The Z Protein of the New World Arenavirus Tacaribe Virus Has Bona Fide Budding Activity That Does Not Depend on Known Late Domain Motifs

The arenavirus small RING finger Z protein has been shown to be the main driving force of budding for several arenaviruses. This Z budding activity was found to be mediated by the late (L)-domain motifs P(T/S)AP and PPXY, located at the C terminus of Z. Here, we show that the Z protein of Tacaribe virus (TACV), a New World arenavirus, buds efficiently from cells despite lacking the canonical L-domain motifs P(T/S)AP and PPXY. Likewise, potential L-domain motifs ASAP and YLCL present in TACV Z did not exhibit any significant contribution to TACV Z budding activity. Budding of TACV Z was Tsg101 independent but required the activity of Vps4A/B. These results indicate that TACV Z utilizes a budding mechanism distinct from that reported for other arenaviruses.Arenaviruses are enveloped viruses with a bisegmented, negative-strand (NS) RNA genome and a life cycle restricted to the cell cytoplasm (1). Each RNA segment uses an ambisense coding strategy to direct the expression of two genes that are in opposite orientations and separated by a noncoding intergenic region. The large segment (7.2 kb) encodes the late (L) protein, an RNA-dependent RNA polymerase, and the small RING finger protein Z, which is the counterpart of the matrix (M) protein found in many enveloped NS RNA viruses. The small segment (3.5 kb) encodes the viral nucleoprotein (NP) and the glycoprotein precursor (GPC). The GPC is processed by the cellular protease S1P into GP1 and GP2 (13). Trimers of GP1/GP2 form the spikes that decorate the virus surface and mediate cell entry via receptor-mediated endocytosis (12).Arenaviruses merit significant interest both as tractable experimental model systems to study acute and persistent viral infections (18, 28) and as clinically important human pathogens. The Old World virus Lassa virus (LASV) and several New World (NW) arenaviruses cause hemorrhagic fever (HF) disease in humans, posing a serious public health problem (1). LASV is estimated to infect several hundred thousand individuals yearly in the regions of West Africa where it is endemic, resulting in a high number of Lassa fever (LF) cases associated with significant mortality and high morbidity. Notably, increased travel to and from regions of endemicity has led to the importation of LF into metropolitan areas, where the virus is not endemic, around the globe (11). Likewise, the NW arenavirus Junin virus causes Argentine HF, a severe illness with hemorrhagic and neurological manifestations and a fatality rate of 15 to 30% (7, 22, 27), while the NW Machupo and Guanarito arenaviruses have emerged as causative agents of HF in Bolivia and Venezuela, respectively (22). Public health concerns about HF arenavirus infections are exacerbated by the lack of licensed vaccines and by the fact that current antiarenavirus therapies are limited to the use of the nucleoside analogue ribavirin, which is only partially effective and is associated with significant side effects. Therefore, it is important to develop novel drugs to combat human pathogenic arenaviruses.Similarly to many other enveloped NS RNA viruses, arenavirus infectious particles bud from the plasma membranes of infected cells. Evidence indicates that the M proteins of many enveloped NS RNA viruses play critical roles in virus budding (3). Accordingly, many of these M proteins are, in the absence of any other virus polypeptide, competent in budding and can form virus-like particles (VLPs). Budding of M proteins is often directed by L-domain motifs, which most frequently correspond to one of the following sequences: P(T/S)AP, PPXY, YXXL, or FPIV (3). Efficient M-mediated budding requires interactions between viral L domains and host factors, many of them involved in the cellular multivesicular body (MVB) sorting pathway (3). MVB formation requires the activity of a network of cytoplasmic protein complexes known as endosomal sorting complexes required for transport (ESCRT). Tsg101 is a component of the ESCRT-1 complex and plays a key role in the biogenesis of MVB. An AAA ATPase, Vps4, which is present in humans as two isoforms, Vps4A and Vps4B, binds to components of ESCRT-3 and mediates dissociation of ESCRT-3 complex from the endosomal membrane. We (20, 26) and others (24) have documented that Z is the driving force of arenavirus budding. As with many other bona fide viral budding proteins, all known arenavirus Z proteins contain P(T/S)AP, PPXY, or both, L-domain motifs that play a critical role in Z budding (20, 24). Tacaribe virus (TACV) Z protein, however, constitutes a unique exception, as it lacks both P(T/S)AP and PPXY L-domain motifs (Fig. ​(Fig.1A1A).Open in a separate windowFIG. 1.Arenavirus Z protein and its budding efficiency. (A) Schematic representation of Z proteins from LCMV, LASV, and TACV. G corresponds to the strictly conserved glycine residue found at position 2 of all known arenavirus Z proteins. The locations of bona fide PTAP (LASV), PPPY (LASV and LCMV), and YXXL (LASV and TACV) L-domain motifs are indicated, as well as that of the L-like domain motifs STAP (LCMV) and ASAP (TACV). (B) Budding activity of TACV Z protein. 293T cells were transfected with 0.25 μg of either pC-TACV-Z-HA or pC-LASV-Z-HA. At 36 h posttransfection, tissue culture supernatants were collected, and VLPs and total cell lysates were prepared as described previously (2). Levels of Z proteins in VLPs and cell lysates were determined by WB using an antibody to HA (sc-7392; Santa Cruz). The budding efficiency of LASV Z was set at 1.0. The data are averages and standard deviations from three independent experiments.     

Degradation of myelin proteins by cathepsin B and inhibition by E-64 analogue

Purified human brain myelin was isolated, heat-treated to inactivate the endogenous proteolytic activity and incubated with cathepsin B purified from rat liver, at pH 6.0. Incubation resulted in a marked reduction of myelin basic protein (BP) and partial breakdown of proteolipid protein or Wolfgram protein. Degradation of myelin proteins was inhibited by E-64 analogue (E-64-a). E-64 is a specific thiol protease inhibitor isolated from a solid culture of Aspergillus japonicus. The present study suggests that cathepsin B may play some role in demyelination.     

Conversion of myelin-associated glycoprotein (MAG) to a smaller derivative by calcium activated neutral protease (CANP)-like enzyme in myelin and inhibition by E-64 analogue

Using the immunoblot technique, we found that an incubation of purified human myelin in 10 mM Tris-HCl buffer at pH 7.5 resulted in the conversion of the myelinassociated glycoprotein (MAG) to a smaller derivative (dMAG). Exogenously added 5 mM CaCl₂ accelerated the conversion of MAG. In buffer containing more than 100 M of EGTA, the conversion was inhibited. In addition, the existence of endogenous calcium in purified myelin was confirmed using atomic absorption spectroscopy. The conversion was also inhibited partially by one of the thiol protease inhibitors, E-64 analogue (E-64-a). These observations suggest that the conversion of MAG is mediated by calcium-activated neutral protease (CANP)-like enzyme.     

Effects of chronic AICAR treatment on fiber composition, enzyme activity, UCP3, and PGC-1 in rat muscles.

This study was designed to determine the histological and metabolic effects of the administration of 5'-AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) for 14 successive days. AICAR treatment caused a significant decrease in the percentage of type IIB fibers and the concomitant increase in the percentage of type IIX fibers in extensor digitorum longus (EDL) muscle. The capillary density and the capillary-to-fiber ratio were not altered by AICAR. AICAR treatment increased the glycolytic and oxidative enzyme activities but not the antioxidant enzyme activities. The AICAR treatment increased the uncoupling protein 3 (UCP3) level in EDL and the peroxisome proliferator-activated receptor-gamma coactivator-1alpha protein level in the soleus and EDL muscles, whereas the myogenin level was not altered by AICAR. These results seem to imply that the chronic activation of AMPK alters such muscle histochemical and metabolic characteristics.     

Effects of Trypsin and Plasmin Treatment of Myelin on the Myelin-Associated Glycoprotein and Basic Protein

Human and rat myelin preparations were incubated with varying concentrations of trypsin and plasmin to determine the effects of these proteolytic enzymes on myelin-associated glycoprotein (MAG), basic protein, and other myelin proteins and to compare the effects with those of the neutral protease that was reported to be endogenous in myelin. Basic protein was most susceptible to degradation by both trypsin and plasmin, whereas MAG was relatively resistant to their actions. Under the assay conditions used, the highest concentrations of trypsin and plasmin degraded greater than 80% of the basic protein but less than 30% of the MAG, and lower concentrations caused significant loss of basic protein without appreciably affecting MAG. Neither trypsin nor plasmin caused a specific cleavage of MAG to a derivative of MAG (dMAG) in a manner analogous to the endogenous neutral protease. Thus the endogenous protease appears unique in converting human MAG to dMAG much more rapidly than it degrades basic protein. MAG is slowly degraded along with other proteins when myelin is treated with trypsin or plasmin, but it is less susceptible to their action than is basic protein.     

Suppressive effect of camostat mesilate (FOY 305) on acute experimental allergic encephalomyelitis (EAE)

Camostat mesilate (FOY305), a synthetic serine protease inhibitor and has been developed as a crug for pancreatitis, is effective in suppressing acute experimental allergic encephalomyelitis in Lewis rats. Loss of weight, clinical score and yield of myelin protein from brain stem were improved by daily injection of FOY305 compared with saline from day 6 after inoculation with homogenate of guinea pig spinal cord. A significant decrease of yield of myelin has been shown here for the first time in acute EAE in Lewis rat. This is in accord with myelin breakdown demonstrated morphologically. Our study also demonstrates a significant improvement of yield of myelin protein by FOY305. Our results suggest the possibility of a clinical application of this protease inhibitor for human demyelinating diseases such as multiple sclerosis.     

Expression in Escherichia coli of the Extrinsic 18-kDa Protein of Photosystem II of Spinach

A cDNA encoding the precursor for the 18-kDa protein of PSIIof spinach was expressed in Escherichia coli. When the celllysate was incubated at 7°C, the precursor was degradedby proteases of E. coli to a polypeptide of 18 kDa (P18) thatconsisted of the mature protein moiety plus the last four residuesof the transit peptide. P18 was able to reconstitute the water-oxidizingcomplex of NaCl-treated PSII membranes supplemented with the23-kDa protein. Moreover, P18 was cleaved by the prolyl endoproteinaseof spinach specifically at the Pro-12-Leu-13 bond, as was theauthentic 18-kDa protein. These properties of P18 indicate thatthe present expression system is potentially useful for studiesof the substrate specificity of the endoproteinase, as wellas of the structure-function relationships of the 18-kDa protein. (Received November 12, 1994; Accepted January 11, 1995)     

CD4 mimics targeting the mechanism of HIV entry

A structure–activity relationship study was conducted of several CD4 mimicking small molecules which block the interaction between HIV-1 gp120 and CD4. These CD4 mimics induce a conformational change in gp120, exposing its co-receptor-binding site. This induces a highly synergistic interaction in the use in combination with a co-receptor CXCR4 antagonist and reveals a pronounced effect on the dynamic supramolecular mechanism of HIV-1 entry.