AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 8, Number 3, 1992
Mary
Ann
Liebert, Inc., Publishers
Macaque CD4+ T-Cell Subsets: Influence of Activation on Infection by Simian Immunodeficiency Viruses (SIV) P. POLACINO
FIRPO, I. AXBERG, M. SCHEIBEL, and E.A. CLARK
ABSTRACT Simian immunodeficiency virus (SIV) infects a small number of CD4+ T cells including "memory" T cells. The following describes the cell surface markers which may delineate subsets of CD4+ memory T cells and reviews how memory CD4+ T cells are activated and regulated through the T-cell receptor and such accessory receptors as CD28. The factors which may influence initial expression and infection of T cells by CD4 are discussed. Unlike activated and infected T cells, unstimulated CD4+ T cells have little or no SIV DNA detectable in the genomic fraction, but key activation signals may promote integration of viral DNA in memory T cells. Bacterial superantigens (SuperAg) can promote increased levels of SIV viral DNA in mature and immature T cells. Immunodeficiency virus products such as gpl20, Nef, and Tat can affect CD4+ T-cell function. Whereas Nef can reduce expression of CD4, Tat reduces the expression of CD28. We hypothesize that the lack of expression of key accessory molecules on CD4 lineage T cells infected with immunodeficiency viruses may make infected T cells more
susceptible to recall-antigen-induced programmed cell death.
instance, host cell factors that influence uncoating, entry,
INTRODUCTION
transcription, integration, and virus reinfection may affect the establishment of a persistent or lytic infection. As the role of CD4-gpl20 interactions in AIDS has been reviewed recently,3 we will emphasize only a few recent findings. The natural ligands for CD4 are class II major histocompatibility complex (MHC) molecules on antigen-presenting cells (APC). Cross-linking CD4 can trigger a cellular response mediated in part by the p56lck protein tyrosine kinase (PTK) associated with CD4." While binding of gpl20 to CD4 is required for viral entry, signaling via CD4 is not:12-14 after binding to CD4, gpl20can induce increases in intracellularfree calcium ((Ca2+]i) and in interleukin-2 (IL-2) receptor expression and cell motility.13 Binding of HIV to CD4 also induces rapid and sustained phosphorylation of CD4, which could involve protein kinase C. This phosphorylation can be blocked by antibody against CD4 and antibody against HIV gpl20, indicating that a specific interaction between CD4 and gpl20 is reverse
CD4
immunodeficiency viruses (HIV) and simian immunodeficiency viruses (SIV). '"3 Both HIV and SIV are highly tropic for cells expressing CD4 on the cell surface and they induce cytopathic effects in CD4+ cells in vitro.4~7 Although the cellular tropism of both HIV and SIV is determined largely by the affinity of the major virus envelope component, gpl20, for CD4, relatively few is the receptor FOR both
CD4+ T cells
are
human
infected in HIV-1+ individuals.8'9 The
frequency of provirus-carrying cells also correlates with the progression of the disease,10 from one provirus molecule per 6,000-80.000 cells on the early stages to one per 700-3,300 cells in the advanced stages. In SIV-infected macaques, SIV has been estimated to be in approximately one per 1,000 to 20,000 mononuclear cells.10" Thus, factors other than the presence or amount of CD4 receptors clearly influence whether retroviruses such as HIV or SIV establish a productive infection. For
Regional
Primate Research Center,
University of Washington, Seattle WA 98195. 357
FIRPO ET AL.
358
required for phosphorylation to occur.12 However, when selected inhibitors of signal transduction (e.g., H7, cyclosporin A)
evaluated for their effects on penetration, none of them affected viral penetration. Not only are CD4-related signal transduction events not required for entry, but also the CD4 receptor does not internalize with HIV.I4 The binding of virus to the CD4+ cell is followed by rapid fusion of the viral membrane with the plasma membrane of the target cell leading to viral entry. A detailed analysis of cells bearing various altered forms of CD4 has been performed to determine which parts of CD4 are critical for HIV-1 and HIV-2 to enter cells and replicate.3'1'' In addition to the known binding region of CD4, which is sufficient for viral infectivity, the proximal membrane portion of CD4 appears to facilitate viral fusion and entry. Furthermore, a strong correlation between the down-modulation of the surface CD4 and HIV entry was observed.lfi A temporal association between CD4 down-modulation and the accumulation of intracellular HIV-1 gp 160/120 also has been found in a chronically infected cell line, OM-10.1, and surface CD4 expression by OM-10.1 cells returned once viral activation ceased."' Modulation of CD4 expression also occurs after stimulation with phorbol myristate acetate (PMA), antigen, or HIV, all of which induce phosphorylation of serine residues on the cytoplasmic tail of CD4.317 CD4 can probably be down-regulated at the level of mRNA synthesis as well as were
protein expression (see below). These early binding, fusion, and entry steps may be influenced by the activation state of the CD4+ T cell; although activated CD4+ T cells rapidly form syncytia when cultured with HIV-1 envelope glycoprotein-expressing cells, unstimulated CD4+ T cells do so more slowly. CD4+ T cells become susceptible to HIV-1 envelope-dependent syncytia formation after 2 h of exposure to immunological stimuli and the activation of protein kinase C (PKC) may play a critical role in this process.I8 SIVs do not replicate in CD4 CD8 lymphocytes2 f
and also have a restricted host range among human CD4+ cells when compared with HIV-1 and HIV-2. This restricted tropism has been associated with the inability of the SIV envelope glycoprotein to induce membrane fusion in cells not susceptible In a study comparto productive exogenous infection by SIV. ing the expression of HIV and SIV in six human cell lines infectable by HIV, we found that SIV could infect only three of the lines.20 Only cell lines expressing the B-cell-associated activation markers B7/BB1 and CD40 were susceptible to infection, but by no means demonstrating, that an additional cell surface molecule may be required for efficient SIV infection. Once HIV enters the host cell, reverse transcription, RNA expression, and production of virus particles is about as efficient in monocytes as in T cells.21 Thus, a major limiting step for establishing infection may be the steps regulating viral entry into the host cell cytoplasm. Two recent studies are consistent with this hypothesis: Using a molecularly cloned HIV-1 restricted for replication in T-cell lines, Cann and co-workers22 found that little or no viral DNA or RNA was synthesized in nonpermissive cells after infection. At least one block to infection in nonpermissive T cells occurs at a point in entry or uncoating before provirus formation.22 Another study, analyzing HIV-1 replication in four T-cell lines, showed that a major determinant of the permissiveness of growing T cells for HIV-1 is the rate and efficiency of virus entry,23 a result consistent with our studies
with SIV.20 Although a variety of murine retroviruses appear to exhibit cellular tropism on the basis of specific alterations in their long terminal repeats (LTRs), the LTR does not appear to play a major role in determining cellular tropism of HIV.24
SIV INFECTS A SUBPOPULATION OF CD4+ T CELLS
Collectively, the above studies suggest that HIV/SIV infectivity can be greatly influenced at the entry step, perhaps by
receptors distinct from CD4. Thus, we examined whetherCD4+ T-cell subsets differing in their expression of other markers are more or less susceptible to SIV infection. We found that a subset ofCD4+ T cells expressing high levels of the adhesion receptor, CD44 (CD44hl), are selectively lost in SIV-infected macaques prior to the onset of AIDS25 and that SIV selectively replicates in this subset in vitro. Based on their expression of CD44 and a form of the CD45 (T200) membrane protein tyrosine phosphatase (PTPase) marker, CD45RA, macaque CD4 subsets can+ be divided into at least three populations:26 CD441", CD45RA cells, which contain principally resting cells not in cycle, CD44hi, CD45RA+ subset, which contain virtually all the cells in S phase, and CD44hi, CD45RA~ cells, which contain most of the memory T cells, also subseted as CD45RO+ cells.27'28 We isolatedeitherCD44hi""orCD45RA+/RA CD4+ subsetsfrom SIV-infected macaques; using a polymerase chain reaction (PCR) method with oligonucleotide probes to the SIV gag region, we were not able to detect SIV DNA in CD441" cells, but we could detect it in CD44hi, CD45RA+, orCD45RA~ cells.2" These results demonstrated that SIV infection is restricted to a definable subset of CD4+ T cells which includes memory T cells. Similar results have been obtained with HIV: HIV was found in CD45RO+ cells30 and the memory response of CD45RO ' T cells was shown to be defective in HIV-infected individuals. These results suggest that memory T cells or cells in the process of becoming memory T cells are an initial target for SIV and HIV infection. However, whether or not CD45RA and CD45RO are in fact good markers for "naive" and "'memory" T cells is controversial, particularly in light of recent findings demonstrating that CD45RO+ cells can "revert" to a CD45RA + phenotype.3l'31a Furthermore, CD4+ CD45RO+ generally comprise about 5-10% of CD4+ cells in blood (Fig. 1), but HIV and SIV DNA are found in
Mary
Ann
Liebert, Inc., Publishers
Macaque CD4+ T-Cell Subsets: Influence of Activation on Infection by Simian Immunodeficiency Viruses (SIV) P. POLACINO
FIRPO, I. AXBERG, M. SCHEIBEL, and E.A. CLARK
ABSTRACT Simian immunodeficiency virus (SIV) infects a small number of CD4+ T cells including "memory" T cells. The following describes the cell surface markers which may delineate subsets of CD4+ memory T cells and reviews how memory CD4+ T cells are activated and regulated through the T-cell receptor and such accessory receptors as CD28. The factors which may influence initial expression and infection of T cells by CD4 are discussed. Unlike activated and infected T cells, unstimulated CD4+ T cells have little or no SIV DNA detectable in the genomic fraction, but key activation signals may promote integration of viral DNA in memory T cells. Bacterial superantigens (SuperAg) can promote increased levels of SIV viral DNA in mature and immature T cells. Immunodeficiency virus products such as gpl20, Nef, and Tat can affect CD4+ T-cell function. Whereas Nef can reduce expression of CD4, Tat reduces the expression of CD28. We hypothesize that the lack of expression of key accessory molecules on CD4 lineage T cells infected with immunodeficiency viruses may make infected T cells more
susceptible to recall-antigen-induced programmed cell death.
instance, host cell factors that influence uncoating, entry,
INTRODUCTION
transcription, integration, and virus reinfection may affect the establishment of a persistent or lytic infection. As the role of CD4-gpl20 interactions in AIDS has been reviewed recently,3 we will emphasize only a few recent findings. The natural ligands for CD4 are class II major histocompatibility complex (MHC) molecules on antigen-presenting cells (APC). Cross-linking CD4 can trigger a cellular response mediated in part by the p56lck protein tyrosine kinase (PTK) associated with CD4." While binding of gpl20 to CD4 is required for viral entry, signaling via CD4 is not:12-14 after binding to CD4, gpl20can induce increases in intracellularfree calcium ((Ca2+]i) and in interleukin-2 (IL-2) receptor expression and cell motility.13 Binding of HIV to CD4 also induces rapid and sustained phosphorylation of CD4, which could involve protein kinase C. This phosphorylation can be blocked by antibody against CD4 and antibody against HIV gpl20, indicating that a specific interaction between CD4 and gpl20 is reverse
CD4
immunodeficiency viruses (HIV) and simian immunodeficiency viruses (SIV). '"3 Both HIV and SIV are highly tropic for cells expressing CD4 on the cell surface and they induce cytopathic effects in CD4+ cells in vitro.4~7 Although the cellular tropism of both HIV and SIV is determined largely by the affinity of the major virus envelope component, gpl20, for CD4, relatively few is the receptor FOR both
CD4+ T cells
are
human
infected in HIV-1+ individuals.8'9 The
frequency of provirus-carrying cells also correlates with the progression of the disease,10 from one provirus molecule per 6,000-80.000 cells on the early stages to one per 700-3,300 cells in the advanced stages. In SIV-infected macaques, SIV has been estimated to be in approximately one per 1,000 to 20,000 mononuclear cells.10" Thus, factors other than the presence or amount of CD4 receptors clearly influence whether retroviruses such as HIV or SIV establish a productive infection. For
Regional
Primate Research Center,
University of Washington, Seattle WA 98195. 357
FIRPO ET AL.
358
required for phosphorylation to occur.12 However, when selected inhibitors of signal transduction (e.g., H7, cyclosporin A)
evaluated for their effects on penetration, none of them affected viral penetration. Not only are CD4-related signal transduction events not required for entry, but also the CD4 receptor does not internalize with HIV.I4 The binding of virus to the CD4+ cell is followed by rapid fusion of the viral membrane with the plasma membrane of the target cell leading to viral entry. A detailed analysis of cells bearing various altered forms of CD4 has been performed to determine which parts of CD4 are critical for HIV-1 and HIV-2 to enter cells and replicate.3'1'' In addition to the known binding region of CD4, which is sufficient for viral infectivity, the proximal membrane portion of CD4 appears to facilitate viral fusion and entry. Furthermore, a strong correlation between the down-modulation of the surface CD4 and HIV entry was observed.lfi A temporal association between CD4 down-modulation and the accumulation of intracellular HIV-1 gp 160/120 also has been found in a chronically infected cell line, OM-10.1, and surface CD4 expression by OM-10.1 cells returned once viral activation ceased."' Modulation of CD4 expression also occurs after stimulation with phorbol myristate acetate (PMA), antigen, or HIV, all of which induce phosphorylation of serine residues on the cytoplasmic tail of CD4.317 CD4 can probably be down-regulated at the level of mRNA synthesis as well as were
protein expression (see below). These early binding, fusion, and entry steps may be influenced by the activation state of the CD4+ T cell; although activated CD4+ T cells rapidly form syncytia when cultured with HIV-1 envelope glycoprotein-expressing cells, unstimulated CD4+ T cells do so more slowly. CD4+ T cells become susceptible to HIV-1 envelope-dependent syncytia formation after 2 h of exposure to immunological stimuli and the activation of protein kinase C (PKC) may play a critical role in this process.I8 SIVs do not replicate in CD4 CD8 lymphocytes2 f
and also have a restricted host range among human CD4+ cells when compared with HIV-1 and HIV-2. This restricted tropism has been associated with the inability of the SIV envelope glycoprotein to induce membrane fusion in cells not susceptible In a study comparto productive exogenous infection by SIV. ing the expression of HIV and SIV in six human cell lines infectable by HIV, we found that SIV could infect only three of the lines.20 Only cell lines expressing the B-cell-associated activation markers B7/BB1 and CD40 were susceptible to infection, but by no means demonstrating, that an additional cell surface molecule may be required for efficient SIV infection. Once HIV enters the host cell, reverse transcription, RNA expression, and production of virus particles is about as efficient in monocytes as in T cells.21 Thus, a major limiting step for establishing infection may be the steps regulating viral entry into the host cell cytoplasm. Two recent studies are consistent with this hypothesis: Using a molecularly cloned HIV-1 restricted for replication in T-cell lines, Cann and co-workers22 found that little or no viral DNA or RNA was synthesized in nonpermissive cells after infection. At least one block to infection in nonpermissive T cells occurs at a point in entry or uncoating before provirus formation.22 Another study, analyzing HIV-1 replication in four T-cell lines, showed that a major determinant of the permissiveness of growing T cells for HIV-1 is the rate and efficiency of virus entry,23 a result consistent with our studies
with SIV.20 Although a variety of murine retroviruses appear to exhibit cellular tropism on the basis of specific alterations in their long terminal repeats (LTRs), the LTR does not appear to play a major role in determining cellular tropism of HIV.24
SIV INFECTS A SUBPOPULATION OF CD4+ T CELLS
Collectively, the above studies suggest that HIV/SIV infectivity can be greatly influenced at the entry step, perhaps by
receptors distinct from CD4. Thus, we examined whetherCD4+ T-cell subsets differing in their expression of other markers are more or less susceptible to SIV infection. We found that a subset ofCD4+ T cells expressing high levels of the adhesion receptor, CD44 (CD44hl), are selectively lost in SIV-infected macaques prior to the onset of AIDS25 and that SIV selectively replicates in this subset in vitro. Based on their expression of CD44 and a form of the CD45 (T200) membrane protein tyrosine phosphatase (PTPase) marker, CD45RA, macaque CD4 subsets can+ be divided into at least three populations:26 CD441", CD45RA cells, which contain principally resting cells not in cycle, CD44hi, CD45RA+ subset, which contain virtually all the cells in S phase, and CD44hi, CD45RA~ cells, which contain most of the memory T cells, also subseted as CD45RO+ cells.27'28 We isolatedeitherCD44hi""orCD45RA+/RA CD4+ subsetsfrom SIV-infected macaques; using a polymerase chain reaction (PCR) method with oligonucleotide probes to the SIV gag region, we were not able to detect SIV DNA in CD441" cells, but we could detect it in CD44hi, CD45RA+, orCD45RA~ cells.2" These results demonstrated that SIV infection is restricted to a definable subset of CD4+ T cells which includes memory T cells. Similar results have been obtained with HIV: HIV was found in CD45RO+ cells30 and the memory response of CD45RO ' T cells was shown to be defective in HIV-infected individuals. These results suggest that memory T cells or cells in the process of becoming memory T cells are an initial target for SIV and HIV infection. However, whether or not CD45RA and CD45RO are in fact good markers for "naive" and "'memory" T cells is controversial, particularly in light of recent findings demonstrating that CD45RO+ cells can "revert" to a CD45RA + phenotype.3l'31a Furthermore, CD4+ CD45RO+ generally comprise about 5-10% of CD4+ cells in blood (Fig. 1), but HIV and SIV DNA are found in
