Jonathan Silver

HIV-associated nephropathy in transgenic mice expressing HIV-1 genes

Transgenic mice were produced that bore copies of a defective HIV provirus. The transgenic offspring from three independently derived mouse lines manifested renal disease associated with proteinuria, a high mortality rate, and HIV-specific gene expression in the kidney. An early histopathological lesion in the kidney was focal glomerulosclerosis. Moribund animals had diffuse glomerulosclerosis with prominent microcystic tubular dilatation, tubular epithelial degeneration, and interstitial nephritis. Electron microscopy revealed ultrastructural features consistent with the glomerulosclerosis: effacement of the foot processes of visceral epithelium and an increase in mesangial cell matrix. Transgenic mice variably expressed 6-, 4.3-, and 2-kb HIV-specific RNAs and HIV-related polypeptides in several tissues including kidney. Immunocytostaining revealed the presence of HIV-related protein in the glomeruli of affected animals. Glomerulopathy in these transgenic mice and HIV-associated nephropathy in man have similar features.

The mouse homolog of the Gibbon ape leukemia virus receptor: Genetic mapping and a possible receptor function in rodents

The mouse homolog of the Gibbon ape leukemia virus (GALV) receptor (Glvr-1) was mapped to mouse Chromosome 2 (Chr 2) by Southern blot analysis of somatic cell hybrids and positioned on this chromosome using an interspecies genetic cross. Mouse Chr 2 also encodes a receptor (Rec-2) for the wild mouse virus M813. To investigate whether Glvr-1 and Rec-2 could be the same gene, we sought evidence for sequence homology between the env- genes of their respective viruses. Southern blot hybridization with GALV-derived env and pol-env probes failed to detect any homology between GALV and M813, but did show that all mouse species tested carry numerous copies of GALV-related sequences. We speculate that a functional receptor for GALV-related viruses was expressed during Mus evolution.

Statistical Equilibrium Wealth Distributions in an Exchange Economy with Stochastic Preferences

Abstract We describe an exchange market consisting of many agents with stochastic preferences for two goods. When individuals are indifferent between goods, statistical mechanics predicts that goods and wealth will have steady-state gamma distributions. Simulation studies show that gamma distributions arise for a broader class of preference distributions. We demonstrate this mathematically in the limit of large numbers of individual agents. These studies illustrate the potential power of a statistical mechanical approach to stochastic models in economics and suggest that gamma distributions will describe steady-state wealths for a class of stochastic models with periodic redistribution of conserved quantities. Journal of Economic Literature Classification Numbers: C15, C62, C73, D3, D5.

A syncytia assay for human immunodeficiency virus type I(H IV-I) envelope protein and its use in studying HIV-I mutations

We describe a syncytia assay that utilizes a noninfectious plasmid (pEVd1443) derived from human immunodeficiency virus type I (HIV-I). This plasmid carries a large deletion of gag-pol sequences but expresses HIV-I envelope proteins and induces syncytia following transfection into HeLa-CD4 cells. This plasmid was used to study the effects of mutations in the gp 120 and gp41 portions of the envelope gene of HIV-I.

Asymmetric requirement for cholesterol in receptor-bearing but not envelope-bearing membranes for fusion mediated by ecotropic murine leukemia virus.

ABSTRACT We show that fusion mediated by ecotropic murine leukemia virus envelope is dependent on cholesterol in receptor-bearing membranes. The effect is >10 times larger in insect cells than mammalian cells, probably because the former can be more extensively depleted of cholesterol. The fact that cholesterol is apparently not needed in envelope-bearing membranes suggests that it plays a role in an asymmetric step in membrane fusion and argues against a class of models in which cholesterol is important in symmetric fusion intermediates. The insect cell system has promise for clarifying the role of membrane rafts in other aspects of cell physiology.

Internal strain drives spontaneous periodic buckling in collagen and regulates remodeling

Significance Collagen fibrils resemble nanoscale cables that self-assemble and constitute the most prevalent protein structure in the body. Our experiments reveal unanticipated defects that form along collagen fibrils. These defects are the initiation sites of collagenase activity and represent a strain-sensitive mechanism for regulating tissue remodeling. The emergence of defects, their spatial periodicity, and fluctuations are quantitatively accounted for with a buckling model in which defects spontaneously form, repulsively interact, and self-heal. Fibrillar collagen, an essential structural component of the extracellular matrix, is remarkably resistant to proteolysis, requiring specialized matrix metalloproteinases (MMPs) to initiate its remodeling. In the context of native fibrils, remodeling is poorly understood; MMPs have limited access to cleavage sites and are inhibited by tension on the fibril. Here, single-molecule recordings of fluorescently labeled MMPs reveal cleavage-vulnerable binding regions arrayed periodically at ∼1-µm intervals along collagen fibrils. Binding regions remain periodic even as they migrate on the fibril, indicating a collective process of thermally activated and self-healing defect formation. An internal strain relief model involving reversible structural rearrangements quantitatively reproduces the observed spatial patterning and fluctuations of defects and provides a mechanism for tension-dependent stabilization of fibrillar collagen. This work identifies internal–strain-driven defects that may have general and widespread regulatory functions in self-assembled biological filaments.

Detection and quantitation of hepatitis C virus RNA in serum using the polymerase chain reaction and a colorimetric enzymatic detection system.

A sensitive, non-isotopic method for detecting and quantifying hepatitis C virus (HCV) RNA in serum using the reverse transcriptase-polymerase chain reaction (RT-PCR) and a hybridization specific, colorimetric biotin-avidin peroxidase detection system has been developed. The sensitivity of the PCR-colorimetric system was determined using RNA synthesized from cloned HCV cDNA. The assay could detect as few as 10 molecules of HCV RNA, comparable to the sensitivity achieved with double PCR using nested primers. Thus, this colorimetric assay can detect low levels of HCV RNA in serum and appears to be quantitative, suggesting that this technique may be applied to rapid screening of large numbers of samples and to monitor the effect of antiviral therapy.

Role of a conserved amino-terminal sequence in the ecotropic MLV receptor mCAT1

The amino-terminus of mCAT1 and homologous proteins is predicted to form a positively charged, amphipathic alpha helix on the cytoplasmic side of the plasma membrane. Peptides with similar sequence motifs often provide membrane anchors, protein-protein interaction domains, or intracellular transport-targeting signals. Deleting most of the cytoplasmic N-terminal sequence of mCAT1 led to reduced expression on the cell surface and accumulation in the endoplasmic reticulum but did not abrogate receptor function. Surprisingly, when the N-terminal 36 or 18 amino acids of mCAT1 were fused to green fluorescent protein (gfp), gfp accumulated almost exclusively in mitochondria. Mitochondrial targeting depended on arginines at positions 15 and 16 and was inhibitable by downstream transmembrane sequences. Although the full-length mCAT1 was not detected in mitochondria, the mitochondrial-targeting property of the N-terminal sequence fused to gfp is conserved in orthologous and paralogous proteins that diverged approximately 80 million years ago, suggesting a conserved biological function. We propose that the conserved N-terminal motif of CAT proteins provides a regulatable signal for transport to, or retention in, different cell membrane compartments.

Secretion of a murine retroviral Env associated with resistance to infection.

Fv4 is an endogenous defective murine leukaemia virus (MuLV) which expresses high levels of an envelope protein (Env) closely related to that of the ecotropic class of MuLVs. Mice bearing the natural Fv4 gene or a transgenic version are resistant to infection by ecotropic MuLVs. Fv4 mice secrete the surface peptide (SU) of the Fv4 Env in their serum and this secreted Env can block infection of NIH3T3 cells. To study the secretion of Fv4, we metabolically labelled cells expressing Fv4 Env or Env from infectious MuLVs and followed synthesis, glycosylation, proteolytic processing and secretion of Env species. We found no difference in the kinetics of synthesis or processing of Fv4 Env compared to the envelopes of infectious MuLVs, but Fv4 Env associated more weakly with its transmembrane anchor and was shed from the surface of cells.

The transmission and activation of endogenous mouse retroviral genomes

Abstract Proviral copies of the mouse leukemia viruses behave as classical Mendelian loci and as mobile genetic elements. As mobile elements they can reinsert in somatic and germ cells; these insertions may lead to cancer or heritable mutations. The mobility of these proviral elements results in great diversity among mice in the number and chromosomal position of proviral genes. We discuss factors which may influenced the rate of acquisition of novel proviruses in the germ line.