Peter Ventura

A Trojan Horse mechanism for the spread of visna virus in monocytes

Visna virus is the prototype of the lentivirus subfamily of retroviruses that cause slow infections of sheep and goats. These viruses persist and can be isolated from blood and cerebrospinal fluid for years despite neutralizing antibody. In the studies reported here we have used quantitative in situ hybridization to analyze infected leukocytes. We show that (1) monocytes harbor the visna genome; and (2) virus gene expression is as constrained in this cell as it is in glial and epithelial cells. These results are in accord with a Trojan Horse mechanism of virus dissemination in an immunologically responsive host.

Visna DNA synthesis and the tempo of infection in vitro

Visna virus is the prototype of the subfamily of nontransforming retroviruses that cause slow infections in vivo, and lytic infections in vitro. In this paper we present the first quantitative single cell analysis of the synthesis of viral nucleic acids, using improved methods of in situ hybridization; we identify early visna virus DNA synthesis as the rate-limiting step in transcription, virus production, and cell death in vitro; and we show that by manipulating the extent of early DNA synthesis we can slow the tempo of infection in vitro from 3 days to 3 weeks.

Visna virus DNA: Discovery of a novel gapped structure

Abstract We have analyzed the structure of visna virus DNA synthesized during the life cycle of this lytic virus in confluent cultures of sheep choroid plexus cells. Viral DNA is of predominantly two forms: A linear duplex of about 6 million daltons corresponding to a transcript of viral RNA of subunit size; and, a structure with a gap in the center comprised of a full-length minus strand, and two long plus strands that together are about 300–500 bases shorter than the minus strand. A physical map of the linear DNA derived with restriction enzymes was ordered with respect to viral RNA, and the DNA was shown to bear terminal repeats of 350–450 nucleotides. Two circular forms of visna DNA differing only by the length of one terminal repeat also were detected, but constituted a very minor population. We discuss the relationship of these observations with this prototype of the lentiviruses to those recorded for transforming retroviruses.

Quantitative analysis of visna virus replication in vivo.

Visna virus is the prototype of the lentivirus subfamily, a group of nontransforming retroviruses that cause slow infections in sheep and goats. In nature, virus is acquired primarily by the respiratory route and subsequently spreads to several organ systems. These viruses persist for years in their hosts despite a vigorous immune response because of a block in virus gene expression. This report continues the analysis of persistence in vivo, and specifically examines a gene dosage hypothesis that has been advanced as an explanation for the decrease in transcription and virus production in the cells in infected animals. For this analysis a new pulmonary model has been developed that, in conjunction with quantitative in situ hybridization, provides an opportunity to examine in animals the molecular events that occur in the course of the viral life cycle. We establish the feasibility of such a longitudinal analysis in vivo, document restriction in gene expression in alveolar macrophages and provide evidence that this restriction cannot be accounted for simply by gene dosage. The approach illustrated with visna should be of general applicability to other dynamic and molecular investigations of virus infection.

Detection by Hybridization of Viral Infection of the Human Central Nervous System

Studies of animal models of demyelination and investigation of the epidemiology of multiple sclerosis (MS) both point to the possibility of virus infection as one inciting factor in the disease, but until recently there has been little evidence for the central prediction of this hypothesis, persistence of viruses in the human brain. Advances in technology, particularly in methods for detecting virus genes by hybridization, have now provided us with our first glimpse of an indigenous viral flora of the brain, and

Combined macroscopic and microscopic detection of viral genes in tissues

A hybridization technique has been devised for detecting and quantitating viral genes in tissues that combines macroscopic and microscopic analyses in the same section. The method is based on dual labeling virus-specific probes with 125I and 35S to generate signals that can be detected both with X-ray films and nuclear track emulsions. The regions of increased hybridization evident in the X-ray film serve as a guide to the portion of the section that warrants microscopic examination. Detection of viral RNA in tissues with visna virus and viral DNA with hepatitis B virus are illustrated, and potential applications of this technique in virology and other disciplines are discussed.

Infectivity of visna virus DNA

Abstract Deoxyribonucleic acid isolated from cells infected with the RNA containing slow virus visna confers on uninfected cells the capacity to synthesize new virus. The cytopathic agent isolated in these experiments has the biological and biochemical properties of visna virus, and the active material is DNA by several criteria, the most important of which is the loss of infectivity with prior treatment of the DNA with deoxyribonuclease. The efficiency of infection with DNA is markedly enhanced by presenting DNA to the cell as a calcium DNA phosphate precipitate. Because of the lytic nature of visna virus infection, the infectivity of visna DNA can be assayed quantitatively by plaque formation. Our major findings with this infectivity assay include the following: (1) Duplex DNA is infectious while denatured DNA is not. (2) At least one complete copy of proviral DNA is integrated in the host genome. (3) The minimal infective size of duplex DNA corresponds to a transcript of one subunit of viral RNA. (4) However, the concentration dependence of infectivity is two hit over the size range of 10–30 million molecular weight. These findings suggest that the unique genetic information of the viral genome is distributed over more than one subunit and that the proviral DNA transcripts are unlinked in the cell. The usefulness of the DNA infectivity assay and the implications of the visna provirus for persistent viral infections are discussed.