Miles W. Cloyd

Spectrum of biological properties of human immunodeficiency virus (HIV-1) isolates

In vitro assessment of biological properties of 14 independent isolates of human immunodeficiency virus type 1 (HIV-1) was performed in order to gain insight into the spectrum of behavioral diversity of HIV-1s and to attempt to identify phenotypic traits that may be eventually correlated with in vivo pathogenesis. All of these biologically cloned isolates were found to spread very slowly in most cell cultures, requiring 8-10 weeks for virus to spread from a few infected cells to around 10(5) cells. If viral synergistic activity was also present, as in HTLV-1-infected cells, HIV-1 spread was greatly accelerated. The isolates varied in their cellular tropisms, having as much as 100,000-fold difference in their tropisms for various human CD4-positive cell lines. Several HIV isolates were dual-tropic for both T and promonocytic cells, but some of these isolates did not readily infect U937 promonocytes while readily infecting THP-1 promonocytes. Both the slow spread and extreme tropisms of HIV-1 isolates have practical implications for titering HIVs and for initiating any studies examining the interaction between a given isolate and any given cell. Some isolates did not score readily by reverse transcriptase assays while others did and this did not reflect the amount of infectious virus produced. These findings raise questions about the reliability of HIV quantitation by RT assay. The HIV isolates further varied in their ability to kill and/or fuse cells, whereas some induced cytopathology more efficiently in a given cell line than others, even though the latter appeared to replicate as well. Finally, most isolates killed cells without syncytia formation, demonstrating that cell-to-cell fusion is a minor mechanism of cytopathology. The properties observed for each HIV isolate appeared to be stable phenotypes for that virus and the diversity of biological behavior raises the possibility that independent HIV isolates may differ in their virulence properties in vivo as well.

Human immunodeficiency virus (HIV-1) cytotoxicity: Perturbation of the cell membrane and depression of phospholipid synthesis

The molecular mechanism(s) by which human immunodeficiency virus (HIV-1) injures a T-cell line was studied. A pathological role for viral env proteins, which are inserted into the plasma membrane, has been previously demonstrated for HIV as well as other retroviruses which are cytopathic. We therefore initiated studies examining whether perturbations of the cell membrane or membrane-associated biochemical events may be occurring in cells acutely infected with HIV and whether such perturbations, if present, may be responsible for cytopathology. A human T-cell line (ERIC), which is sensitive to the cytopathic effects of HIVs, was infected with HTLV-IIIB and its membrane permeability to cations and its lipid metabolism were studied coincident with the peak expression of viral p24 and with the first sign of cytopathology (slowing of cell division) 72 to 96 hr after infection. It was found that the rate of influx of Ca2+ into the cell increased over that of uninfected cells and that phospholipid synthesis, primarily phosphatidylcholine, became depressed. Diacylglycerol, which serves both as an intermediate for synthesis of phospholipids and as a second-messenger for lymphocyte activation, was also greatly reduced. However, triglyceride synthesis was enhanced, indicating that not all lipid metabolic pathways were being shut down. This decreased membrane-synthetic ability and reduced second-messenger for cell division are likely to be important causes of HIV-1 cytopathology in ERIC cells. This hypothesis was supported by our finding that HIV cytopathology of ERIC cells could be partially prevented by treatment with compounds (diacylglyceride or PMA and transiently by oleic acid) which either replenish diacylglycerol in the infected cell and/or activate protein kinase C or phosphocholine cytidyltransferase, the latter being the rate-limiting step in synthesis of the major structural phospholipid in most cells.

Polymorphic human gene(s) determines differential susceptibility of CD4 lymphocytes to infection by certain HIV-1 isolates

Independent isolates of HIV-1 differ widely in their tropisms for CD4-positive T-cell lines. This study demonstrates that tropisms of 10 different HIV-1 isolates also differ widely, as much as 1000-fold, for normal peripheral blood lymphocytes (PBLs) cultured from any given donor. This could only be reproducibly demonstrated by end point titrations. In addition, the degree of susceptibility of PBLs from 12 random donors to productive infection by any given HIV-1 isolate also varied in a reproducible pattern from donor to donor, with some donors relatively resistant to one or a few isolates. The HIV susceptibility pattern of each donor was manifested at the level of the CD4 lymphocyte and it segregated within a family, conclusively demonstrating that it was genetically determined.

Dynamics of Naive and Memory CD4+ T Lymphocytes in HIV-1 Disease Progression

Understanding the dynamics of naive and memory CD4+ T cells in the immune response to HIV-1 infection can help elucidate typical disease progression patterns observed in HIV-1 patients. Although infection markers such as CD4+ T-cell count and viral load are monitored in patient blood, the lymphatic tissues (LT) have been shown to be an important viral reservoir. Here, we introduce the first comprehensive theoretical model of disease progression based on T-cell subsets and virus circulating between the two compartments of LT and blood. We use this model to predict several trademarks observed in adult HIV-1 disease progression such as the establishment of a setpoint in the asymptomatic stage. Our model predicts that both host and viral elements play a role in determining different disease progression patterns. Viral factors include viral infectivity and production rates, whereas host factors include elements of specific immunity. We also predict the effect of highly active antiretroviral therapy and treatment cessation on cellular and viral dynamics in both blood and LT.

Model of HIV-1 disease progression based on virus-induced lymph node homing and homing-induced apoptosis of CD4+ lymphocytes.

Several proposed theories have described the progression of HIV infection. Even so, no concrete evidence supports any as comprehensive, including, for example, why the CD4+ T-cell counts fall from 1000/mm3 of blood to roughly 100/mm3 over an average 10-year period, whereas concomitant viral loads are relatively constant, increasing by several orders of magnitude in late-stage disease. Here, we develop and validate a theoretical model that altered lymphocyte circulation patterns between the lymph system and blood due to HIV-induced enhanced lymph-node homing and subsequent apoptosis of resting CD4+ T cells can explain many aspects of HIV-1 disease progression. These results lead to a recalculation of the CD4+ lymphocyte dynamics during highly active antiretroviral therapy, and also suggest new targets for therapy.

Perturbation of host-cell membrane is a primary mechanism of HIV cytopathology

Cytopathic viruses injure cells by a number of different mechanisms. The mechanism by which HIV-1 injures T cells was studied by temporally examining host-cell macromolecular syntheses, stages of the cell cycle, and membrane permeability following acute infection. T cells cytopathically infected at an m.o.i. of 1-5 grew normally for 24-72 hr, depending on the cell line, followed by the first manifestation of cell injury, slowing of cell division. At that time significant amounts of unintegrated HIV DNA and p24 core protein became detectable, and acridine orange flow cytometric cell cycle studies demonstrated the presence of fewer cells in the G2/M stage of the cell cycle. There was no change in the frequency of cells in the S-stage, and metabolic pulsing with radioactive precursors demonstrated that host-cell DNA, RNA, and protein syntheses were normal at that time and normal up to the time cells started to die (approximately 24 hr later), when all three decreased. Cellular lipid synthesis, however, was perturbed when cell multiplication slowed, with phospholipid synthesis reduced and neutral lipid synthesis enhanced. Permeability of the host-cell membrane to small molecules, such as Ca2+ and sucrose, was slightly enhanced early postinfection, and by the time of slowing of cell division, host membrane permeability was greatly increased to both Ca2+ and sucrose (Stokes radius 5.2 A) but not to inulin (Stokes radium 20 A). These changes in host-cell membrane permeability and phospholipid synthesis were not observed in acutely infected H9 cells, which are not susceptible to HIV cytopathology. Thus, HIV-1 appeared to predominantly injure T cells by perturbing host-cell membrane permeability and lipid synthesis, which is similar to the cytopathic mechanisms of paramyxoviruses.

HIV-1 binding to CD4 on CD4+CD25+ regulatory T cells enhances their suppressive function and induces them to home to, and accumulate in, peripheral and mucosal lymphoid tissues: an additional mechanism of immunosuppression

The establishment and persistence of many chronic infections have been demonstrated to depend on restraint of the vigor of the anti-microbial immune responses by CD4+CD25+ regulatory T (Treg) cells. In HIV-infected individuals, Treg cells suppress both HIV-specific and general CD4+ and CD8+ T cell responses. Increases of CD4+CD25+ Treg cell function during viral infections might be mediated by host-derived pro-inflammatory molecules or directly by viral infection or binding. We examined the effect HIV has upon binding to CD4+CD25+ Treg cells by exposing human purified CD4+CD25+ T cells from healthy donors to HIV-1 in vitro and assessing their Treg-associated functional marker profile and suppressive activities. We found that HIV-1 binding increased their suppressor activities by 2- to 5-fold, which was accompanied by enhanced expression of Treg-associated functional markers sCTLA-4, glucocorticoid-induced tumor necrosis factor receptor and FoxP3. Moreover, HIV-1 binding extended the survival of CD4+CD25+ Treg cells and up-regulated the expression of homing receptors CD62L and integrin alpha4beta7, which in turn would result in Treg cells migrating more rapidly to the peripheral lymph nodes and mucosal lymphoid tissues where anti-HIV immune responses are occurring. Importantly, CD4+CD25+ Treg cells exposed to HIV were not susceptible to homing-induced apoptosis like are other resting CD4+ cells following HIV-1 binding. We show that CD4+CD25+ Treg cells respond directly to HIV-1 itself through HIV gp120 interactions with CD4 molecules. Collectively, our findings explain a mechanism that contributes to the abnormal accumulation of intensified Treg cells in lymphoid and mucosal tissues in HIV patients, resulting in impairment of immune responses which would greatly help HIV persistence.

Low‐level plasma HIVs in patients on prolonged suppressive highly active antiretroviral therapy are produced mostly by cells other than CD4 T‐cells

The cellular source(s) and the clinical significance of persistent low‐level viremia, below 50 HIV RNA copies per ml of plasma, achieved in many patients with high adherence to highly active antiretroviral therapy (HAART) remain unclear. Also, it is not clear if residual plasma HIVs during HAART can become predominant populations in the rebounding plasma viral loads after therapy interruption. Since, different HIV quasispecies tend to compartmentalize in various cell types and tissue locations in patients during chronic infection, the phylogenetic relationships between HIV sequences amplified from residual plasma viruses and CD4 T cells of five patients on long‐term suppressive therapy were examined. Three of these patients stopped therapy voluntarily for 3 weeks, but only one of them demonstrated viral load rebound in plasma. In phylogenetic analyses, the residual plasma viruses were found to be distinct genetically from the majority of CD4 T cell‐associated virus populations in four of five patients. The compartmental analyses revealed that in all patients, plasma‐ and CD4 T cell‐derived viral sequences were compartmentalized separately. Interestingly, the plasma sequences obtained before and after HAART‐off in two patients were produced apparently from the same compartment, which was different from the circulating CD4 T cell‐compartment. These results suggest the possibility that residual plasma viruses in patients on long‐term suppressive HAART may be produced persistently from a cellular source yet to be identified, and are capable of spreading quickly in vivo, accounting for the rapid rebound of viral loads in plasma after therapy interruption. J. Med. Virol. 81:9–15, 2009.

Interference patterns of human immunodeficiency viruses HIV-1 and HIV-2

The ability of cells infected with a retrovirus to interfere with superinfection by another retrovirus usually involves blockage, by the primary virus, of the receptors for the superinfecting virus. Retroviruses using different receptors do not interfere with each other, and this property has been used to classify various types of retroviruses. Different isolates of human immunodeficiency virus (HIV) were subjected to this type of analysis, and it was found that all HIV-1s cross-interfere with each other in T cells as well as in U937 promonocytic cells, substantiating further that all isolates use the same receptor on these cells. An HIV-2 isolate was found to interfere with HIV-1s, but HIV-1s only partially interfered with HIV-2 superinfection, indicating that inherent differences in receptor interactions exist between HIV-1s and HIV-2. For comparison, interference patterns of D-type primate retroviruses (SRVs) and murine amphotropic and xenotropic retroviruses revealed that each virus fell within distinct interference groups demonstrating that human T cells possess at least four distinct receptors for retroviruses. The mechanism of HIV interference was found to be due to receptor blockage in productively infected cells and to receptor elimination in latently infected T cells. Our findings that all HIV-1s completely interfere with each other and that interference occurs rapidly following acute infection suggests that a cell infected with HIV-1 will not permit reinfection by progeny or by other exogenous HIVs. This, in turn, suggests that progeny reinfection may not be the source of the large amount of unintegrated viral DNA observed following HIV cytopathic infection.

CD4 lymphocytes in the blood of HIV+ individuals migrate rapidly to lymph nodes and bone marrow: support for homing theory of CD4 cell depletion

The mechanism(s) by which human immunodeficiency virus (HIV) causes depletion of CD4 lymphocytes remains unknown. Evidence has been reported for a mechanism involving HIV binding to (and signaling) resting CD4 lymphocytes in lymphoid tissues, resulting in up‐regulation of lymph node homing receptors and enhanced homing after these cells enter the blood, and induction of apoptosis in many of these cells during the homing process, caused by secondary signaling through homing receptors. Supportive evidence for this as a major pathogenic mechanism requires demonstration that CD4 lymphocytes in HIV+ individuals do migrate to lymph nodes at enhanced rates. Studies herein show that freshly isolated CD4 lymphocytes labeled with 111Indium and intravenously reinfused back into HIV+ human donors do home to peripheral lymph nodes at rates two times faster than normal. They also home at enhanced rates to iliac and vertebral bone marrow. In contrast, two hepatitis B virus‐infected subjects displayed less than normal rates of blood CD4 lymphocyte migration to peripheral lymph nodes and bone marrow. Furthermore, the increased CD4 lymphocyte homing rates in HIV+ subjects returned to normal levels after effective, highly active antiretroviral therapy treatment, showing that the enhanced homing correlated with active HIV replication. This is the first direct demonstration of where and how fast CD4 lymphocytes in the blood traffic to tissues in normal and HIV‐infected humans. The results support the theory that the disappearance of CD4 lymphocytes from the blood of HIV+ patients is a result of their enhanced migration out of the blood (homing) and dying in extravascular tissues.