The C terminus of foamy retrovirus Gag contains determinants for encapsidation of Pol protein into virions

Foamy viruses (FV) differ from orthoretroviruses in many aspects of their replication cycle. A major difference is in the mode of Pol expression, regulation, and encapsidation into virions. Orthoretroviruses synthesize Pol as a Gag-Pol fusion protein so that Pol is encapsidated into virus particles through Gag assembly domains. However, as FV express Pol independently of Gag from a spliced mRNA, packaging occurs through a distinct mechanism. FV genomic RNA contains cis-acting sequences that are required for Pol packaging, suggesting that Pol binds to RNA for its encapsidation. However, it is not known whether Gag is directly involved in Pol packaging. Previously our laboratory showed that sequences flanking the three glycine-arginine-rich (GR) boxes at the C terminus of FV Gag contain domains important for RNA packaging and Pol expression, cleavage, and packaging. We have now shown that both deletion and substitution mutations in the first GR box (GR1) prevented neither the assembly of particles with wild-type density nor packaging of RNA genomes but led to a defect in Pol packaging. Site-directed mutagenesis of GR1 indicated that the clustered positively charged amino acids in GR1 play important roles in Pol packaging. Our results suggest that GR1 contains a Pol interaction domain and that a Gag-Pol complex is formed and binds to RNA for incorporation into virions.     

The carboxyl terminus of RNA helicase A contains a bidirectional nuclear transport domain

Human RNA helicase A was recently identified to be a shuttle protein which interacts with the constitutive transport element (CTE) of type D retroviruses. Here we show that a domain of 110 amino acids at the carboxyl terminus of helicase A is both necessary and sufficient for nuclear localization as well as rapid nuclear export of glutathione S-transferase fusion proteins. The import and export activities of this domain overlap but are separable by point mutations. This bidirectional nuclear transport domain (NTD) has no obvious sequence homology to previously identified nuclear import or export signals. However, the Ran-dependent nuclear import of NTD was efficiently competed by excess amounts of the nuclear localization signal (NLS) peptide from simian virus 40 large T antigen, suggesting that import is mediated by the classical NLS pathway. The nuclear export pathway accessed by NTD is insensitive to leptomycin B and thus is distinct from the leucine-rich nuclear export signal pathway mediated by CRM1.     

Nuclear localization of foamy virus Gag precursor protein.

All foamy viruses give rise to a strong nuclear staining when infected cells are reacted with sera from infected hosts. This nuclear fluorescence distinguishes foamy viruses from all other retroviruses. The experiments reported here indicate that the foamy virus Gag precursor protein is transiently located in the nuclei of infected cells and this is the likely reason for the typical foamy virus nuclear fluorescence. By using the vaccinia virus expression system, a conserved basic sequence motif in the nucleocapsid domain of foamy virus Gag proteins was identified to be responsible for the nuclear transport of the gag precursor molecule. This motif was also found to be able to direct a heterologous protein, the Gag protein of human immunodeficiency virus, into the nucleus.     

Expression and maturation of human foamy virus Gag precursor polypeptides.

In this report, we address the processing of the Gag polypeptides of human foamy virus previously reported to be atypical. In the cytoplasm or the nucleus of infected cells as well as in free virus particles, two Gag precursor polypeptides were identified at approximately 72 and 68 kDa, p72 giving rise to p68 by a maturation process. Efficient maturation of Gag precursors was observed only in two situations: (i) during the early steps of virus adsorption and (ii) under experimental conditions, including treatment with DNase I, known to dissociate actin polymers associated with high ionic strength and ionic detergents. Rather than being a defective viral protease function, an association of Gag precursors with a cytoskeleton network might be responsible for the low rate of Gag protein maturation through inhibition of their cleavage by the protease.     

Role of the C terminus of foamy virus Gag in RNA packaging and Pol expression

Foamy viruses (FV) are complex retroviruses that possess several unique features that distinguish them from all other retroviruses. FV Gag and Pol proteins are expressed independently of one another, and both proteins undergo single cleavage events. Thus, the mature FV Gag protein does not consist of the matrix, capsid, and nucleocapsid (NC) proteins found in orthoretroviruses, and the putative NC domain of FV Gag lacks the hallmark Cys-His motifs or I domains. As there is no Gag-Pol fusion protein, the mechanism of Pol packaging is different but unknown. FV RNA packaging is not well understood either. The C terminus of FV Gag has three glycine-arginine motifs (GR boxes), the first of which has been shown to have nucleic acid binding properties in vitro. The role of these GR boxes in RNA packaging and Pol packaging was investigated with a series of Gag C-terminal truncation mutants. GR box 1 was found to be the major determinant of RNA packaging, but all three GR boxes were required to achieve wild-type levels of RNA packaging. In addition, Pol was packaged in the absence of GR box 3, but GR boxes 1 and 2 were required for efficient Pol packaging. Interestingly, the Gag truncation mutants demonstrated decreased Pol expression levels as well as defects in Pol cleavage. Thus, the C terminus of FV Gag was found to be responsible for RNA packaging, as well as being involved in the expression, cleavage, and incorporation of the Pol protein.     

Investigating the intercellular spreading properties of the foamy virus Gag protein

Small regions called protein transduction domains (PTDs), identified in cellular and viral proteins, have been reported to efficiently cross biological membranes. Here we show that the structural Gag protein of the prototypic foamy virus (PFV) is apparently able to move from cell to cell and to transport the green fluorescent protein (GFP) from few transfected cells to the nuclei of the entire monolayer. Deletion studies showed that this property lies within the second glycine/arginine (GRII) box in the C-terminus of the protein. We also found that uptake and nuclear accumulation of Gag GRII expressed as GFP-fusion protein in recipient cells was observed only following methanol fixation, but never in living cells or when cells were fixed with glutaraldehyde or treated with trichloroacetic acid prior to methanol fixation. Absence of intercellular spreading in vivo was further confirmed using a sensitive luciferase activity assay based on transactivation of the PFV long terminal repeats. Thus, we conclude that intercellular spreading of PFV Gag represents an artificial diffusion event occurring during cell fixation, followed by nuclear retention mediated by the chromatin-binding sequence within the Gag GRII box. In light of these results, we advise caution before defining a peptide as PTD on the basis of intercellular spreading observed by fluorescence microscopy.     

Identification of human immunodeficiency virus type 1 Gag protein domains essential to membrane binding and particle assembly.

Assembly of human immunodeficiency virus type 1 (HIV-1) particles occurs at the plasma membrane of infected cells. Myristylation of HIV-1 Gag precursor polyprotein Pr55Gag is required for stable membrane binding and for assembly of viral particles. We expressed a series of proteins representing major regions of the HIV-1 Gag protein both with and without an intact myristyl acceptor glycine and performed subcellular fractionation studies to identify additional regions critical for membrane binding. Myristylation-dependent binding of Pr55Gag was demonstrated by using the vaccinia virus/T7 hybrid system for protein expression. Domains within the matrix protein (MA) region downstream of the initial 15 amino acids were required for membrane binding which was resistant to a high salt concentration (1 M NaCl). A myristylated construct lacking most of the matrix protein did not associate with the plasma membrane but formed intracellular retrovirus-like particles. A nonmyristylated construct lacking most of the MA region also was demonstrated by electron microscopy to form intracellular particles. Retrovirus-like extracellular particles were produced with a Gag protein construct lacking all of p6 and most of the nucleocapsid region. These studies suggest that a domain within the MA region downstream from the myristylation site is required for transport of Gag polyprotein to the plasma membrane and that stable plasma membrane binding requires both myristic acid and a downstream MA domain. The carboxyl-terminal p6 region and most of the nucleocapsid region are not required for retrovirus-like particle formation.     

Human foamy virus Bel1 transactivator contains a bipartite nuclear localization determinant which is sensitive to protein context and triple multimerization domains.

The Bel1 protein of human foamy virus is a 300-amino-acid nuclear regulatory protein which transactivates the gene expression directed by the homologous long terminal repeat and the human immunodeficiency virus type 1 long terminal repeat. While previous reports suggested that the single basic domain of Bel1 from residues 211 to 222 and/or 209 to 226 is necessary and sufficient for efficient nuclear localization (L. K. Venkatesh, C. Yang, P. A. Theodorakis, and G. Chinnandurai, J. Virol. 67:161-169, 1993; F. He, J. D. Sun, E. D. Garrett, and B. R. Cullen, J. Virol. 67:1896-1904, 1993), our recent data showed that another basic domain, from amino acid residues 199 to 200, is also required for nuclear localization of Bel1 (C. W. Lee, C. Jun, K. J. Lee, and Y. C. Sung, J. Virol. 68:2708-2719, 1994). To clarify this discrepancy, we constructed various bel1-lacZ chimeric constructs and several linker insertion mutants and determined their subcellular localization. When the region of Bel1 containing basic domains was placed at an internal site of the lacZ gene, the nuclear localization signal (NLS) of Bel1 consisted of two discontinuous basic regions separated by an intervening sequence. Moreover, insertion of specific amino acids between two basic regions disrupted the activity of the Bel1 NLS. On the other hand, Bel1 residues 199 and 200 were not required to direct the Bel1-beta-galactosidase chimeric protein to the nucleus when the Bel1 NLS was appended to the amino terminus of beta-galactosidase. These results indicate that the function of the Bel1 NLS is sensitive to the protein context within which the sequence is present. In addition, we demonstrated that the Bel1 protein forms a multimeric complex in the nuclei of mammalian cells by using a sensitive in vivo protein-protein interaction assay. Mutational analyses revealed that the regions which mediate multimer formation map to three domains of Bel1, i.e., residues 1 to 31, 42 to 82, and 82 to 111. Furthermore, our results show that the region of Bel1 from residues 202 to 226 prevents Bel1 from forming a multimeric complex.     

Chromatin tethering of incoming foamy virus by the structural Gag protein

Retroviruses hijack cellular machineries to productively infect their hosts. During the early stages of viral replication, proviral integration relies on specific interactions between components of the preintegration complex and host chromatin-bound proteins. Here, analyzing the fate of incoming primate foamy virus, we identify a short domain within the C-terminus of the structural Gag protein that efficiently binds host chromosomes, by interacting with H2A/H2B core histones. While viral particle production, virus entry and intracellular trafficking are not affected by mutation of this domain, chromosomal attachment of incoming subviral complexes is abolished, precluding proviral integration. We thus highlight a new function of the structural foamy Gag protein as the main tether between incoming subviral complexes and host chromatin prior to integration.     

Human foamy virus capsid formation requires an interaction domain in the N terminus of Gag

Retroviral Gag expression is sufficient for capsid assembly, which occurs through interaction between distinct Gag domains. Human foamy virus (HFV) capsids assemble within the cytoplasm, although their budding, which mainly occurs in the endoplasmic reticulum, requires the presence of homologous Env. Yet little is known about the molecular basis of HFV Gag precursor assembly. Using fusions between HFV Gag and a nuclear reporter protein, we have identified a strong interaction domain in the N terminus of HFV Gag which is predicted to contain a conserved coiled-coil motif. Deletion within this region in an HFV provirus abolishes viral production through inhibition of capsid assembly.     

Mutations in the amino terminus of foamy virus Gag disrupt morphology and infectivity but do not target assembly

Foamy viruses (FVs) assemble using pathways distinct from those of orthoretroviruses. FV capsid assembly takes place near the host microtubule-organizing center (MTOC). Assembled capsids then migrate by an unknown mechanism to the trans-Golgi network to colocalize with the FV glycoprotein, Env. Interaction with Env is required for FV capsid egress from cells; the amino terminus of FV Gag contains a cytoplasmic targeting/retention signal that is responsible for targeting assembly to the MTOC. A mutant Gag was constructed by addition of a myristylation (M) signal in an attempt to target assembly to the plasma membrane and potentially overcome the dependence upon Env for budding (S. W. Eastman and M. L. Linial, J. Virol. 75:6857-6864, 2001). Using this and additional mutants, we now show that assembly is not redirected to the plasma membrane. Addition of an M signal leads to gross morphological defects. The aberrant particles still assemble near the MTOC but do not produce infectious virus. Although extracellular Gag can be detected in a pelletable form in the absence of Env, the mutant particles contain very little genomic RNA and are less dense. Our analyses indicate that the amino terminus of Gag contains an Env interaction domain that is critical for bona fide egress of assembled capsids.     

The Pol32 subunit of DNA polymerase delta contains separable domains for processive replication and proliferating cell nuclear antigen (PCNA) binding

We have carried out a domain analysis of POL32, the third subunit of Saccharomyces cerevisiae DNA polymerase delta (Pol delta). Interactions with POL31, the second subunit of Pol delta, are specified by the amino-terminal 92 amino acids, whereas interactions with the replication clamp proliferating cell nuclear antigen (PCNA, POL30) reside at the extreme carboxyl-terminal region. Pol32 binding, in vivo and in vitro, to the large subunit of DNA polymerase alpha, POL1, requires the carboxyl-proximal region of Pol32. The amino-terminal region of Pol32 is essential for damage-induced mutagenesis. However, the presence of its carboxyl-terminal PCNA-binding domain enhances the efficiency of mutagenesis, particularly at high loads of DNA damage. In vitro, in the absence of effector DNA, the PCNA-binding domain of Pol32 is essential for PCNA-Pol delta interactions. However, this domain has minimal importance for processive DNA synthesis by the ternary DNA-PCNA-Pol delta complex. Rather, processivity is determined by PCNA-binding domains located in the Pol3 and/or Pol31 subunits. Using diagnostic PCNA mutants, we show that during DNA synthesis the carboxyl-terminal domain of Pol32 interacts with the carboxyl-terminal region of PCNA, whereas interactions of the other subunit(s) of Pol delta localize largely to a hydrophobic pocket at the interdomain connector loop region of PCNA.     

Mutational analysis of the carboxyl terminus of the Moloney murine leukemia virus integration protein.

The integration protein (IN) is required for retrovirus DNA integration into the host DNA. The function of the C terminus of the Moloney murine leukemia virus IN protein was examined. The terminal 28 amino acids were found to be nonessential. A linker insertion at position 6025, within a 36-amino-acid insertion not found in any other retrovirus, also produced viable virus.     

A premature termination codon mutation at the C terminus of foamy virus Gag downregulates the levels of spliced pol mRNA

Foamy viruses (FV) comprise a subfamily of retroviruses. Orthoretroviruses, such as human immunodeficiency virus type 1, synthesize Gag and Pol from unspliced genomic RNA. However, FV Pol is expressed from a spliced mRNA independently of Gag. FV pol splicing uses a 3′ splice site located at the 3′ end of gag, resulting in a shared exon between gag and pol. Previously, our laboratory showed that C-terminal Gag premature termination codon (PTC) mutations in the 3′ shared exon led to greatly decreased levels of Pol protein (C. R. Stenbak and M. L. Linial, J. Virol. 78:9423-9430, 2004). To further characterize these mutants, we quantitated the levels of unspliced gag and spliced pol mRNAs using a real-time PCR assay. In some of the PTC mutants, the levels of spliced pol mRNA were reduced as much as 30-fold, whereas levels of unspliced gag RNA were not affected. Substitutions of a missense codon in place of a PTC restored normal levels of spliced pol mRNA. Disrupting Upf proteins involved in nonsense-mediated mRNA decay (NMD) did not affect Pol protein expression. Introduction of an exonic splicing enhancer downstream of the PTC mutation restored pol splicing to the wild-type level. Taken together, our results show that the PTC mutation itself is responsible for decreased levels of pol mRNA but that mechanisms other than NMD might be involved in downregulating Pol expression. The results also suggest that normal pol splicing utilizes a suboptimal splice site seen for other spliced mRNAs in most retroviruses, in that introduced exonic enhancer elements can increase splicing efficiency.     

The two biological activities of human immunodeficiency virus type 1 Vpu protein involve two separable structural domains.

The human immunodeficiency virus type 1 (HIV-1) Vpu protein is an integral membrane phosphoprotein that induces CD4 degradation in the endoplasmic reticulum and enhances virus release from the cell surface. CD4 degradation is specific, requires phosphorylation of Vpu, and involves the interaction between Vpu and the CD4 cytoplasmic domain. In contrast, regulation of virus release is less specific and not restricted to HIV-1 and may be mechanistically-distinct from CD4 degradation. We show here that a mutant of Vpu, Vpu35, lacking most of its cytoplasmic domain has residual biological activity for virus release but is unable to induce CD4 degradation. This finding suggests that the N terminus of Vpu encoding the transmembrane (TM) anchor represents an active domain important for the regulation of virus release but not CD4 degradation. To better define the functions of Vpu's TM anchor and cytoplasmic domain, we designed a mutant, VpuRD, containing a scrambled TM sequence with a conserved amino acid composition and alpha-helical structure. The resulting protein was integrated normally into membranes, was able to form homo-oligomers, and exhibited expression levels, protein stability, and subcellular localization similar to those of wild-type Vpu. Moreover, VpuRD was capable of binding to CD4 and to induce CD4 degradation with wild-type efficiency, confirming proper membrane topology and indicating that the alteration of the Vpu TM domain did not interfere with this function of Vpu. However, VpuRD was unable to enhance the release of virus particles from infected or transfected cells, and virus encoding VpuRD had replication characteristics in T cells indistinguishable from those of a Vpu-deficient HIV-1 isolate. Mutation of the phosphorylation sites in VpuRD resulted in a protein which was unable to perform either function of Vpu. The results of our experiments suggest that the two biological activities of Vpu operate via two distinct molecular mechanisms and involve two different structural domains of the Vpu protein.     

Identification of a receptor binding site in the carboxyl terminus of human interleukin-6.

To identify a receptor binding site of human interleukin-6 (IL-6), we created a library of IL-6 variants with single amino acid substitutions in the last 15 residues (171-185) in the COOH terminus of IL-6. Twenty-seven IL-6 variants were tested for biological activity on a human hepatoma and a mouse hybridoma cell line. Most variants were additionally tested in a receptor binding assay using a human myeloma cell line. Several single amino acid substitutions in the COOH terminus of IL-6 were found to decrease biological activity significantly. This is especially seen in variants with amino acid substitutions that alter the postulated amphipathical alpha-helix structure between residues 178 and 183. The two highly conserved Arg residues at positions 180 and 183 seem to play a very important role in biological activity. The loss of biological activity in all inactive variants is completely paralleled by a decrease of IL-6 receptor binding, as determined by competition binding experiments. One mutant (Leu171) displayed a higher activity on human cells and a higher binding affinity to the receptor and can be considered an IL-6 agonist. It is concluded that the amphipathical alpha-helix structure in the COOH terminus of IL-6 is critical for ligand receptor interaction. Furthermore, the region between residues Ser178 and Arg183 (Ser-Leu-Arg-Ala-X-Arg) is identified as a receptor binding site in the COOH terminus of human IL-6.