from individuals in Africa) and two HIV-1 subtype-E isolates (HIV-19466 and HIV-1CAR402 from AIDS patients in Thailand and Africa), respectively. These four SHIV clones, designated SHIV-A-170, SHIV-A-Q23, SHIV-9466.33, and SHIV-E-CAR, contain envelope (env) genes from the subtype-A or -E viruses. Interestingly, SHIV-A-170, SHIV-A-Q23, and SHIV-9466.33 were restricted for replication in cultures of macaque lymphoid cells, whereas SHIV-E-CAR replicated efficiently in these cells. Additional studies to define the block to replication in macaque cells were focused on the subtype-E clone SHIV-9466.33. A SHIV intragenic env clone, containing sequence-encompassing V1/V2 regions of HIV-1CAR402 and V3/V4/V5 regions of SHIV-9466.33, infected and replicated in macaque lymphoid cells. These results indicated that the sequence-encompassing V1/V2 region of HIV-19466 was responsible for the block of the SHIV-9466.33 replication in macaque cells. Analysis of viral DNA in acutely infected macaque cells revealed that SHIV-9466.33 was blocked at a step at/or before viral DNA synthesis, presumably during the process of virion entry into cells. In a fluorescence-based cell–cell fusion assay, fusion pore formation readily took place in cocultures of cells expressing the SHIV-9466.33 env glycoprotein with macaque T-lymphoid cells. Taken together, these results demonstrated that the block of SHIV-9466.33 replication in macaque cells is at an early step after fusion pore formation but before reverse transcription

Multiplexed measurements of immunomodulator levels in peripheral blood of healthy subjects: Effects of analytical variables based on anticoagulants, age, and gender

Multiplex microbead immunoassay (MMIA) is a powerful technology for a wide range of biomedical and clinical applications. It is important to study the normal concentration ranges of immunomodulators under different sample preparation conditions and age groups of subjects in order to more precisely determine their reference values for use in assessing alterations of their levels in disease. The aim of this study was to determine the plasma concentrations of immunomodulators (cytokines, chemokines, and growth factors) in the peripheral blood from healthy subjects by the use of a large multiplex panel, and to determine the effects of different anticoagulants, age, and gender on the immunomodulator levels. In addition, the assay precision for these biomarker analytes was determined. Plasma samples from 107 healthy subjects, aged 18 to 85 years, were collected in three different anticoagulants (sodium citrate, EDTA, Heparin); corresponding serum samples were also obtained. Multiplex microbead immunoassays were performed for measuring a total of 23 analytes including chemokines, cytokines, and growth factors (IL-1β, IL-1ra, IL-2, IL-6, IL-7, IL-8, IL-12 p70, IL-17, IFN-γ, IP-10, MCP-1, PDGF-BB, RANTES, TNF-α, IL-1a, IL-16, HGF, MIG, TNF-β, PDGF-ABBB, EGF, Flt-3 Ligand, VEGF). For these analytes, our results showed that the anticoagulant affected the concentration measurements and the coefficients of variation. However, the relative levels of the analytes (profiles) of samples collected in a particular anticoagulant are consistent. The analytes IL-1β, IL-7, Flt-3 Ligand, and IL-12p70 show the largest variation (up to fourfold) between the age groups. In addition, no statistically significant differences in the level of the analytes were found between the sexes

Activation of PAK by HIV and SIV Nef: importance for AIDS in rhesus macaques

AbstractBackground The primate lentiviruses, human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV), encode a conserved accessory gene product, Nef. In vivo, Nef is important for the maintenance of high virus loads and progression to AIDS in SIV-infected adult rhesus macaques. In tissue culture cells expressing Nef, this viral protein interacts with a cellular serine kinase, designated Nef-associated kinase.Results This study identifies the Nef-associated kinase as a member of the p21-activated kinase (PAK) family of kinases and investigates the role of this Nef-associated kinase in vivo. Mutants of Nef that do not associate with the cellular kinase are unable to activate the PAK-related kinase in infected cells. To determine the role of cellular kinase association in viral pathogenesis, macaques were infected with SIV containing point-mutations in Nef that block PAK activation. Virus recovered at early time points after inoculation with mutant virus was found to have reverted to prototype Nef function and sequence. Reversion of the kinase-negative mutant to a kinase-positive genotype in macaques infected with the mutant virus preceded the induction of high virus loads and disease progression.Conclusions Nef associates with and activates a PAK-related kinase in lymphocytes infected in vitro. Moreover, the Nef-mediated activation of a PAK-related kinase correlates with the induction of high virus loads and the development of AIDS in the infected host. These findings reveal that there is a strong selective pressure in vivo for the interaction between Nef and the PAK-related kinase

Fatal Immunopathogenesis by SIV/HIV-1 (SHIV) Containing a Variant Form of the HIV-1sf33 env Gene in Juvenile and Newborn Rhesus Macaques

AbstractSIV/HIV-1 (SHIV) chimeric clones, constructed by substituting portions of the pathogenic molecular clone SIVmac239 with counterpart portions from HIV-1 clones, provide a means to analyze functions of selected HIV-1 genes in vivo in nonhuman primates. Our studies focused on SHIVsf33, which contains the vpu, tat, rev, and env genes of the cytopathic, T-cell line tropic clone HIV-1sf33 (subtype-B); this clone has a premature stop codon in the vpu gene. In three juvenile macaques inoculated intravenously with SHIVsf33, low-level persistent infection was established; no disease was observed for a period of >2 years. However, at ∼16 months p.i., one of four SHIVsf33-infected juvenile macaques exhibited an increase in virus load, depletion of CD4+ T cells in peripheral blood and lymph nodes, and other symptoms of simian AIDS (SAIDS). Virus recovered from this animal in the symptomatic stage was designated SHIVsf33a (A, adapted); this virus displayed multiple amino acid sequence changes throughout the HIV-1 env gene compared with the input SHIVsf33 clone. Additionally, a mutation in all clones from SHIVsf33a restored the open reading frame for the vpu gene. In vitro evaluations in tissue-culture systems revealed that SHIVsf33a replicated to higher levels and exhibited greater cytopathicity than SHIVsf33. Furthermore cloned env genes for SHIVsf33a were more fusogenic in a cell-fusion assay compared with the env gene of the SHIVsf33. Intravenous inoculation of SHIVsf33a into juvenile and newborn macaques resulted in a rapid decline in CD4+ T cells to very low levels and development of a fatal AIDS-like disease. A cell-free preparation of this pathogenic chimeric virus also established persistent infection when applied to oral mucosal membranes of juvenile macaques and produced a fatal AIDS-like disease. These studies on pathogenic SHIVsf33a establish the basis for further investigations on the role of the HIV-1 env gene in virus adaptation and in mechanism(s) of immunodeficiency in primates; moreover, the chimeric virus SHIVsf33a can play a role in elucidating mucosal membrane transmission and development of antiviral vaccines in newborns as well as juvenile and adult macaques

Mass Spectrometry Imaging Reveals Heterogeneous Efavirenz Distribution within Putative HIV Reservoirs

ABSTRACT Persistent HIV replication within active viral reservoirs may be caused by inadequate antiretroviral penetration. Here, we used mass spectrometry imaging with infrared matrix-assisted laser desorption–electrospray ionization to quantify the distribution of efavirenz within tissues from a macaque dosed orally to a steady state. Intratissue efavirenz distribution was heterogeneous, with the drug concentrating in the lamina propria of the colon, the primary follicles of lymph nodes, and the brain gray matter. These are the first imaging data of an antiretroviral drug in active viral reservoirs

A Recombinant Attenuated Mycobacterium tuberculosis Vaccine Strain Is Safe in Immunosuppressed Simian Immunodeficiency Virus-Infected Infant Macaques

ABSTRACT Many resource-poor countries are faced with concurrent epidemics of AIDS and tuberculosis (TB) caused by human immunodeficiency virus (HIV) and Mycobacterium tuberculosis , respectively. Dual infections with HIV and M. tuberculosis are especially severe in infants. There is, however, no effective HIV vaccine, and the only licensed TB vaccine, the Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine, can cause disseminated mycobacterial disease in HIV-infected children. Thus, a pediatric vaccine to prevent HIV and M. tuberculosis infections is urgently needed. We hypothesized that a highly attenuated M. tuberculosis strain containing HIV antigens could be safely administered at birth and induce mucosal and systemic immune responses to protect against HIV and TB infections, and we rationalized that vaccine safety could be most rigorously assessed in immunocompromised hosts. Of three vaccine candidates tested, the recombinant attenuated M. tuberculosis strain mc 2 6435 carrying a simian immunodeficiency virus (SIV) Gag expression plasmid and harboring attenuations of genes critical for replication ( panCD and leuCD ) and immune evasion ( secA2 ), was found to be safe for oral or intradermal administration to non-SIV-infected and SIV-infected infant macaques. Safety was defined as the absence of clinical symptoms, a lack of histopathological changes indicative of M. tuberculosis infection, and a lack of mycobacterial dissemination. These data represent an important step in the development of novel TB vaccines and suggest that a combination recombinant attenuated M. tuberculosis -HIV vaccine could be a safe alternative to BCG for the pediatric population as a whole and, more importantly, for the extreme at-risk group of HIV-infected infants