NeuroAids Vol. 1, Issue 6 (October 98) |
Role of Viral Proteins in HIV-1 Neuropathogenesis with Emphasis on Tat
A. Nath1,2,
J.D. Geiger4,
M.P. Mattson3,
D.S.K. Magnuson5,
M. Jones2,
and J.R. Berger1
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Abstract |
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Abstract | Intro | Pathogenesis | Limitations | Conclusions | Refs |
HIV-1 infection is the most common CNS infection in the world and the most common cause of dementia in people & lt; 60 years of age in North America. HIV-1 associated dementia (AIDS dementia complex) has a poor prognosis and it is marked by deficits in motor control, cognition and behavior (1). Neuropathological changes include infiltration of mononuclear cells, glial cell activation and loss of select populations of neurons (2)(3). Although neuronal functions are adversely affected during HIV-1 infection, direct infection of neurons with HIV-1 has only rarely been demonstrated (4)(5). Neuronal dysfunction and neuronal cell death by apoptosis and necrosis can be caused by neurotoxic viral proteins and, thus, these proteins may represent important contributors to HIV-1 neuropathogenesis (6). However, the presence of most of these proteins in the brains of patients with HIV-1 infection have yet to be demonstrated and, of those detected, their levels in the extracellular compartment, where their neurotoxic actions are thought to be mediated, remain to be determined. Therefore, in studies focused on establishing the pathogenic mechanisms occurring as a consequence of HIV-1 viral protein-mediated neurotoxicity and on their pharmacological amelioration, the doses/concentrations of these neurotoxins relevant to the human condition is a concern. Our position, espoused here, is that Neuro-AIDS researchers should look past the question of doses and concentrations, and instead address mechanistic questions. |
Introduction |
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Several HIV proteins have been shown to have neurotoxic properties in vitro. However, their presence in autopsy brain, particularly in extracellular compartments, has been difficult to demonstrate using conventional histopathological techniques. In this manuscript we examine the existing experimental evidence to help determine if these proteins play a role in the pathogenesis of HIV dementia. |
Pathogenesis |
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Abstract | Intro | Pathogenesis | Limitations | Conclusions | Refs |
The HIV-1 viral coat glycoproteins gp41, gp120, and their precursor gp160 have all been shown to be neurotoxic (6). gp120, a protein involved in regulating viral entry and in determining viral tropism, is indirectly neurotoxic. Its actions are mediated via microglia and astrocytes (13), although some have suggested direct actions on neurons (14). The mechanisms implicated in gp120-induced neurotoxicity include decreased glutamate re-uptake by astrocytes, increased arachidonic acid release which then inhibits re-uptake of glutamate by neurons and astrocytes (15)(16), and increased levels of intracellular calcium in astrocytes and neurons (17). gp41 causes neurotoxicity in the presence of glial cells. Its mechanism of action includes nitric oxide formation (10). Attempts to detect gp120 protein in HIV-1 autopsy brain or in brains of transgenic mouse models of HIV-1 infection have not been successful (18) possibly due to technical factors (see below). But levels in the picomolar range have been demonstrated to result in neurotoxicity (19). Given the presence of gp120 mRNA, the well documented presence of gp41 protein (10), and the fact that both gp41 and gp120 are formed from gp160, the absence of gp120 in HIV-1 infected brain is highly unlikely. However, the duration of expression of gp120 will depend upon its rate of degradation which is yet to be determined. Recently, attention has shifted somewhat towards the study of the
neurotoxic properties of Tat, a non-structural viral protein essential
for viral replication. The first exon of Tat encodes for the initial
72 amino acids and the second exon forms the remaining 14 to 32 amino
acids (20). Tat transcripts are elevated in HIV-1
demented brain (8) and both Tat1-72 (derived
from the first exon) and Tat1-86 to 101 (full length) are
produced by HIV-1 infected cells (21). Mechanisms
implicated in the neurotoxic actions of Tat include direct depolarization
of neurons, increased levels of intracellular calcium, increased production/release
of pro-inflammatory cytokines, increased macrophage infiltration,
activation of excitatory amino acid receptors by, as yet, uncharacterized
mechanisms, and increased incidence of programmed cell death (apoptosis)
(6)(22)(23).
Despite, the documented presence of mRNA for Tat in HIV-1 autopsy
brain (8)(9), investigators have
questioned the potential relevance of findings on Tat neurotoxicity
in the absence of evidence of Tat protein in HIV-1 infected brain.
This position appears to be dramatically at odds with the well documented
requirement of Tat protein for HIV-1 trans-activation. In other words,
because the virus is replicating, Tat protein must be present. Here,
we report preliminary findings that Tat protein, as demonstrated immunohistochemically,
is present in HIV-1 infected human brain and in chimeric simian-human
immunodeficiency virus infected brain of macaque monkey (Figure 1)(Nath
et al., unpublished observations). These results show that Tat protein
is present in close proximity to infected cells and neurons, and these
findings bring us one step closer to determining the amounts of Tat
protein present in HIV-1 infected tissues.
Neurotoxicity of other HIV-1 proteins has also been demonstrated. Peptides of HIV-1 Rev, when injected icv, were lethal (25) and in lymphoid cells caused necrotic cell death (26). Furthermore, Vpr was shown to cause neurotoxicity and caused membrane depolarization of neurons (7). Nef, an accessory protein that is over-expressed in HIV-1 infected astrocytes (27), and Nef-derived peptides may be neurotoxic (28). Although mRNA for these accessory proteins has been demonstrated in HIV-1 infected brain, protein expression has yet to be reported. The role of each of these HIV-1 proteins in cell death and apoptosis in specific sub-populations of neurons remains uncertain. There are many factors that affect the levels of these HIV-1 proteins in brain and a discussion of these factors is important because of their obvious relevance to discussions of HIV-1 protein-induced neurotoxicity. These factors include, but are not necessarily limited to (because this field is relatively new and evolving quickly), the requirement for the continuous presence of the proteins, the in vitro and in vivo models used, the physiochemical properties of the viral proteins, and the size of the extracellular space which then dictates the concentrations reached in this compartment. Continued presence of HIV-1 proteins may not be required
for neurotoxicity: Evidence for a hit and run phenomenon
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Limitations |
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Abstract | Intro | Pathogenesis | Limitations | Conclusions | Refs |
Physiochemical properties of HIV-1 viral proteins The neurotoxic properties of gp120 and Tat are also highly dependent
upon their tertiary configuration (39)(Holden et
al., submitted). These proteins rapidly lose their neurotoxic potential
when subjected to freeze-thaw conditions or if heated above physiological
temperatures. For example, a single freeze-thaw cycle may result in
a 50% reduction in Tat-induced neurotoxicity (30).
The transactivation domain and the neurotoxic epitope of Tat contains
6 cysteine residues which are easily oxidized (39).
This is also the most conserved region of Tat amongst all HIV strains
(40). However, Tat looses its functional properties
upon oxidation and because of this, Tat purification procedures require
the presence of reducing agents and the final steps must be performed
anaerobically. While these actions help maintain Tat stability, the
presence of reducing agents would, almost certainly decrease its neurotoxicity
by buffering oxygen free-radical induced cell death. Thus, physiochemical
properties of Tat and gp120 significantly reduce bioavailability and
these properties help explain some of the batch to batch variability
that others and we have noted in determining dose-response profiles
for neurotoxicity (Nath et al., unpublished observations; Amini and
Alexander, Allegheny University, personal communication). The half-life of HIV-1 proteins in extracellular spaces will affect
their neurotoxic actions. Factors that control the half-lives of these
substances include factors that regulate its release into the extracellular
space, inactivation by proteases, and cellular transport. To date,
only the HIV-1 protein Tat has been shown to be secreted from HIV-1
infected cells and this process has been characterized as being energy
dependent (23). Once secreted, into the extracellular
space, HIV-1 proteins may be released into the circulation, may bind
to cell surface proteins on uninfected cells, or may be taken up into
infected and uninfected cells. Both Tat and Vpr have been measured
in the serum of HIV-infected patients (7)(45),
both proteins bind to uninfected cells (7) (Figure
3), and, at least Tat, has been shown to be taken up by non-infected
cells (46).
The cellular uptake of Tat appears to be governed by the region of Tat protein formed by its second exon (46). Interestingly, neurotropic strains of HIV-1 from patients with HIV dementia show glutamate substitutions in the second exon (40) which is likely to decrease its ability to be taken up by cells, increasing its extracellular concentrations. Intracellularly, proteins such as the p6 gag protein bind to ubiquitin and are degraded by energy-dependent processes (47) but mechanisms for degradation of other HIV proteins need to be determined. Another mechanism by which HIV proteins may interact with surrounding uninfected cells is by cell-to-cell contact. For example, gp41 is a transmembrane protein that is expressed on the surface of infected cells which may induce neuronal injury in cells in close proximity (10). |
Conclusions |
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Abstract | Intro | Pathogenesis | Limitations | Conclusions | Refs |
We conclude that all viral proteins are present in the brain at one time or another. Their continued presence may not be necessary to cause neurotoxicity. A transient release of viral proteins may be sufficient to initiate a cascade of events that may last for prolonged periods of time. The in vivo concentrations of these proteins are dependent upon factors that regulate the production and release of the proteins in the extracellular space, the structural and physiochemical properties of the proteins themselves, and the dynamics of the extracellular space in the brain. In vitro assays invariably require large concentrations of proteins again due to their physiochemical properties and practical issues related to the design of these assays. The mechanisms responsible for neurotoxicity in AIDS is the fundamental issue; concentrations of HIV-1 proteins employed in in vitro assays are of secondary importance.
Acknowledgements We would like to thank Dr. O. Narayan at the University of Kansas for providing us with tissues from the SHIV infected animals, and Drs. G. Alexander and S. Armini at Allegheny University for sharing with us their observations regarding physiochemical properties of Tat and for providing us with the protocol for silanizing glass. Martha Cooper assisted with preparation of figure 3. Dr. M. Ma performed the immunostaining in figure 4. Dr. JDG's research program is supported by grants from the National Health Research Development Program/Medical Research Council of Canada AIDS/HIV Research Strategy Program. |
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