Opendra Narayan

Biology and Pathogenesis of Lentiviruses

Lentiviruses are a family of retroviruses linked by similarities in genetic composition, molecular mechanisms of replication and in biological interactions with their hosts. They are best known as agents of slow disease syndromes that begin insidiously after prolonged periods of subclinical infection and progress slowly leading to the degeneration of multiple organ systems, cachexia and death. The viruses are species-specific in host range and several have been recognized as pathogens of domestic animals, non-human primates and humans. The prototypes of the family are the agents causing maedi-visna in sheep and infectious anaemia in horses. These diseases have been known for several decades and studies on the biology of the viruses have provided a fund of information that predicted most of the properties of their human counterpart which was identified only 6 years ago as the aetiological agent of AIDS. Lentiviruses persist indefinitely in their hosts and replicate continuously at variable rates during the course of the lifelong infection.

Slow virus replication: The role of macrophages in the persistence and expression of visna viruses of sheep and goats

Lentiviruses of sheep and goats cause slowly progressive diseases of the central nervous system (visna), lungs (maedi) and joints (arthritis) in their natural hosts. However, the virus target cell(s) in these diseases are still unknown. In this report, using laboratory-adapted Icelandic visna virus and several field strains recently obtained from sheep and goats with natural disease in the U.S.A., we show that macrophages became persistently infected when inoculated in culture. Furthermore, macrophages were an invariable source of virus from experimentally and naturally infected animals. Virus-producing macrophages developed minimal cytopathic changes and virus assembly occurred mainly intracellularly, accumulating in cytoplasmic vacuoles. In contrast to macrophages, sheep choroid plexus fibroblasts developed syncytial cytopathic changes after inoculation and virus maturation occurred at the cell surfaces. Replication of the Icelandic virus was highly productive in this system but that of the field viruses was very inefficient. In some cases these agents failed to replicate in the fibroblasts and no cytopathic effect occurred. This block in the field virus replication was, however, overcome when infected nonproducer fibroblasts were co-cultivated with macrophages. In these cases, virus production with attendant cytopathic effect in the fibroblasts required the continuous presence of macrophages because the cells reverted to a non-productive state when separated from macrophages and became productive again when subcultures were added to new macrophages. The roles of the macrophage as a virus target cell and virus inducer in the virus-macrophage-fibroblast interactions are discussed with inferences to the well-known phenomenon of restricted virus replication in infected animals and the immunopathological aspects of the diseases.

Interaction of Virion Protein Vpr of Human Immunodeficiency Virus Type 1 with Cellular Transcription Factor Sp1 and trans-Activation of Viral Long Terminal Repeat

Acquired immunodeficiency syndrome (AIDS) is a result of replication of the human immunodeficiency virus type 1 (HIV-1) predominantly in CD4 T lymphocytes and macrophages. However, most of these cells in vivo are immunologically quiescent, a condition restricting HIV-1 replication. Vpr is an HIV-1 virion protein suspected to enhance HIV-1 replication in vivo. We demonstrate in this report that Vpr specifically activates HIV-1 long terminal repeat (LTR)-directed transcription. This effect is most pronounced on a minimal promoter from HIV-1 LTR containing the TATA box and binding motifs for the ubiquitous cellular transcription factor Sp1. Evidence is presented that Vpr interacts with Sp1 when Sp1 is bound to the Sp1 motifs within the HIV-1 LTR. Both Vpr-Sp1 interaction and Vpr trans-activation require a central Leu/Ile-rich domain in Vpr. Our findings suggest that Vpr trans-activation through Sp1 is most critical for the immediate early transcription of HIV-1 when other positive regulators, such as NF-κB, are limited or inactive, a condition presumably present in vivo. By interacting with Sp1, Vpr also has the potential to influence cellular gene expression and cellular functions. Thus, therapeutic approaches directed toward blocking the Vpr trans-activation function could prove valuable in treating AIDS.

Detection of the human immunodeficiency virus regulatory protein tat in CNS tissues.

Neuropathologically, human immunodeficiency virus (HIV) is associated with a range of inflammatory disorders, extensive cortical neuronal loss, and dendritic and synaptic damage. Although the mechanisms resulting in these abnormalities are still unclear, the neurotoxic effects are thought to be due in part to viral products including the tat gene product. We have previously shown that Tat when presented to neurons extracellularly interacts with neuronal cell membranes to cause neuronal excitation and toxicity in fmole amounts. To determine the role of Tat in mediating HIV encephalitis (HIVE), we detected tat mRNA and protein in tissue extracts of nine patients with HIVE and seven patients without HIVE. Despite long autopsy times and significant degradation, tat mRNA was detected in 4/9 patients with HIVE but not in any of the seven patients without dementia. Similarly, the env mRNA was also detected in 5/9 patients with HIVE but not in the patients without HIVE. However, vif mRNA was detected in both groups of patients with (5/9) or without (2/7) HIVE. Using protein extracts from the brains of the same groups of patients we were unable to detect Tat by enzyme linked immunosorbant assay (ELISA) (sensitivity of 2 ng Tat/ml of brain tissue). However, Tat could be detected immunohistochemically and in protein extracts from the brains of rhesus macaques with encephalitis due to a chimeric strain of HIV and simian immunodeficiency virus (SHIV). Our observations support the role of Tat in the neuropathogenesis of HIV and SHIV encephalitis.

Oxidative stress in HIV demented patients and protection ex vivo with novel antioxidants

Objective: To determine the role of oxidative stress in mediating HIV dementia and to identify novel therapeutic compounds that may block this oxidative stress. Methods: Brain tissue from patients with HIV encephalitis and macaques with simian immune deficiency virus encephalitis was immunostained for lipid peroxidation. Oxidized proteins in CSF of patients with various stages of HIV dementia were quantitated and we determined whether CSF from these patients could alter mitochondrial function. Several novel compounds with antioxidant effects were screened to determine their relative efficacy in protecting against CSF-induced neurotoxicity. Results: Evidence for oxidative stress was present both in brain and in CSF. The presence of oxidized proteins in the CSF and CSF-induced progressive decrease in mitochondrial activity correlated with the severity of cognitive impairment, but only the group of patients with moderate to severe dementia reached statistical significance. l-deprenyl, didox, imidate, diosgenin, and ebselen blocked the CSF-induced toxicity. No effect of trimidox, ruthenium red, or Quercetin was seen. Conclusions: Increased oxidative stress is present in brain and CSF of HIV-infected patients. There is also an accumulation of toxic substances in the CSF that are capable of inducing oxidative stress. The authors have identified several novel compounds that are capable of blocking the CSF-induced toxicity, the therapeutic potential of which is worthy of further exploration.

Biochemical Characterization of the Virus Causing Leukoencephalitis and Arthritis in Goats

Goat leukoencephalitis-arthritis virus (GLV) has the density of a retrovirus in sucrose and contains an endogenous RNA-dependent DNA polymerase (reverse transcriptase). The virion reverse transcriptase utilizes the synthetic RNA template poly(rA). (dT)12 but not the synthetic DNA template poly(dA). (dT)12. A high mol. wt. RNA similar in size to visna virus RNA was isolated from 3H-uridine-labelled virions. The major structural protein of GLV has the same mol. wt. as that of visna virus. From these data the GLV appears to be a retrovirus.

The neurobiology of human immunodeficiency virus infections.

A variety of diseases of the central and peripheral nervous systems evolves during the course of human immunodeficiency virus (HIV) infections. Most are not related to documented opportunistic infections and may be the direct result of HIV infections, as large proportions of healthy and ill HIV‐infected persons show evidence of nervous system infection. These diseases occur at different times during the infection and have diverse inflammatory, demyelinating, or degenerative pathological features that suggest different pathogenetic mechanisms. The route and determinants of HIV invasion of the nervous system are unknown. Within the brain, viral antigen and RNA are found predominantly in macrophages, but the reason why profound dementia and cortical atrophy result from this infection remains a mystery. By analogy to other lentivirus infections, particularly visna virus in sheep, neuropathological changes may be mediated by cytokines. Other possible pathogenetic mechanisms include toxicity of viral polypeptides, transactivation of viral or cellular genes, autoimmunity, or other opportunistic infections. Clarification of the pathogenesis of HIV‐related diseases is critical to the design of rational therapies.— Johnson, R. T.; McArthur, J. C.; Narayan, O. The neurobiology of human immunodeficiency virus infection. FASEB J. 2: 2970‐2981; 1988.

Etiology of progressive multifocal leukoencephalopathy. Identification of papovavirus.

Abstract Papovaviruses were identified in brains of 13 patients with progressive multifocal leukoencephalopathy, asubacute human demyelinating disease. Fluorescent-antibody staining and electron microscopical agglutination technics demonstrated the JC type of papovavirus in 11 patients. Simian-virus 40 and the virus of progressive multifocal leukoencephalopathy were isolated from the first two patients studied, but all subsequent isolates were of the JC-virus type, and the BK type was not implicated in the disease. The use of monospecific rabbit serums to identify viral antigens in the brain tissue of patients and to serotype by electron microscopy virions extracted directly from brain demonstrates that rapid diagnostic methods can be employed in this disease. (N Engl J Med 289:1278–1282, 1973)

Neuronal Apoptosis Is Mediated by CXCL10 Overexpression in Simian Human Immunodeficiency Virus Encephalitis

Inflammatory mediators play a crucial role in the pathophysiology of several neurodegenerative diseases including acquired immune deficiency syndrome dementia complex. In the present study we identified a link between CXCL10 overexpression in the brain and human immunodeficiency virus dementia and demonstrated the presence of the chemokine CXCL10 and its receptor, CXCR3, in the neurons in the brains of macaques with simian human immunodeficiency virus encephalitis. Using human fetal brain cultures, we showed that treatment of these cells with either SHIV89.6P or viral gp120 resulted in induction of CXCL10 in neurons. Cultured neurons treated with the chemokine developed increased membrane permeability followed by apoptosis via activation of caspase-3. We confirmed the relevance of these findings in sections of human and macaque brains with encephalopathy demonstrating that neurons expressing CXCL10 also expressed caspase-3.

Efficiency of in situ hybridization as a function of probe size and fixation technique

In an attempt to improve fixation technique for viral RNA detection by in situ hybridization, we have quantitatively compared the hybridization signal obtained when measles virus or visna virus infected cell cultures were fixed with eight different fixatives and hybridized with 35S-labeled virus-complementary DNA probes of several size ranges. Small probes (mean length, 70 bases) gave higher signals than larger probes (mean lengths 140, 350, and 780 bases) with all fixatives. This increase in signal was minimal with acetic ethanol or formalin, but was dramatic with fixatives containing glutaraldehyde; with these fixatives the signals with small probes were 6.5- to 22-fold greater than with large probes. The highest signals were obtained with periodate-lysine-paraformaldehyde-glutaraldehyde (PLPG) fixed cells hybridized with small probes, and were 1.5- to 6.7-fold greater than those obtained with the commonly used fixative acetic ethanol. PLPG and other glutaraldehyde based fixatives also greatly improved the preservation of cellular morphology compared to acetic ethanol.