Katherine Staskus

Quantitative Image Analysis of HIV-1 Infection in Lymphoid Tissue

Tracking human immunodeficiency virus-type 1 (HIV-1) infection at the cellular level in tissue reservoirs provides opportunities to better understand the pathogenesis of infection and to rationally design and monitor therapy. A quantitative technique was developed to determine viral burden in two important cellular compartments in lymphoid tissues. Image analysis and in situ hybridization were combined to show that in the presymptomatic stages of infection there is a large, relatively stable pool of virions on the surfaces of follicular dendritic cells and a smaller pool of productively infected cells. Despite evidence of constraints on HIV-1 replication in the infected cell population in lymphoid tissues, estimates of the numbers of these cells and the virus they could produce are consistent with the quantities of virus that have been detected in the bloodstream. The cellular sources of virus production and storage in lymphoid tissues can now be studied with this approach over the course of infection and treatment.

Nucleotide sequence of the visna lentivirus: relationship to the AIDS virus

We have determined the complete 9202 nucleotide sequence of the visna lentivirus. The deduced genetic organization most closely resembles that of the AIDS retrovirus in that there is a novel central region separating pol and env. Moreover, there is a close phylogenetic relationship between the conserved reverse transcriptase and endonuclease/integrase domains of the visna and AIDS viruses. These findings support the inclusion of the AIDS virus in the retroviral subfamily Lentivirinae.

In situ amplification of visna virus DNA in tissue sections reveals a reservoir of latently infected cells.

Maedi and visna are, respectively, the pulmonary and neurological manifestations of slowly progressive infections of sheep caused by retroviruses of the lentivirus subfamily. Lentivirus infections are also persistent infections in which host defenses are generally not successful in eliminating the infectious agent because of restricted viral gene expression in many infected cells. In this report, we describe a method for amplifying and detecting viral DNA in tissue sections which has made it possible to verify experimentally the postulated existence of this reservoir of latently infected cells, as well as to estimate the actual number of cells which harbor viral genomes in infected tissues. In the discussion, we present a simple mathematical model that relates this number to the rate at which inflammatory lesions develop. This model can account for both the slow progression of natural infections and for the rapid accumulation of inflammatory foci in the high dosage experimental system analysed in our studies.

A Pilot Study of Cidofovir in Patients with Kaposi Sarcoma

A clinical trial was conducted to test the activity of cidofovir (CDV), a drug with in vitro activity against Kaposi sarcoma (KS)-associated herpesvirus (KSHV), in KS. Five patients with human immunodeficiency virus-associated KS (4 receiving antiretroviral therapy) and 2 patients with classical KS were administered CDV (5 mg/kg/dose) weekly for 2 weeks and then every other week. All 7 patients had progression of their KS at a median of 8.1 weeks (range, 5-27 weeks). Skin biopsy specimens of KS lesions showed no change in expression of latent or early lytic genes, but, in the 1 assessable patient, there was decreased expression of a late lytic gene. There was no decrease in the virus load of KSHV in peripheral blood mononuclear cells. This study does not provide proof of principle for the treatment of KS with CDV. However, it remains possible that antiherpesvirus therapy can be developed for herpes-induced tumors.

Isolation of replication-competent molecular clones of visna virus

Visna virus is the prototypic member of a subfamily of retroviruses responsible for slow infections of animals and humans. As a part of our investigation of the functions of viral gene products in virus replication, we have isolated three infectious molecular clones and determined the complete nucleotide sequences of two of the clones. We have also characterized the progeny of the biologically cloned viral stocks and of the infectious clones and document considerable heterogeneity in plaque size and antigenic phenotype of the former that is reduced to near homogeneity in the progeny of the infectious clones. It thus should now be possible to trace the emergence of antigenic variants of visna virus as well as ascribe defined functions to structural and regulatory genes of the virus in determining neurovirulence and the slow tempo of infection.

The molecular pathogenesis of astrogliosis in scrapie and Alzheimer's disease

In slow infections caused by scrapie and other unconventional agents, and in Alzheimers disease (AD), the formation of neuritic plaques and the increase in astrocytes and astrocyte-specific protein, glial fibrillary acidic protein (GFAP), are pathological changes common to both conditions. With the rationale that these parallels imply convergent pathogenetic mechanisms, we identified a gene whose expression increases in both. We now report the results of a more extensive analysis of this gene and show that by sequence analysis it is highly homologous and likely identical to GFAP. GFAP mRNA accumulates late in the course of scrapie in subpial and periventricular astrocytes and in cells in foci in the hippocampus. The increased abundance of GFAP mRNA is accompanied by an increase in the corresponding protein. GFAP mRNA is localized by in situ hybridization to the cell body and processes of astrocytes. In AD, the latter pattern predominates, consistent with induction of GFAP mRNA in the sites of synthesis in glial processes in the neuritic plaque.

Initiation of DNA synthesis by the avian oncornavirus RNA-directed DNA polymerase: Structural and functional localization of the major species of primer RNA on the oncornavirus genome

Abstract We have both structurally and functionally localized the site on the avian oncornavirus RNA genome at which the major species of primer RNA is bound. Localization of the primer site involved either determining the minimum size class of poly(A)-containing fragments of the viral RNA genome to which radiolabeled primer RNA would anneal, or determining the minimum size class of poly(A)-containing fragments that could be reconstituted as an active template primer complex for the RNA-directed DNA polymerase subsequent to reannealing to unlabeled purified primer RNA. Both approaches indicate that the primer site appears to be very close to (within 10%), or at the 5′ end of the 35 S RNA genome. The implications of these findings on the transcription of the oncornavirus genome in vitro and in vivo are discussed.

Persistence of Kaposi Sarcoma-Associated Herpesvirus (KSHV)—Infected Cells in KSHV/HIV-1-Coinfected Subjects without KSHV-Associated Diseases

Inguinal lymph nodes from 24 human immunodeficiency virus (HIV) type 1-infected subjects without Kaposi sarcoma-associated herpesvirus (KSHV)-associated diseases were examined for KSHV infection. KSHV-infected cells were detected at a very low frequency in the lymph nodes of 7 subjects (median frequency, 2 infected cells/10(7) lymph node cells). Latent, but not lytic, KSHV gene expression was detected and KSHV-infected cells were located in B cell-rich areas of lymph node follicles. These findings provide evidence that, in the absence of KSHV-associated diseases, latent infection of lymph node cells provides a mechanism for the persistence of KSHV in KSHV/HIV-1-coinfected persons.

Mammalian mRNAs encoding protein closely related to ubiquitin-conjugating enzyme encoded by yeast DNA repair gene RAD6

A clone of about 1 kb has been isolated from a human brain cDNA library. The clone possesses a 151 amino acid open reading frame that exhibits 72% amino acid identity with the E2 ubiquitin-conjugating enzyme encoded by the RAD6 gene of Saccharomyces cerevisiae. A 90% amino acid identity was observed in a central sequence surrounding a cysteine, which most likely contributes the sulfhydryl group involved in the formation of the ubiquitin-E2 thiolester linkage. Northern hybridization analyses have identified a poly(A)-containing mRNA of about 1 kb encoding the E2-like sequence in human CEM lymphoblastoid and HeLa cells, Novikoff rat hepatoma cells and S49 mouse leukemia cells. Southern hybridization analyses indicate the presence of a single gene encoding this sequence in both human cell lines, but of two or more related genes in the rodent cell lines.

PCR Amplification of Viral DNA and Viral Host Cell mRNAs in Situ

The hallmark and power of in situ hybridization methodology are its ability to determine the levels of gene expression in an individual cell in a population or determine which cells in a population have acquired new genes by infection or other processes. In this way one can gain insight into how levels of gene expression can vary within a population with the same complement of genes, how different genes within a cell are coordinately regulated, and how newly introduced genes are regulated and interact with genes in the host cell. In contrast to other techniques that measure the average number of molecules per cell in the population, in situ analyses measure the number of molecules in a particular cell in a particular spatial and temporal context. With double-labeling techniques, one can also appreciate, for example, the relative abundancy of specific nucleic acid and protein in the same cell.