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In the replication of human immunodeficiency virus type 1 (HIV-1), gag MA (matrix), a major structural protein of the virus, carries out opposing targeting functions. During virus assembly, gag MA is cotranslationally myristoylated, a modification required for membrane targeting of gag polyproteins. During virus infection, however, gag MA, by virtue of a nuclear targeting signal at its N terminus, facilitates the nuclear localization of viral DNA and establishment of the provirus. We now show that phosphorylation of gag MA on tyrosine and serine prior to and during virus infection facilitates its dissociation from the membrane, thus allowing it to translocate to the nucleus. Inhibition of gag MA phosphorylation either on tyrosine or on serine prevents gag MA-mediated nuclear targeting of viral nucleic acids and impairs virus infectivity. The requirement for gag MA phosphorylation in virus infection is underscored by our finding that a serine/threonine kinase is associated with virions of HIV-1. These results reveal a novel level of regulation of primate lentivirus infectivity.

Eukaryotic initiation factor-2B (eIF-2B) is a guanine nucleotide exchange factor (GEF) that plays a key role in the regulation of protein synthesis. In this study, we have used the baculovirus-infected Sf9 insect cell system to express and characterize the five dissimilar subunits of rat eIF-2B. GEF activity was detected in extracts of Sf9 cells expressing the ⑀-subunit alone and was greatly increased when all five subunits were coexpressed. In addition, high GEF activity was observed in extracts containing a four-subunit complex lacking the ␣-subunit. Assembly of an eIF-2B holoprotein was confirmed by coimmunoprecipitation of all five subunits. Gel filtration chromatography revealed that recombinant eIF-2B had the same molecular mass as eIF-2B purified from rat liver and that it did indeed possess GEF activity. Phosphorylation of the substrate eIF-2 inhibited the GEF activity of the five-subunit eIF-2B; this inhibition required the eIF-2B ␣-subunit. The results demonstrate that eIF-2B␣ functions as a regulatory subunit that is not required for GEF activity, but instead mediates the regulation of eIF-2B by substrate phosphorylation. Furthermore, eIF-2B⑀ is necessary and is perhaps sufficient for GEF activity in vitro.Protein synthesis in eukaryotic cells is regulated in response to growth factors, hormones, and changes in the external environment (reviewed in Refs. 1 and 2). One of the major control points for protein synthesis is the initiation of mRNA translation, which is mediated by a number of eukaryotic initiation factors (eIFs) 1 (recently reviewed in Ref.3). Translation of mRNA begins with the binding of initiator Met-tRNA i to the 40 S ribosomal subunit and is mediated by eIF-2 as part of the eIF-2⅐GTP⅐Met-tRNA i ternary complex. During the initiation process, the GTP bound to eIF-2 is hydrolyzed, and a binary complex consisting of eIF-2 and GDP is released from the 80 S initiation complex. Since eIF-2 has a 100 -400-fold higher affinity for GDP than for GTP, the guanine nucleotide exchange factor (GEF) known as eIF-2B is required to regenerate the GTP-bound form of eIF-2, which can then participate in another cycle of translation initiation.In cells subjected to a variety of stresses, e.g. heat shock, serum and amino acid deprivation, and heme deprivation in reticulocytes, protein synthesis is inhibited by a mechanism involving phosphorylation of the ␣-subunit of eIF-2 (reviewed in Ref. 4). Phosphorylated eIF-2 efficiently binds to eIF-2B, but cannot undergo nucleotide exchange and therefore competitively inhibits the GEF function of eIF-2B, which is present in limiting amounts in all cell types examined (5-7). In addition, eIF-2B may be regulated by other mechanisms since its activity is modulated under certain conditions in the absence of a change in phosphorylation of eIF-2␣ (8 -12). The ⑀-subunit of eIF-2B is phosphorylated in vitro by glycogen synthase kinase-3, casein kinase-1, and casein kinase-2, which may alter the GEF activity of the holoprotein (13-16). In addition, the activity of eIF-2B ap...

The phs chromosomal locus of Salmonella typhimurium is essential for the dissimilatory anaerobic reduction of thiosulfate to hydrogen sulfide. Sequence analysis of the phs region revealed a functional operon with three open reading frames, designated phsA, phsB, and phsC, which encode peptides of 82.7, 21.3, and 28.5 kDa, respectively. The predicted products of phsA and phsB exhibited significant homology with the catalytic and electron transfer subunits of several other anaerobic molybdoprotein oxidoreductases, including Escherichia coli dimethyl sulfoxide reductase, nitrate reductase, and formate dehydrogenase. Simultaneous comparison of PhsA to seven homologous molybdoproteins revealed numerous similarities among all eight throughout the entire frame, hence, significant amino acid conservation among molybdoprotein oxidoreductases. Comparison of PhsB to six other homologous sequences revealed four highly conserved iron-sulfur clusters. The predicted phsC product was highly hydrophobic and similar in size to the hydrophobic subunits of the molybdoprotein oxidoreductases containing subunits homologous to phsA and phsB. Thus, phsABC appears to encode thiosulfate reductase. Single-copy phs-lac translational fusions required both anaerobiosis and thiosulfate for full expression, whereas multicopy phs-lac translational fusions responded to either thiosulfate or anaerobiosis, suggesting that oxygen and thiosulfate control of phs involves negative regulation. A possible role for thiosulfate reduction in anaerobic respiration was examined. Thiosulfate did not significantly augment the final densities of anaerobic cultures grown on any of the 18 carbon sources tested. On the other hand, washed stationary-phase cells depleted of ATP were shown to synthesize small amounts of ATP on the addition of formate and thiosulfate, suggesting that thiosulfate reduction plays a unique role in anaerobic energy conservation by S. typhimurium.