Evolutionary Modeling of Rate Shifts Reveals Specificity Determinants in HIV-1 Subtypes

A hallmark of the human immunodeficiency virus 1 (HIV-1) is its rapid rate of evolution within and among its various subtypes. Two complementary hypotheses are suggested to explain the sequence variability among HIV-1 subtypes. The first suggests that the functional constraints at each site remain the same across all subtypes, and the differences among subtypes are a direct reflection of random substitutions, which have occurred during the time elapsed since their divergence. The alternative hypothesis suggests that the functional constraints themselves have evolved, and thus sequence differences among subtypes in some sites reflect shifts in function. To determine the contribution of each of these two alternatives to HIV-1 subtype evolution, we have developed a novel Bayesian method for testing and detecting site-specific rate shifts. The RAte Shift EstimatoR (RASER) method determines whether or not site-specific functional shifts characterize the evolution of a protein and, if so, points to the specific sites and lineages in which these shifts have most likely occurred. Applying RASER to a dataset composed of large samples of HIV-1 sequences from different group M subtypes, we reveal rampant evolutionary shifts throughout the HIV-1 proteome. Most of these rate shifts have occurred during the divergence of the major subtypes, establishing that subtype divergence occurred together with functional diversification. We report further evidence for the emergence of a new sub-subtype, characterized by abundant rate-shifting sites. When focusing on the rate-shifting sites detected, we find that many are associated with known function relating to viral life cycle and drug resistance. Finally, we discuss mechanisms of covariation of rate-shifting sites

Identification and characterization of a human herpesvirus 6 gene segment capable of transactivating the human immunodeficiency virus type 1 long terminal repeat in an Sp1 binding site-dependent manner.

The human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) is transactivated by various extracellular signals and viral cofactors that include human herpesviruses. These transactivators are capable of transactivating the HIV-1 LTR through the transactivation response element, NF-kappa B, or other regulatory binding elements. Human herpesvirus 6 (HHV-6) is a potential cofactor of HIV-1. Here, we report that an HHV-6 gene segment, ZVH14, which can neoplastically transform NIH 3T3 and human keratinocytes, is capable of transactivating HIV-1 LTR chloramphenicol acetyltransferase constructs in an Sp1 binding site-dependent manner. Transactivation increased synergistically in the presence of multiple Sp1 sites and was dramatically reduced by cotransfection with oligomers designed to form triplex structures with HIV-1 LTR Sp1 binding sites. HIV-1 LTR NF-kappa B sites were not essential for ZVH14-mediated transactivation. A putative open reading frame in ZVH14, B115, which may encode a highly basic peptide consisting of 115 amino acid residues, showed transactivation capacity similar to that of ZVH14. This open reading frame also transactivated the HIV-1 LTR in an Sp1 site-dependent fashion in African green monkey kidney cells and human T cells. These data suggest that HHV-6 may stimulate HIV-1 replication via transactivation of Sp1 binding sites present in the HIV-1 promoter

Comparison of drug and cell-based delivery: engineered adult mesenchymal stem cells expressing soluble tumor necrosis factor receptor II prevent arthritis in mouse and rat animal models

Rheumatoid arthritis (RA) is a systemic autoimmune disease with unknown etiology where tumor necrosis factor-alpha (TNFα) plays a critical role. Etanercept®, the biologic drug form of soluble tumor necrosis factor receptor-II (sTNFRII) is used to treat RA based on the rational that sTNFRII binds TNFα and blocks inflammation. We compared and benchmarked sTNFRII protein delivery from genetically engineered mesenchymal stem cells (MSCs) to Etanercept®. Blocking TNFα-dependent ICAM-1 surface expression on transduced human MSCs and culture media inhibition of nitric oxide production from ΤΝFα−treated bovine chondrocytes showed functionality of the sTNFRII construction. Implanted TNFRII-transduced MSCs removed mouse serum circulating TNFα generated from either implanted TNFα expressing cells or lipopolysaccharide (LPS) induction better than Etanercept® (TNFα, 100%; IL1α, 90%; and IL6, 60% within 6 hrs.), suggesting faster clearance of the sTNFR:TNFα complex from the animals. In vivo efficacy of sTNFRII-transduced MSCs was illustrated in two (immune-deficient and immune-competent) arthritic rodent models. In the BalbC/SCID mouse antibody-induced arthritis (AbIA) model, intramuscular injection of sTNFR transduced human MSCs reduced joint inflammation by 90% compared to untransduced human MSCs; in the antigen-induced arthritis (AIA) Fisher rat model, both sTNFR transduced rat MSCs and Etanercept® inhibited joint inflammation by 30%. In vitro chondrogenesis assays showed the ability of TNFα and IL1α, but not IFNγ to inhibit human MSC differentiation to chondrocytes, illustrating an additional negative role for inflammatory cytokines in joint repair. The data supports the utility of human MSCs as therapeutic gene delivery vehicles and their potential used in alleviating inflammation within the arthritic joint