scholarly journals Comprehensive Analysis of Nef Functions Selected in Simian Immunodeficiency Virus-Infected Macaques

2004 ◽  
Vol 78 (19) ◽  
pp. 10588-10597 ◽  
Author(s):  
Michael Schindler ◽  
Jan Münch ◽  
Matthias Brenner ◽  
Christiane Stahl-Hennig ◽  
Jacek Skowronski ◽  
...  
Keyword(s):  
Simian Immunodeficiency Virus ◽  
Rhesus Macaques ◽  
Surface Expression ◽  
Viral Fitness ◽  
Cell Surface Expression ◽  
Mhc I ◽  
Mhc Ii ◽  
Immunodeficiency Virus

ABSTRACT A variety of simian immunodeficiency virus (SIVmac) nef mutants have been investigated to clarify which in vitro Nef functions contribute to efficient viral replication and pathogenicity in rhesus macaques. Most of these nef alleles, however, were only functionally characterized for their ability to down-modulate CD4 and class I major histocompatibility complex (MHC-I) cell surface expression and to enhance SIV replication and infectivity. To obtain information on the in vivo relevance of more recently established Nef functions, we examined the ability of a large panel of constructed SIVmac Nef mutants and of variants that emerged in infected macaques to down-regulate CD3, CD28, and MHC-II and to up-regulate the MHC-II-associated invariant chain (Ii). We found that all these four Nef functions were restored in SIV-infected macaques. In most cases, however, the initial mutations and the changes selected in vivo affected several in vitro Nef functions. For example, truncated Nef proteins that emerged in animals infected with SIVmac239 containing a 152-bp deletion in nef efficiently modulated both CD3 and Ii surface expression. Overall, our results suggest that the effect of Nef on each of the six cellular receptors investigated contributes to viral fitness in the infected host but also indicate that modulation of CD3, MHC-I, MHC-II, or Ii surface expression alone is insufficient for SIV virulence.

scholarly journals Simian Immunodeficiency Virus Containing Mutations in N-Terminal Tyrosine Residues and in the PxxP Motif in Nef Replicates Efficiently in Rhesus Macaques

2000 ◽  
Vol 74 (9) ◽  
pp. 4155-4164 ◽  
Author(s):  
Silke Carl ◽  
A. John Iafrate ◽  
Sabine M. Lang ◽  
Nicole Stolte ◽  
Christiane Stahl-Hennig ◽  
...  
Keyword(s):  
Simian Immunodeficiency Virus ◽  
Rhesus Macaques ◽  
High Viral Load ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Tyrosine Residues ◽  
Immunodeficiency Virus ◽  
Endocytic Machinery

ABSTRACT SIVmac Nef contains two N-terminal tyrosines that were proposed to be part of an SH2-ligand domain and/or a tyrosine-based endocytosis signal and a putative SH3-ligand domain (P104xxP107). In the present study, we investigated the effects of combined mutations in these tyrosine and proline residues on simian immunodeficiency virus (SIV) Nef interactions with the cellular signal transduction and endocytic machinery. We found that mutation of Y28F, Y39F, P104A, and P107A (FFAA-Nef) had little effect on Nef functions such as the association with the cellular tyrosine kinase Src, downregulation of cell surface expression of CD4 and class I major histocompatibility complex, and enhancement of virion infectivity. However, mutations in the PxxP sequence reduced the ability of Nef to stimulate viral replication in primary lymphocytes. Three macaques infected with the SIVmac239 FFAA-Nef variant showed high viral loads during the acute phase of infection. Reversions in the mutated prolines were observed between 12 and 20 weeks postinfection. Importantly, reversion of A107→P, which restored the ability of Nef to coprecipitate a 62-kDa phosphoprotein in in vitro kinase assays, did not precede the development of a high viral load. The Y28/Y39→F28/F39substitutions did not revert. In conclusion, mutations in both the tyrosine residues and the putative SH3 ligand domain apparently do not disrupt major aspects of SIV Nef function in vivo.

scholarly journals T-Cell Receptor:CD3 Down-Regulation Is a Selected In Vivo Function of Simian Immunodeficiency Virus Nef but Is Not Sufficient for Effective Viral Replication in Rhesus Macaques

2002 ◽  
Vol 76 (23) ◽  
pp. 12360-12364 ◽  
Author(s):  
Jan Münch ◽  
Ajit Janardhan ◽  
Nicole Stolte ◽  
Christiane Stahl-Hennig ◽  
Peter ten Haaft ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Surface ◽  
Viral Replication ◽  
Simian Immunodeficiency Virus ◽  
Rhesus Macaques ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Unique Region ◽  
Immunodeficiency Virus

ABSTRACT We investigated the function of severely truncated simian immunodeficiency virus (SIV) Nef proteins (tNef) in vitro and in vivo. These variants emerged in rhesus monkeys infected with SIVmac239 containing a 152-bp deletion in the nef-unique region and have been suggested to enhance SIV virulence (E. T. Sawai, M. S. Hamza, M. Ye, K. E. Shaw, and P. A. Luciw, J. Virol. 74:2038-2045, 2000). We found that the tNef proteins were unable to down-regulate the cell surface expression of major histocompatibility complex class I proteins, CD4, and CD28 and neither stimulated SIV replication nor enhanced virion infectivity. The tNef proteins did efficiently down-regulate T-cell receptor (TCR):CD3 cell surface expression. Nevertheless, the SIVmac239 tnef variants were strongly attenuated in six infected juvenile rhesus macaques. Thus, while the ability of SIV Nef to down-modulate TCR:CD3 cell surface expression apparently confers a selective advantage in vivo, it is insufficient for efficient viral replication in infected macaques. Additional mutations elsewhere in SIVmac239 tnef genomes are required for a virulent phenotype.

scholarly journals Intrinsic Susceptibility of Rhesus Macaque Peripheral CD4+ T Cells to Simian Immunodeficiency Virus In Vitro Is Predictive of In Vivo Viral Replication

2000 ◽  
Vol 74 (20) ◽  
pp. 9388-9395 ◽  
Author(s):  
Simoy Goldstein ◽  
Charles R. Brown ◽  
Houman Dehghani ◽  
Jeffrey D. Lifson ◽  
Vanessa M. Hirsch
Keyword(s):  
T Cell ◽  
Simian Immunodeficiency Virus ◽  
Rank Order ◽  
Rhesus Macaques ◽  
Target Cells ◽  
Plasma Viremia ◽  
Immunodeficiency Virus ◽  
Siv Infection

ABSTRACT Previous studies with simian immunodeficiency virus (SIV) infection of rhesus macaques suggested that the intrinsic susceptibility of peripheral blood mononuclear cells (PBMC) to infection with SIV in vitro was predictive of relative viremia after SIV challenge. The present study was conducted to evaluate this parameter in a well-characterized cohort of six rhesus macaques selected for marked differences in susceptibility to SIV infection in vitro. Rank order relative susceptibility of PBMC to SIVsmE543-3-infection in vitro was maintained over a 1-year period of evaluation. Differential susceptibility of different donors was maintained in CD8+T-cell-depleted PBMC, macrophages, and CD4+ T-cell lines derived by transformation of PBMC with herpesvirus saimiri, suggesting that this phenomenon is an intrinsic property of CD4+target cells. Following intravenous infection of these macaques with SIVsmE543-3, we observed a wide range in plasma viremia which followed the same rank order as the relative susceptibility established by in vitro studies. A significant correlation was observed between plasma viremia at 2 and 8 weeks postinoculation and in vitro susceptibility (P < 0.05). The observation that the two most susceptible macaques were seropositive for simian T-lymphotropic virus type 1 may suggests a role for this viral infection in enhancing susceptibility to SIV infection in vitro and in vivo. In summary, intrinsic susceptibility of CD4+ target cells appears to be an important factor influencing early virus replication patterns in vivo that should be considered in the design and interpretation of vaccine studies using the SIV/macaque model.

scholarly journals Effect of the Attenuating Deletion and of Sequence Alterations Evolving In Vivo on Simian Immunodeficiency Virus C8-Nef Function

1999 ◽  
Vol 73 (4) ◽  
pp. 2790-2797 ◽  
Author(s):  
Silke Carl ◽  
A. John Iafrate ◽  
Jacek Skowronski ◽  
Christiane Stahl-Hennig ◽  
Frank Kirchhoff
Keyword(s):  
Viral Replication ◽  
Simian Immunodeficiency Virus ◽  
Point Mutations ◽  
Residual Activity ◽  
Intermediate Phenotype ◽  
Cell Surface Expression ◽  
In Vitro Assays ◽  
Immunodeficiency Virus

ABSTRACT The simian immunodeficiency virus macC8 (SIVmacC8) variant has been used in a European Community Concerted Action project to study the efficacy and safety of live attenuated SIV vaccines in a large number of macaques. The attenuating deletion in the SIVmacC8nef-long terminal repeat region encompasses only 12 bp and is “repaired” in a subset of infected animals. It is unknown whether C8-Nef retains some activity. Since it seems important to use only well-characterized deletion mutants in live attenuated vaccine studies, we analyzed the relevance of the deletion, and the duplications and point mutations selected in infected macaques for Nef function in vitro. The deletion, affecting amino acids 143 to 146 (DMYL), resulted in a dramatic decrease in Nef stability and function. The initial 12-bp duplication resulted in efficient Nef expression and an intermediate phenotype in infectivity assays, but it did not significantly restore the ability of Nef to stimulate viral replication and to downmodulate CD4 and class I major histocompatibility complex cell surface expression. The additional substitutions however, which subsequently evolved in vivo, gradually restored these Nef functions. It was noteworthy that coinfection experiments in the T-lymphoid 221 cell line revealed that even SIVmac nef variants carrying the original 12-bp deletion readily outgrew an otherwise isogenic virus containing a 182-bp deletion in the nef gene. Thus, although C8-Nef is unstable and severely impaired in in vitro assays, it maintains some residual activity to stimulate viral replication.

scholarly journals A novel lineage-tracing mouse model for MmuPV1 infection enables in vivo studies in the absence of cytopathic effects

2021 ◽  
Author(s):  
Vural Yilmaz ◽  
Panayiota Louca ◽  
Katerina Strati
Keyword(s):  
Mouse Model ◽  
Laboratory Mouse ◽  
Surface Expression ◽  
Lineage Tracing ◽  
Human Papillomaviruses ◽  
In Vivo Studies ◽  
Cell Surface Expression ◽  
Mhc I

Human papillomaviruses (HPVs) are DNA viruses that ubiquitously infect humans and have been associated with hyperproliferative lesions. The recently discovered mouse specific papillomavirus, MmuPV1, provides the opportunity to study papillomavirus infections in vivo in the context of a common laboratory mouse model (Mus musculus). To date, a major challenge in the field has been the lack of tools to identify, observe and characterize individually the papillomavirus hosting cells and also trace the progeny of these cells over time. Here, we present the successful generation of an in vivo lineage-tracing model of MmuPV1-harboring cells and their progeny by means of genetic reporter activation. Following the validation of the system both in vitro and in vivo, we used it to provide a proof-of-concept of its utility. Using flow-cytometry analysis, we observed increased proliferation dynamics and decreased MHC-I cell surface expression in MmuPV1-treated tissues which could have implications in tissue regenerative capacity and ability to clear the virus. This model is a novel tool to study the biology of the MmuPV1 host-pathogen interactions.

scholarly journals Importance of the N-Distal AP-2 Binding Element in Nef for Simian Immunodeficiency Virus Replication and Pathogenicity in Rhesus Macaques

2006 ◽  
Vol 80 (9) ◽  
pp. 4469-4481 ◽  
Author(s):  
Matthias Brenner ◽  
Jan Münch ◽  
Michael Schindler ◽  
Steffen Wildum ◽  
Nicole Stolte ◽  
...  
Keyword(s):  
Viral Replication ◽  
Rhesus Macaques ◽  
Point Mutations ◽  
Mhc I ◽  
Viral Loads ◽  
Mhc Ii ◽  
Histocompatibility Complex ◽  
Immunodeficiency Virus ◽  
Clathrin Adaptor

ABSTRACT Point mutations in SIVmac239 Nef disrupting CD4 downmodulation and enhancement of virion infectivity attenuate viral replication in acutely infected rhesus macaques, but changes selected later in infection fully restore Nef function (A. J. Iafrate et al., J. Virol. 74:9836-9844, 2000). To further evaluate the relevance of these Nef functions for viral persistence and disease progression, we analyzed an SIVmac239 Nef mutant containing a deletion of amino acids Q64 to N67 (Δ64-67Nef). This mutation inactivates the N-distal AP-2 clathrin adaptor binding element and disrupts the abilities of Nef to downregulate CD4, CD28 and CXCR4 and to stimulate viral replication in vitro. However, it does not impair the downmodulation of CD3 and class I major histocompatibility complex (MHC-I) or MHC-II and the upregulation of the MHC-II-associated invariant chain, and it has only a moderate effect on the enhancement of virion infectivity. Replication of the Δ64-67Nef variant in acutely infected macaques was intermediate between grossly nef-deleted and wild-type SIVmac239. Subsequently, three of six macaques developed moderate to high viral loads and developed disease, whereas the remaining animals efficiently controlled SIV replication and showed a more attenuated clinical course of infection. Sequence analysis revealed that the deletion in nef was not repaired in any of these animals. However, some changes that slightly enhanced the ability of Nef to downmodulate CD4 and moderately increased Nef-mediated enhancement of viral replication and infectivity in vitro were observed in macaques developing high viral loads. Our results imply that both the Nef functions that were disrupted by the Δ64-67 mutation and the activities that remained intact contribute to viral pathogenicity.

scholarly journals A Marine Diterpenoid Modulates the Proteasome Activity in Murine Macrophages Stimulated with LPS

Biomolecules ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 109 ◽  
Author(s):  
Yisett González ◽  
Deborah Doens ◽  
Héctor Cruz ◽  
Ricardo Santamaría ◽  
Marcelino Gutiérrez ◽  
...  
Keyword(s):  
Ubiquitin Proteasome System ◽  
Surface Expression ◽  
Nuclear Factor Κb ◽  
Cell Surface Expression ◽  
Mhc I ◽  
Docking Simulations ◽  
Murine Macrophages ◽  
Future Evaluation

The proteasome is an intracellular complex that degrades damaged or unfolded proteins and participates in the regulation of several processes. The immunoproteasome is a specialized form that is expressed in response to proinflammatory signals and is particularly abundant in immune cells. In a previous work, we found an anti-inflammatory effect in a diterpenoid extracted from the octocoral Pseudopterogorgia acerosa, here called compound 1. This compound prevented the degradation of inhibitor κB α (IκBα) and the subsequent activation of nuclear factor κB (NFκB), suggesting that this effect might be due to inhibition of the ubiquitin-proteasome system. Here we show that compound 1 inhibits the proteasomal chymotrypsin-like activity (CTL) of murine macrophages in the presence of lipopolysaccharide (LPS) but not in its absence. This effect might be due to the capacity of this compound to inhibit the activity of purified immunoproteasome. The compound inhibits the cell surface expression of major histocompatibility complex (MHC)-I molecules and the production of proinflammatory cytokines induced by LPS in vitro and in vivo, respectively. Molecular docking simulations predicted that compound 1 selectively binds to the catalytic site of immunoproteasome subunits β1i and β5i, which are responsible for the CTL activity. Taken together these findings suggest that the compound could be a selective inhibitor of the immunoproteasome, and hence could pave the way for its future evaluation as a candidate for the treatment of inflammatory disorders and autoimmune diseases.

scholarly journals Simian Immunodeficiency Virus in Which nef and U3 Sequences Do Not Overlap Replicates Efficiently In Vitro and In Vivo in Rhesus Macaques

2001 ◽  
Vol 75 (17) ◽  
pp. 8137-8146 ◽  
Author(s):  
Jan Münch ◽  
Nadia Adam ◽  
Nathaly Finze ◽  
Nicole Stolte ◽  
Christiane Stahl-Hennig ◽  
...  
Keyword(s):  
Simian Immunodeficiency Virus ◽  
Rhesus Macaques ◽  
Point Mutations ◽  
Regulatory Elements ◽  
Coding Sequences ◽  
Reading Frame ◽  
Immunodeficiency Virus ◽  
Primate Lentiviruses

ABSTRACT The nef genes of human immunodeficiency virus and simian immunodeficiency virus (SIV) overlap about 80% of the U3 region of the 3′ long terminal repeat (LTR) and contain several essentialcis-acting elements (here referred to as the TPI region): a T-rich region, the polypurine tract, and attachment (att) sequences required for integration. We inactivated the TPI region in the nef reading frame of the pathogenic SIVmac239 clone (239wt) by 13 silent point mutations. To restore viral infectivity, intact cis-regulatory elements were inserted just downstream of the mutatednef gene. The resulting SIV genome contains U3 regions that are 384 bp shorter than the 517-bp 239wt U3 region. Overall, elimination of the duplicated Nef coding sequences truncates the proviral genome by 350 bp. Nonetheless, it contains all known coding sequences and cis-acting elements. The TPI mutant virus expressed functional Nef and replicated like 239wt in all cell culture assays and in vivo in rhesus macaques. Notably, these SIVmac constructs allow us to study Nef function in the context of replication-competent viruses without the restrictions of overlapping LTR sequences and important cis-acting elements. The genomes of all known primate lentiviruses contain a large overlap between nefand the U3 region. We demonstrate that this conserved genomic organization is not obligatory for efficient viral replication and pathogenicity.

scholarly journals Downregulation of cell-surface CD4 expression by simian immunodeficiency virus Nef prevents viral super infection.

1993 ◽  
Vol 177 (6) ◽  
pp. 1561-1566 ◽  
Author(s):  
R E Benson ◽  
A Sanfridson ◽  
J S Ottinger ◽  
C Doyle ◽  
B R Cullen
Keyword(s):  
Cell Surface ◽  
Simian Immunodeficiency Virus ◽  
State Level ◽  
Surface Expression ◽  
Biologically Active ◽  
Cell Surface Expression ◽  
Viral Spread ◽  
Immunodeficiency Virus ◽  
Hiv 1

The nef gene product encoded by the mac239 proviral clone of simian immunodeficiency virus (SIV) markedly enhances viral replication and pathogenesis in vivo. We have used this biologically active nef isolate to examine the phenotype of Nef in retrovirally transduced human T cells in culture. SIV Nef is shown to dramatically inhibit cell-surface expression of the CD4 glycoprotein without significantly affecting the total steady-state level of cellular CD4. This downregulation of the cell-surface CD4 receptor for human immunodeficiency virus type 1 (HIV-1) infection correlated with the acquisition of resistance to superinfection by HIV-1. However, SIV Nef did not affect the level of gene expression directed by the HIV-1 long terminal repeat. It is hypothesized that downregulation of cell-surface CD4 by Nef facilitates the efficient release of infectious progeny virions and, hence, viral spread in vivo.

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