In vitro polymerization of microtubules from HeLa cells.

Although the purification of microtubules from brain by alternate cycles of polymerization and depolymerization in vitro has become routine, the application of this method to non-neural cultured cells has been less successful. Previous investigations have suggested that it was necessary to use substrate-grown cells and 4 M glycerol to obtain microtubules from cultured cells. We have developed a method for preparing microtubules from HeLa cells in spinner cultures without the use of glycerol. Microtubules can be readily carried through two complete cycles of polymerization at 37 degrees C and depolymerization at 4 degrees C in vitro. The microtubules obtained are morphologically similar to brain microtubules in electron micrographs, and the tubulin subunits have mobilities similar to those of brain tubulins on polyacrylamide gels. Typical yields in the second polymerization pellet are about 1 mg protein/ml of packed cells or 2.5-3.0% of the total protein in the soluble cell extract. The major nontubulin protein present after two cycles of polymerization and depolymerization has an apparent mol wt of 68,000 daltons. If glycerol is used during polymerization, this band is virtually absent.

Microtubule-associated proteins of HeLa cells: heat stability of the 200,000 mol wt HeLa MAPs and detection of the presence of MAP-2 in HeLa cell extracts and cycled microtubules.

One of the major groups of microtubule-associated proteins (MAPs) found associated with the microtubules isolated from HeLa cells has a molecular weight of just over 200,000. Previous work has demonstrated that these heLa MAPs are similar in several properties to MAP-2, one of the major MAPs of mammalian neural microtubules, although the two types of proteins are immunologically distinct. The 200,000 mol wt HeLa MAPs have now been found to remain soluble after incubation in a boiling water bath and to retain the ability to promote tubulin polymerization after this treatment, two unusual properties also shown by neural MAP-2. This property of heat stability has allowed the development of a simplified procedure for purification of the 200,000 HeLa MAPs and has provided a means for detection of these proteins, even in crude cell extracts. These studies have also led to the detection of a protein in crude extracts of HeLa cells and in cycled HeLa microtubules which has been identified as MAP-2 on the basis of (a) comigration with calf brain MAP-2 on SDS PAGE, (b) presence in purified microtubules, (c) heat stability, and (d) reaction with two types of antibodies prepared against neural high molecular weight-MAPs, one of these a monoclonal antibody against hog brain MAP-2, although present in HeLa cells, is at all stages of microtubule purification a relatively minor component in comparison to the 200,000 HeLa MAP's.

Positive and negative aspects of the human immunodeficiency virus protease: development of inhibitors versus its role in AIDS pathogenesis.

In this review we summarize multiple aspects of the human immunodeficiency virus (HIV) protease from both structural and functional viewpoints. After an introductory overview, we provide an up-to-date status report on protease inhibitors (PI). This proceeds from a discussion of PI structural design, to how PI are optimally utilized in highly active antiretroviral triple therapy (one PI along with two reverse transcriptase inhibitors), the emergence of PI resistance, and the natural role of secretory leukocyte PI. Then we switch to another focus: the interaction of HIV protease with other genes in acute and persistent infection, which in turn may have an effect on AIDS pathogenesis. We conclude with a discussion on future directions in HIV treatment, involving multiple-target anti-HIV therapy, vaccine development, and novel reactivation-inhibitory reagents.

Viral interactions with the host-cell cytoskeleton: the role of retroviral proteases.

A surprisingly large number of animal viruses interact with cytoskeletal elements inside infected cells at different stages of replication. For example, a viral protease is activated during assembly of murine leukemia viruses at the cell surface, which causes both the morphological conversion of 'immature' to 'mature' particles and a drastic reduction of the actin stress fiber network.

A dominant epitope of HIV-1 protease recognized by hamster monoclonal antibodies.

Monoclonal antibodies (mAbs) against HIV-1 protease (HIV PR), the essential protease of human immunodeficiency virus type 1 (HIV-1), were produced in Armenian hamsters. Studies of direct binding to synthetic peptides and inhibition of binding to intact protease by peptide competition showed that five mAbs recognized an epitope that includes the sequence LPGRWKPK (residues 38-45), which lies near the region of the protease called the flap. All of the mAbs react specifically with HIV PR in Western blots. Because of structural conservation of the epitope in the proteases of many HIV-1 isolates, mAbs to this epitope are likely to be useful for detection of HIV PR in field isolates of HIV-1. Also, mAbs specific for this epitope, which lies close to the flap of HIV PR, may be useful for functional studies of HIV PR and possibly for the design of inhibitors of protease activity that bind outside the enzyme's catalytic site.

Increased visualization of microtubules by an improved fixation procedure.

We have found that when a buffer utilized for in vitro polymerization of microtubules, i.e., 1 mM guanosine triphosphate, 1 mM MgSO4, 2 mM ethylene glycol bis(beta-aminoethyl ether)-N, N'-tetraacetic acid 100 mM piperazine-N,N'-bis(2-ethanesulfonic acid), pH 6.9 polymerization mix, was used in the glutaraldehyde prefixation regimen instead of classical fixative buffers, i.e., isotonic cacodylate or phosphate buffer, the following features were observed in thin-sections of the cytoplasm of interphase HeLa cells: (a) a greater than 2-fold increase in total microtubule contour length, (b) a 2-fold increase in a number of microtubules greater than or equal to 1 mu long, (c) an enhanced association of microtubules with cytoplasmic organelles, and (d) an increased clustering of 100 A filaments located in a perinuclear region of the cell. Furthermore, we found that after we incubated purified chick brain microtubules on a Sephadex G-25 column pre-equilibrated with polymerization mix, cacodylate or phosphate buffer at 37 degrees C, and then eluted the microtubules at 37 degrees C, the exposure to cacodylate or phosphate buffer caused extensive depolymerization, but exposure to polymerization mix buffer allowed reisolation of highly polymerized microtubules. Our results imply that prefixation with cacodylate or phosphate buffered glutaraldenyde destabilizes microtubules leading to the decreased visualization of microtubules.

Stabilization of actin filaments at early times after adenovirus infection and in heat-shocked cells.

Human cells (HEp-2) infected with adenovirus type 5 (Ad5) at early times (5-7 h) after infection exhibit stabilization of the filamentous actin network against disruption by latrunculin (300-2000 ng), a potent microfilament toxin. This protection is abrogated by pretreatment of infected cells with cycloheximide, suggesting that it is due to a protein induced early after Ad5 infection. Support for a role of HSP70 (heat shock protein of Mr = 70 kDa) in actin stabilization is based on several findings; (i) HSP70 is induced at early times post-infection in Ad5-infected HEp-2 cells, (ii) heat shock treatment (42 degrees C) of uninfected HEp-2 or HeLa cells results in a rearrangement of actin filaments around the nucleus, that is resistant to disruption by latrunculin, (iii) using a DNase I inhibition assay, the percentage of filamentous actin increases from 50 to 65% of total following heat shock of uninfected cells, and (iv) HSP70 induces actin polymerization from monomers in vitro.

Antigenic differences among multiply charged Moloney murine leukemia virus p30 polypeptides found inside infected cells.

At least three Moloney murine leukemia virus (M-MuLV) p30 polypeptides (p30's), viz., a major species at pI 6.3 and two minor ones at pI 6.1 and pI 6.6, have previously been identified in purified virions by 2-dimensional gel electrophoresis and chromatofocusing (Katoh, I., Yoshinaka, Y. and Luftig, R.B. (1984) J. Gen. Virol. 65, 733-741). We have observed a similar, but distinctive pI pattern for [35S]methionine-labeled MuLV p30's in lysates from chronically infected (MuLV) cells. The variation in pI pattern of the intracellular MuLV p30's was dependent on the type of p30 reactive antibody used for immunoprecipitation. Specifically: a p30 spot with pI 6.3 was always precipitated as the major spot with three different antibodies, minor spots with pI 6.0 and 6.6 were variably seen dependent on the antibody used, and an intracellular p30 spot at pI 6.1 was only precipitated with a rat p30 monoclonal antibody but not with monospecific mouse or intact MuLV cross-reacting p30 sera. These results indicate that first, there are differences between the pI pattern of virion and intracellular MuLV p30's, and second, the antigenic determinants of intracellular p30's vary dependent on the antibody used for immunoprecipitation.

An unexpected effect of divalent cations on the adenovirus endogenous protein kinase: alteration in the specificity of phosphorylation.

The adenovirus endogenous protein kinase specifically phosphorylates capsid protein IIIa in the presence of Mg2+, utilizing either ATP or GTP as a phosphate donor. When Mn2+ is substituted for Mg2+, in the presence of ATP, phosphorylation of IIIa is enhanced by 2-3 fold. However, in addition to IIIa phosphorylation, the core proteins V and VII are now phosphorylated. A similar finding is made when Co2+ is used instead of Mg2+. Further, when Mn2+ or Co2+ is substituted for Mg2+, the phosphoamino acid residue profile is changed, viz., instead of only phosphoserine being labeled, phosphothreonine is now extensively labeled. These results are specific for the above endogenous viral proteins, since when the viral protein kinase is used to phosphorylate added exogenous substrates such as casein, no enhancement of phosphorylation nor any change in phosphoamino acid profile is achieved by substituting Mn2+ for Mg2+. It is thus likely that MnATP or CoATP somehow interacts differently with adenovirus structural proteins than does MgATP and this facilitates their accessibility to the enzyme.

Evidence for the existence of a thermolabile element in adenovirus type 5 that plays a role during an early stage of infection.

In an attempt to learn the molecular mechanism behind the initial steps (before genome expression) of adenovirus infection, the effect of temperature treatment of adenovirus type 5 particles on their infectivity was studied. It was found that adenovirus type 5 could be inactivated by temperature treatment at between 43 and 44 degrees C. Both biochemical and biophysical examination of adenovirus particles failed to disclose any significant changes after exposure to temperatures up to 45 degrees C. Further, viral particles treated with the same temperature range retained their ability to attach to cell surface receptors and to penetrate the plasma membrane. Application of a temperature pulse of 44 degrees C to adenovirus-infected HeLa cells indicates that the infection can be inhibited by exposure of infected cells to a brief temperature pulse of 44 degrees C between 10 and 50 min postadsorption with a mid-point of 30 min postadsorption. In addition, electron microscopic examination of HeLa cells following a temperature pulse of 44 degrees C suggests that there is a failure to accumulate viral particles at the perinuclear region. These findings support the hypothesis that adenovirus contains a thermolabile element which plays a crucial role during an early stage of infection. Possible correlation of this element with the adenovirus endogenous protein kinase is also examined and discussed.

The effect of cerulenin on Moloney murine leukemia virus morphogenesis.

Cerulenin is an antibiotic that interferes with fatty acid synthesis in eukaryotic cells. It had been shown by Schultz and Oroszlan (1983), that murine leukemia virus (MuLV) Pr65gag, the polyprotein precursor to the virion core proteins contains the fatty acid myristate at its NH2 terminus. We showed that when 20 micrograms/ml of cerulenin is added for 3 h to mouse fibroblasts chronically infected with Moloney (M)-MuLV it causes a greater than 4-fold decrease in virus production. This is accompanied by an accumulation of uncleaved Pr65gag in the infected cells. Further, thin-section electron micrographs of cerulenin-treated cells show a 2-fold increase in the number of nascent-budding forms, as well as the appearance of aberrant viral forms at the cell membrane. This suggests that the failure to add myristic acid to Pr65gag prevents their proper assembly into viral particles.

AIDS pathogenesis: the role of accessory gene mutations, leading to formation of long-lived persistently infected cells and/or apoptosis-inducing HIV-1 particles.

Human immunodeficiency virus type 1 (HIV-1) infection indirectly induces activation-dependent apoptosis in bystander immune CD4+ T-cells, a hallmark of AIDS pathogenesis. It is well known that this pathogenetic event is significantly correlated with a high virus load. Active viral replication occurs in HIV-1 asymptomatic carriers throughout all stages of clinical disease. Most of the HIV-1 in plasma is derived from short-lived infected cells with a half life of a few days; however, a minor population of virus is derived from long-lived persistently and latently infected cells. Recently, the importance of such latent reservoirs for HIV-1 has come to the forefront because of studies with potent antiretroviral inhibitors that block only new rounds of infection. An initial large drop in viral load occurs within two weeks as noted by a decrease in plasma viremia. This is then followed by a slower second-phase decay, since only a small fraction of latently infected resting CD4+ T-cells carry replication-competent, integrated provirus. This review highlights the mechanisms of apoptosis induction in bystander immune cells by both protease-defective, gp120-containing HIV-1 particles, as well as by wild-type virus that appears to be derived predominantly from long-lived infected cells. A model involving the NH2-terminal Nef domain (p7) in this 'bystander apoptosis' event is also presented.

Human adenovirus type 41 contains two fibers.

DNA sequencing of the subgroup F human adenovirus serotype 41 (TAK, Ad41) fiber gene revealed the presence of two adjacent open reading frames encoding information for proteins with molecular weights of 60.6 kDa and 41.4 kDa (Pieniazek, et al; Nucleic Acids Res. 18: p. 1901, 1990). In this paper, various approaches were used to characterize the two proteins and determine whether both fibers were expressed in infected cells as well as on viral particles. We initially used a reverse transcriptase-polymerase chain reaction with primers for the short and long fiber genes to amplify mRNA from Ad41 infected HEp-2 cells at 48 h post-infection. Two distinct DNA bands; one slightly larger than 1.1 kbp and the other at about 1.7 kbp were identified. Second, we used polyclonal anti-Ad41 virion and monoclonal anti-Ad5 fiber antibodies to demonstrate that at both 24 and 36 h post-infection, Ad41 expressed two fiber proteins of the expected size. Specifically, by SDS-PAGE, one fiber (short) had a molecular weight of 40 kDa, while the other (long) had a molecular weight of 60 kDa. Third, by electron microscopy, two sizes of fibers were released from CsCl purified virions, both having a characteristic adenovirus morphology, with a knob at one end. The long fiber measured 315A in length and the short fiber was 250A long. These measurements are consistent with the two Ad41 fibers being encoded by the above open reading frames. We also performed a computer search to compare fiber sequences from other human adenovirus serotypes with that of the Ad41 short and long fiber proteins. The primary structure of both Ad41 fibers were found to be similar in that they contained tail, shaft and knob regions. Further, the tail region of both fibers (amino acids 1-42) showed a 74% overall homology to each other and contained the Ad conserved sequence NH2-F-N-P-V-Y-P-Y-COOH. An interesting difference, however, was observed in the shaft region where the long fiber (amino acids 43-389) had twenty-two 16-amino acid repeat motifs, while the short fiber (amino acids 43-233) had only twelve. Finally, we noted that the long fiber knob region was about 15% longer than that of the short fiber, and showed little overall homology. In conclusion, human adenovirus subgroup F (type 41) virions appear to differ from those of all other human adenoviruses (subgenera A-E) in that they contain two fiber genes and correspondingly, two different sized fibers.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential susceptibility of resting CD4(+) T lymphocytes to a T-tropic and a macrophage (M)-tropic human immunodeficiency virus type 1 is associated with their surface expression of CD38 molecules.

Recent evidence has accumulated which definitively shows that chemokine receptors CCR5 and CXCR4 play an essential role as coreceptors for human immunodeficiency virus type 1 (HIV-1) infection. Flow cytometric analysis permitted us to detect CD38, a surface marker of early differentiation, as well as activation of T cells, on about half of healthy donor-derived CD4(+) T cells. In this study, we focused on the susceptibility of CD38(+) and CD38(-) subsets of CD4(+) T cells to HIV-1 infection with different coreceptor tropisms. About 20% of peripheral blood mononuclear cell-derived resting CD4(+) T cells were recovered into the CD38(+) subset fraction by panning with a monoclonal antibody to CD38. Most of the cells in this CD38(high) fraction also expressed CD45RA and CD62L at higher intensities compared with those of CD38(low) fraction. CCR5(+) T cells predominated in the CD38(-) subset, although cell surface expression of CD4 and CXCR4 was almost similar between both subsets. This difference was consistent with a significantly higher susceptibility of the CD38(-) subset to a macrophage (M)-tropic HIV-1 strain. In contrast, it was shown that a T-tropic strain of HIV-1 could replicate more efficiently in the CD38(+) subset, although viral adsorption rates were similar between both subsets. Thus, the differential susceptibility of CD4(+) T cells to M(-) and T-tropic HIV-1 was associated with their surface expression of CD38.

Fusion of uninfected T-cells occurs with immature HIV-1 protease-mutant, but not morphologically similar protease inhibitor derived particles.

Protease inhibitors are widely used in the treatment of human immunodeficiency virus type 1 (HIV-1)-infected individuals and show a drastic effect on the reduction of virus load. We previously reported that doughnut-shaped, protease-defective gp120-containing HIV-1 particles from an L-2 cell clone, carrying a provirus with mutations at the pol (protease), env (gp41) and nef genes, rapidly and more effectively induces virus particle-mediated syncytia formation of uninfected T-cells, than a parental wild-type laboratory strain of HIV-1 (LAI). In this study, we examined the possibility of whether enhanced syncytia formation is mediated by morphologically similar doughnut-shaped particles obtained after treatment of LAI-infected cells with the protease inhibitors L-689, 502, DMP-323, RO-31-8959, and KNI-272. Utilizing such protease inhibitor-induced particles and a clone of MOLT-4 cells, we could not detect any enhancement of syncytia formation, over that seen with wild-type LAI particles. This result should alleviate concerns of patients on highly active antiretroviral therapy (HAART), that protease inhibitors might accelerate progression of the disease through enhanced production of defective, 'immature'-appearing particles.

Serial passage of human immunodeficiency virus type 1 generates misalignment deletions in non-essential accessory genes.

Human immunodeficiency virus type 1 (HIV-1) derived from an infectious molecular clone pNL432 was extensively passaged in tissue culture by repeated rounds of acute infection. We previously showed the natural occurrence of a nonsense mutation in the vpr gene during continued passage of this virus. In this report, we show that two forms of large deletions (561 and 518 base pairs containing short direct repeats at the deletion junctions) occur after passage 50 in the region that spans the vif and vpr open reading frames. One model to explain the occurrence of these deletion regions is that such mutations result from misalignment of the growing point at a limited number of nucleotide positions. Infection of CD4+ T-cells with a recombinant HIV-1 construct containing the same vif to vpr deletion showed virtually no cytopathogenic phenotype. Thus, misalignment deletions at non-essential accessory genes of HIV-1 might be induced during replication, which result in the generation of virus with a low cytopathogenic potential.

A specific T-cell subset with CD4+/CD38- markers derived from HIV-1 carriers induces apoptosis in healthy donor-derived T-lymphocytes.

Apoptosis is an important mechanism of human immunodeficiency virus type 1 (HIV-1)-induced T-cell depletion. Our recent findings revealed mitogenic stimulation-dependent apoptosis induction in healthy donor-derived peripheral blood T-lymphocytes after adsorption with defective HIV-1 particles through acquirement by a subset of CD4+/CD38- cells of specific killer function. Based on these in vitro observations, we have extended the significance of this killing activity of CD4+/CD38- cells directly derived from HIV-1 carriers. The CD4+/CD38- cells from HIV-1-positive individuals showed significantly higher cell-killing activities than those from HIV-1-negative donors by co-culture with allogeneic resting T-cells after mitogenic stimulation. Furthermore, most of the samples induced apoptosis in a Fas-dependent manner. Thus, it is suggested that HIV-1 infection-related apoptosis is triggered by inappropriate activation of a certain resting T-cell subset, presumably due to adsorption with HIV-1 particles.

Inhibition of bacterially expressed HIV protease activity determined by an in vitro cleavage assay with MuLV Pr65gag.

HIV protease is a virally coded enzyme that cleaves gag as well as gag-pol precursor polyproteins into functional products needed for virus assembly. A pUC plasmid containing an HIV insert starting at the 5' end of the pol gene and ending just inside the intergrase coding sequence was expressed in E. coli. It provided an 11 kD gene product (protease) that specifically cleaved the Gazdar MuLV Pr65gag precursor into Pr40gag (p30 + p10) and Pr27gag (p15 + p12) intermediates, as well as lower molecular weight gag-encoded products. These were detected by immunoblotting with either MuLV anti-p30 or p12 sera. Using cleavage of MuLV Pr65gag as an assay system, pepstatin A, fusidic acid, and cerulenin were observed to inhibit HIV protease cleavage by greater than 50% at concentrations of 0.1, 0.2-0.5, and 0.5 mM, respectively.