Palmitoylation of the Rous sarcoma virus transmembrane glycoprotein is required for protein stability and virus infectivity.

The Rous sarcoma virus (RSV) transmembrane (TM) glycoprotein is modified by the addition of palmitic acid. To identify whether conserved cysteines within the hydrophobic anchor region are the site(s) of palmitoylation, and to determine the role of acylation in glycoprotein function, cysteines at residues 164 and 167 of the TM protein were mutated to glycine (C164G, C167G, and C164G/C167G). In CV-1 cells, palmitate was added to env gene products containing single mutations but was absent in the double-mutant Env. Although mutant Pr95 Env precursors were synthesized with wild-type kinetics, the phenotypes of the mutants differed markedly. Env-C164G had properties similar to those of the wild type, while Env-C167G was degraded faster, and Env containing the double mutant C164G/C167G was very rapidly degraded. Degradation occurred after transient plasma membrane expression. The decrease in steady-state surface expression and increased rate of internalization into endosomes and lysosomes paralleled the decrease in palmitoylation observed for the mutants. The phenotypes of mutant viruses were assessed in avian cells in the context of the pATV8R proviral genome. Virus containing the C164G mutation replicated with wild-type kinetics but exhibited reduced peak reverse transcriptase levels. In contrast, viruses containing either the C167G or the C164G/C167G mutation were poorly infectious or noninfectious, respectively. These phenotypes correlated with different degrees of glycoprotein incorporation into virions. Infectious revertants of the double mutant demonstrated the importance of cysteine-167 for efficient plasma membrane expression and Env incorporation. The observation that both cysteines within the membrane-spanning domain are accessible for acylation has implications for the topology of this region, and a model is proposed.

Type D retrovirus Gag polyprotein interacts with the cytosolic chaperonin TRiC.

The carboxy terminus-encoding portion of the gag gene of Mason-Pfizer monkey virus (M-PMV), the prototype immunosuppressive primate type D retrovirus, encodes a 36-amino-acid, proline-rich protein domain that, in the mature virion, becomes the p4 capsid protein. The p4 domain has no known role in M-PMV replication. We found that two mutants with premature termination codons that remove half or all of the p4 domain produced lower levels of stable Gag protein and of self-assembled capsids. Interestingly, yeast two-hybrid screening revealed that p4 specifically interacted with TCP-1gamma, a subunit of the chaperonin TRiC (TCP-1 ring complex). TRiC is a cytosolic chaperonin that is known to be involved in both folding and subunit assembly of a variety of cellular proteins. TCP-1gamma also associated with high specificity with the M-PMV pp24/16-p12 domain and human immunodeficiency virus p6. Moreover, in cells, Gag polyprotein associated with the TRiC chaperonin complex and this association depended on ATP hydrolysis. In the p4 truncation mutants, the Gag-TRiC association was significantly reduced. These results strongly suggest that cytosolic chaperonin TRiC is involved in Gag folding and/or capsid assembly. We propose that TRiC associates transiently with nascent M-PMV Gag molecules to assist in their folding. Consequently, properly folded Gag molecules carry out the intermolecular interactions involved in self-assembly of the immature capsid.

Activation of monocytes, T-lymphocytes and plasma inflammatory markers in angina patients.

Inflammation and activation of immune cells have important roles in the pathogenesis of atherosclerosis. We analyzed the plasma levels of inflammatory markers and the degree of activation of peripheral blood monocytes and T-lymphocytes isolated from 12 unstable angina, 12 stable angina, and 12 normal subjects. In 20%-33% of patients, monocytes expressed high basal levels of IL-8, tissue factor, IL-1beta, and monocyte chemoattractant protein-1 mRNA. Furthermore, basal mRNA levels of these cytokines showed strong correlation with each other (p < 0.01 in all combination) but not with tumor necrosis factor-alpha or transforming growth factor-beta1. Plasma level of C-reactive protein was highest in the unstable angina patients (1.63+/-0.70 mg/l) and lowest in the control subjects (0.22+/-0.08 mg/l) (P = 0.03). We also observed a high correlation between C-reactive protein level and the occurrence of minor and major coronary events during 6 months of follow-up. Activation status of T-cells, assessed by the percentage of HLA-DR positive cells, was highest in the unstable angina patients (26.8+/-1.4%) compared with that in the control (14.7+/-1.2%) (P = 0.0053). Our data represent the first case showing that the circulating monocytes in angina patients are activated to a state express numerous proatherogenic cytokines. These results may help to diagnose angina patients according to the inflammatory markers and evaluate the prognosis of the disease.

Inhibition of protein phosphatase 1 decreases PTH secretion from isolated dispersed parathyroid cells.

To investigate the regulation of parathyroid hormone secretion by phosphatases we examined the effect of okadaic acid, a selective inhibitor of protein phosphatases (PP)-1 and -2A, on isolated, dispersed parathyroid cells. Okadaic acid inhibited secretion from intact bovine, intact human and streptolysin-O permeabilized bovine cells. Approximately 10(-6) M okadaic acid resulted in a 50% decrease in parathyroid hormone (PTH) secretion from both intact and permeabilized cells, consistent with PP-1 being the target of inhibition. Upon subcellular fractionation, PP-1 overlapped but was not identical to either PTH, a marker of the secretory granule, or Na+/K+-ATPase, a plasma membrane marker. In summary, PP-1 activity is involved in Ca2+-dependent but not basal PTH secretion.

Identification of a cytoplasmic targeting/retention signal in a retroviral Gag polyprotein.

Retroviral capsid assembly can occur by either of two distinct morphogenic processes: in type C viruses, the capsid assembles and buds at the plasma membrane, while in type B and D viruses, the capsid assembles within the cytoplasm and is then transported to the plasma membrane for budding. We have previously reported that a single-amino-acid substitution of a tryptophan for an arginine in the matrix protein (MA) of Mason-Pfizer monkey virus (MPMV) converts its capsid assembly from that of a type D retrovirus to that of the type C viruses (S. S. Rhee and E. Hunter, Cell 63:77-86, 1990). Here we identify a region of 18 amino acids within the MA of MPMV that is responsible for type D-specific morphogenesis. Insertion of these 18 amino acids into the MA of type C Moloney murine leukemia virus causes it to assemble an immature capsid in the cytoplasm. Furthermore, fusion of the MPMV MA to the green fluorescent protein resulted in altered intracellular targeting and a punctate accumulation of the fusion protein in the cytoplasm. These 18 amino acids, which are necessary and sufficient to target retroviral Gag polyproteins to defined sites in the cytoplasm, appear to define a novel mammalian cytoplasmic targeting/retention signal.

HIV-1 Nef plays an essential role in two independent processes in CD4 down-regulation: dissociation of the CD4-p56(lck) complex and targeting of CD4 to lysosomes.

Human immunodeficiency virus type 1 (HIV-1) Nef down-regulates CD4 by triggering rapid endocytosis of cell surface CD4. To better understand how Nef induces CD4 down-regulation, we generated a series of Nef mutants with small in-frame deletions in the coding region. Three classes of mutants were obtained. The first class produces neither CD4 down-regulation nor dissociation of the CD4-p56(lck) complex. The second class induces CD4 down-regulation in cells lacking p56(lck) expression, but not in cells with p56(lck);these mutants fail to dissociate CD4 from p56lck. These results show that Nef-mediated CD4 dissociation from p56(lck) is important for CD4 down-regulation. The third class of mutants is able to dissociate the CD4-p56(lck) complex but fails to down-regulate surface CD4; internalized CD4 molecules are recycled back to the cell surface. This result suggests that Nef diverts the CD4 recycling pathway to a degradative pathway. We also demonstrate that Nef associates with phosphatidylinositol-3-kinase (PI3K) activity, which is known to be involved in several aspects of membrane trafficking. However, Nef mutants that cause internalized CD4 to be recycled do not associate with PI3K activity; thus Nef-associated PI3K activity might be involved in the latter process of targeting CD4 to a degradative pathway. We conclude that HIV-1 Nef plays a critical role in multiple processes in CD4 down-regulation: (i) disrupting the CD4-p56(lck) complex on the cell surface to allow CD4 internalization and (ii) diverting the internalized CD4 to a lysosomal pathway for its degradation, likely through a PI3K activity.

Reconstitution of calcium-regulated parathyroid hormone secretion from streptolysin-O-permeabilized parathyroid cells by guanosine 5'-O-(thio)triphosphate.

Intracellular Ca2+ levels determine the amount of PTH secretion from parathyroid cells. Dissociated calf parathyroid cells were permeabilized with streptolysin-O (SLO) to provide an in vitro model system to examine Ca(2+)-dependent regulation of hormone secretion. PTH release from these cells was energy dependent and increased by cytosolic cofactors. Guanosine 5'-O-(thio)triphosphate (GTP gamma S) increased PTH secretion from SLO-permeabilized cells in a dose-dependent manner from 0.1-100 microM. In the absence of GTP gamma S there was no relationship between the ambient Ca2+ concentration and the rate of PTH secretion. However, in the presence of GTP gamma S, intracellular Ca2+ inhibited PTH secretion with an EC50 of approximately 0.1 microM, corresponding to physiological intracellular Ca2+ levels. Thus, the addition of GTP gamma S to SLO-permeabilized parathyroid cells reconstituted the inverse relationship between extracellular Ca2+ and PTH secretion that is observed in vivo and in intact cells. The data indicate that this effect is mediated at least in part by heterotrimeric guanosine triphosphatases. In addition, calcium/calmodulin-dependent protein kinase II appears to mediate low Ca(2+)-dependent PTH secretion from these cells.

Efficient in vivo and in vitro assembly of retroviral capsids from Gag precursor proteins expressed in bacteria.

The capsid precursor protein (Gag) of Mason-Pfizer monkey virus, the prototype type D retrovirus, has been expressed to high levels in bacteria under the control of the phage T7 promoter. Electron microscopic studies of induced cells revealed the assembly of capsid-like structures within inclusion bodies that formed at the poles of the cells 6 h after induction with isopropyl-beta-D-thiogalactopyranoside (IPTG). The inclusion bodies and enclosed capsid-like structures were solubilized completely in 8 M urea, but following renaturation, we observed assembly in vitro of capsid-like structures that demonstrated apparent icosahedral symmetry. These results demonstrate for the first time that retroviral capsid precursors have the propensity to self-assemble in vitro and point to new approaches for the analysis of retroviral assembly and structure.

Human immunodeficiency virus type 1 Nef-induced down-modulation of CD4 is due to rapid internalization and degradation of surface CD4.

Human immunodeficiency virus type 1 (HIV-1) Nef is a myristylated protein with a relative molecular mass of 27 kDa, is localized to the cytoplasmic surfaces of cellular membranes, and has been reported to down-modulate CD4 in human T cells. To understand the mechanism of HIV-1 Nef-mediated down-modulation of cell surface CD4, we expressed Nef protein in human T-cell line VB. Expression of HIV-1 Nef protein down-modulated surface CD4 molecules. In pulse-chase experiments, CD4 molecules in Nef-expressing cells were synthesized at normal levels. However, the bulk of newly synthesized CD4 protein was degraded with a half-life of approximately 6 h, compared with the 24-h half-life in control cells. This Nef-induced acceleration of CD4 turnover was inhibited by lysosomotropic agents NH4Cl and chloroquine as well as by the protease inhibitor leupeptin. Surface CD4 biotinylation experiments demonstrated that CD4 molecules in Nef-expressing T cells are transported to the plasma membrane with normal kinetics but are then rapidly internalized. Therefore, HIV-1 Nef-induced down-modulation of CD4 is due to rapid internalization of surface CD4 and subsequent degradation by an acid-dependent process, potentially lysosomal. Additionally, in a Nef-expressing cell, we find accelerated dissociation of the T-cell tyrosine kinase p56lck and CD4 but only after the complex reaches the plasma membrane. This implies that HIV-1 Nef protein might play a role in triggering a series of T-cell activation-like events, which contribute to p56lck dissociation and internalization of surface CD4 molecules.

Postassembly cleavage of a retroviral glycoprotein cytoplasmic domain removes a necessary incorporation signal and activates fusion activity.

Viral protease-mediated cleavage within the cytoplasmic domain of the transmembrane (TM) glycoprotein of the type D retrovirus, Mason-Pfizer monkey virus, removes approximately 16 amino acids from the carboxy terminus of the protein. To determine the functional significance of this cleavage in the virus life cycle, we introduced premature stop codons into the TM coding domain, resulting in the production of truncated glycoproteins. Progressive truncated of the cytoplasmic domain identified the carboxy-terminal third as being required for efficient incorporation of the glycoprotein complex into budding virions and profoundly increased the fusogenic capability of the TM glycoprotein. These results, together with the ability of matrix protein mutations to suppress TM cleavage, imply that this portion of the glycoprotein interacts specifically with the capsid proteins during budding, suppressing glycoprotein fusion function until virus maturation has occurred.

HIV-1 Nef activity in murine T cells. CD4 modulation and positive enhancement.

Immediately after infection of the targeted cell by HIV-1, proviral gene expression is limited to the three regulatory proteins, Tat, Rev, and Nef, with the nef transcript representing nearly 80% of total expression. Additionally, simian immunodeficiency virus Nef has been shown to be essential for high in vivo titer and the development of immunodeficiency. Recent findings demonstrate that the negative effects of Nef expression, as first defined in transformed T cell lines, are not present when Nef is expressed in primary human T cells or in T cells from transgenic mice, in which one sees moderate positive enhancements of HIV replication and the T cell activation process, respectively. We find that Nef expression in an Ag-specific murine T cell hybridoma results in both the down-modulation of CD4, as seen in primary cells and human T cell lines, and a positive enhancement of the TCR response to stimuli. Examination of a CD4- cell demonstrated that the positive enhancement is independent of CD4 expression or modulation. CD4 down-modulation is shown to be caused by a post-Golgi, acid-dependent process, which dramatically decreases the lifespan of the CD4 molecule. The TCR, Thy Ag, and CD45 remained unchanged in their surface expression. These findings suggest that Nef alters the normal routing and residencies of the CD4 molecule and that the positive effect of Nef on T cell activation is independent of this modulation.

Importance of p12 protein in Mason-Pfizer monkey virus assembly and infectivity.

Mason-Pfizer monkey virus (M-PMV) represents the prototype type D retrovirus, characterized by the assembly of intracytoplasmic A-type particles within the infected-cell cytoplasm. These immature particles migrate to the plasma membrane, where they are released by budding. The gag gene of M-PMV encodes a novel protein, p12, just 5' of the major capsid protein (CA) p27 on the polyprotein precursor. The function of p12 is not known, but an equivalent protein is found in mouse mammary tumor virus and is absent from the type C retroviruses. In order to determine whether the p12 protein plays a role in the intracytoplasmic assembly of capsids, a series of in-frame deletion mutations were constructed in the p12 coding domain. The mutant gag genes were expressed by a recombinant vaccinia virus-T7 polymerase-based system in CV-1 cells or in the context of the viral genome in COS-1 cells. In both of these high-level expression systems, mutant Gag precursors were competent to assemble but were not infectious. In contrast, when stable transfectant HeLa cell lines were established, assembly of the mutant precursors into capsids was drastically reduced. Instead, the polyprotein precursors remained predominantly soluble in the cytoplasm. These results show that while p12 is not required for the intracytoplasmic assembly of M-PMV capsids, under the conditions of low-level protein biosynthesis seen in virus-infected cells, it may assist in the stable association of polyprotein precursors for capsid assembly. Moreover, the presence of the p12 coding domain is absolutely required for the infectivity of M-PMV virions.

A viral protease-mediated cleavage of the transmembrane glycoprotein of Mason-Pfizer monkey virus can be suppressed by mutations within the matrix protein.

The envelope glycoprotein precursor of retroviruses undergoes proteolytic cleavage in the Golgi complex to yield the mature surface and transmembrane (TM) glycoproteins of the virus. We report here that the TM glycoprotein of Mason-Pfizer monkey virus undergoes a second proteolytic processing event during a late maturation step that can follow virus release and Gag polyprotein cleavage. Cleavage results in the conversion of the cell-associated TM glycoprotein (gp22) to a virus-associated gp20. Processing continues after virus release and yields virions that contain predominantly gp20. A mutation within the active site of the Mason-Pfizer monkey virus aspartyl protease was shown to block both TM glycoprotein cleavage and the processing of the Gag polyprotein precursor. The role of the viral protease in cleavage of the TM glycoprotein localizes the cleavage site to the cytoplasmic domain of this protein. Surprisingly, point mutations within the matrix (MA) coding region of the gag gene can affect the extent to which gp22 is processed to gp20 and in one case [p10(MA)-A79V] results in greater than 90% inhibition of gp22 cleavage. The data provide genetic evidence of a specific interaction between the capsid proteins and the cytoplasmic domain of the TM glycoprotein of a retrovirus. This interaction is required for cytoplasmic domain cleavage to occur and may play a critical role in virus assembly and viral infectivity.

Amino acid substitutions within the matrix protein of type D retroviruses affect assembly, transport and membrane association of a capsid.

The functional roles of the matrix (MA) protein in the assembly and maturation of retroviruses was investigated with a series of MA mutants of Mason-Pfizer monkey virus (M-PMV), an immunosuppressive type D retrovirus. The mutants we describe here were generated by the introduction of random point mutations within the MA coding domain by use of sodium bisulphite mutagenesis. Studies of these mutants show that the MA protein plays a critical role in three different, sequential events in the final stages of type D retrovirus replication: (i) folding of the gag gene-encoded precursor poly-proteins into a stable conformation for capsid assembly in the cytoplasm of infected cells; (ii) capsid transport from the site of assembly to the plasma membrane; and (iii) capsid association with, and extrusion of the membrane during virus budding. The mutants described here interfere with or block M-PMV replication at each of these stages. Large numbers of preassembled capsids accumulate within the cytoplasm of transport-defective mutant-infected cells, suggesting that transport of M-PMV capsids to the plasma membrane is an active and specific intracellular targeting process. The initial association of the capsid with the membrane may depend upon this intracytoplasmic transport process but additional protein-lipid interactions that involve the MA protein are required for membrane extrusion around the preformed capsids; in cells infected with the budding-defective mutant, assembled capsids accumulate under the inner surface of the cell plasma membrane, and are retarded in their release from the infected cell.

A single amino acid substitution within the matrix protein of a type D retrovirus converts its morphogenesis to that of a type C retrovirus.

Two different morphogenic processes of retroviral capsid assembly have been observed: the capsid is either assembled at the plasma membrane during the budding process (type C), or preassembled within the cytoplasm (types B and D). We describe here a gag mutant of Mason-Pfizer monkey virus, a type D retrovirus, in which a tryptophan substituted for an arginine in the matrix protein results in efficient assembly of capsids at the plasma membrane through a morphogenic process similar to that of type C retroviruses. We conclude that a type D retrovirus Gag polyprotein contains an additional, dominant signal that prevents immediate transport of precursors from the site of biosynthesis to the plasma membrane. Instead, they are directed to and retained at a cytoplasmic site where a concentration sufficient for self-assembly into capsids occurs. Thus, capsid assembly processes for different retroviruses appear to differ only in the intracellular site to which capsid precursors are directed.

Structural role of the matrix protein of type D retroviruses in gag polyprotein stability and capsid assembly.

To obtain a better understanding of the role of the gag gene-encoded matrix (MA) protein in the assembly and maturation of type D retroviruses, we have made five mutants with specific in-frame deletions within the p10-coding region by the use of oligonucleotide-directed mutagenesis. The changes in the Gag polyprotein made by these mutations resulted in almost identical phenotypes. In cells expressing mutant genomes, the mutant Gag polyproteins were synthesized and modified with myristic acid in a normal manner. However, they were so unstable that the bulk of the newly synthesized polyproteins was degraded within 1 h without being processed into mature structural polypeptides. In contrast, wild-type polyproteins have a processing half-life of 3.0 to 3.5 h. The mutant Gag polyproteins were assembled with very low efficiency into capsids in the cytoplasm of the mutant-infected cells. Moreover, the few capsids that formed were neither released from nor accumulated in the cells. These results suggest that the matrix protein plays an important role in guiding the correct folding of the Gag polyprotein, which is presumably crucial for both stabilizing the molecule and facilitating the intermolecular interactions that occur during assembly of immature capsids.

Preassembled capsids of type D retroviruses contain a signal sufficient for targeting specifically to the plasma membrane.

The capsids of Mason-Pfizer monkey virus (M-PMV), an immunosuppressive type D retrovirus, are preassembled in the infected cell cytoplasm and are then transported to the plasma membrane, where they are enveloped in a virus glycoprotein-containing lipid bilayer. The role of viral glycoprotein in intracellular transport of M-PMV capsids was investigated with a spontaneous mutant (5A) of M-PMV, which we show here to be defective in envelope glycoprotein biosynthesis. DNA sequence analysis of the env gene of mutant 5A reveals a single nucleotide deletion in the middle of the gene, which results in the synthesis of a truncated form of the envelope glycoprotein. Evidence is presented showing that the mutant glycoprotein is not expressed at the cell surface but is retained in the endoplasmic reticulum. Normal levels of gag-pro-pol precursor polyproteins are made and processed in mutant genome-transfected cells, and high levels of noninfectious particles lacking viral glycoprotein are released with normal kinetics into the culture medium. No intracisternal budding of capsids is observed. We conclude that viral glycoprotein is required neither for targeting preassembled capsids of M-PMV to the plasma membrane for final maturation nor for the budding process. Since the presence or absence of M-PMV glycoprotein at the site of budding does not affect the efficiency or kinetics of the targeting process, the preassembled capsid of M-PMV, in contrast to those of intracisternal type A particles, appears to have an intrinsic signal for intracellular transport to the plasma membrane.

Myristylation is required for intracellular transport but not for assembly of D-type retrovirus capsids.

The role of myristylation, a fatty acid modification of nascent polypeptides, in the assembly and intracellular transport of D-type retroviral capsids was investigated through the use of oligonucleotide-directed mutagenesis. Myristic acid is normally esterified through an amide linkage to a glycine residue at the amino terminus of the Mason-Pfizer monkey virus gag gene products. Mutant pA-1, which has a codon for valine substituted for that of the normally myristylated glycine, is completely noninfectious. While the mutant gag polyprotein precursors are synthesized at normal levels, they are not myristylated and are not cleaved to the mature virion proteins. No extracellular virus particles are released from mutant pA-1-infected cells, but intracytoplasmic A-type particles (capsids) accumulate in the cytoplasm. Since none of the intracellular capsids can be found associated with the plasma membrane, these results strongly suggest that myristylation is a critical signal for intracytoplasmic transport of completed viral capsids to their normal site of budding and release.