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.     

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.     

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.     

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.     

泡沫病毒Gag蛋白的研究进展

Gag蛋白在逆转录病毒复制周期的许多阶段中发挥重要作用。泡沫病毒基因组结构与其它逆转录病毒类似,但它们的基因组成分和生活周期存在明显差异,这在一定程度上是由其Gag蛋白的结构和功能所决定的。本文对18种不同株型的泡沫病毒gag基因序列进行进化树分析,探究不同来源的泡沫病毒的亲缘关系;并以典型的原型泡沫病毒为代表,阐述泡沫病毒Gag蛋白的结构和功能以及对病毒复制不同阶段的影响。     

Localization of the Vpx packaging signal within the C terminus of the human immunodeficiency virus type 2 Gag precursor protein.

Viral protein X (Vpx) is a human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus accessory protein that is packaged into virions in molar amounts equivalent to Gag proteins. To delineate the processes of virus assembly that mediate Vpx packaging, we used a recombinant vaccinia virus-T7 RNA polymerase system to facilitate Gag protein expression, particle assembly, and extracellular release. HIV genes were placed under control of the bacteriophage T7 promoter and transfected into HeLa cells expressing T7 RNA polymerase. Western immunoblot analysis detected p55gag and its cleavage products p39 and p27 in purified particles derived by expression of gag and gag-pol, respectively. In trans expression of vpx with either HIV-2 gag or gag-pol gave rise to virus-like particles that contained Vpx in amounts similar to that detected in HIV-2 virus produced from productively infected T cells. Using C-terminal deletion and truncation mutants of HIV-2 Gag, we mapped the p15 coding sequence for determinants of Vpx packaging. This analysis revealed a region (residues 439 to 497) downstream of the nucleocapsid protein (NC) required for incorporation of Vpx into virions. HIV-1/HIV-2 gag chimeras were constructed to further characterize the requirements for incorporation of Vpx into virions. Chimeric HIV-1/HIV-2 Gag particles consisting of HIV-1 p17 and p24 fused in frame at the C terminus with HIV-2 p15 effectively incorporate Vpx, while chimeric HIV-2/HIV-1 Gag particles consisting of HIV-2 p17 and p27 fused in frame at the C terminus with HIV-1 p15 do not. Expression of a 68-amino-acid sequence of HIV-2 containing residues 439 to 497 fused to the coding regions of HIV-1 p17 and p24 also produced virus-like particles capable of packaging Vpx in amounts similar to that of full-length HIV-2 Gag. Sucrose gradient analysis confirmed particle association of Vpx and Gag proteins. These results demonstrate that the HIV-2 Gag precursor (p55) regulates incorporation of Vpx into virions and indicates that the packaging signal is located within residues 439 to 497.     

The carboxyl terminus of the human foamy virus Gag protein contains separable nucleic acid binding and nuclear transport domains.

The Gag protein of human foamy virus (HFV) lacks Cys-His boxes present in the nucleocapsid (NC) domains of other retroviruses; instead it contains three glycine-arginine-rich motifs (GR boxes). We have expressed the carboxyl end of HFV Gag containing the GR boxes (the NC domain equivalent) and analyzed its nucleic acid binding properties. Our results show that the NC domain of HFV Gag binds with high affinity to both RNA and DNA, in a sequence-independent manner, as determined by filter binding assays. Analysis of a mutant containing a heterologous sequence in place of GR box I indicates that this motif is required for nucleic acid binding and for viral replication. A mutant in GR box II still binds to RNA and DNA in vitro, but virus containing this mutation does not replicate and no nuclear staining of the Gag protein is found in transfected cells. Surprisingly, a revertant from this mutant that completely lacks GR box II and exhibits very little nuclear transport of Gag can readily replicate in tissue culture. This finding thus provides a direct evidence that although the sequences in GR box II can serve as a nuclear transport signal, they are not required for HFV replication and it is unlikely that nuclear localization of Gag protein plays any critical role during viral infection. Taken together, our results suggest that the Gag protein of HFV may be more analogous to the core protein of the hepatitis B virus family than to conventional retroviral Gag 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.     

Improved primate foamy virus vectors and packaging constructs

Foamy virus (FV) vectors that have minimal cis-acting sequences and are devoid of residual viral gene expression were constructed and analyzed by using a packaging system based on transient cotransfection of vector and different packaging plasmids. Previous studies indicated (i) that FV gag gene expression requires the presence of the R region of the long terminal repeat and (ii) that RNA from packaging constructs is efficiently incorporated into vector particles. Mutants with changes in major 5' splice donor (SD) site located in the R region identified this sequence element as responsible for regulating gag gene expression by an unidentified mechanism. Replacement of the FV 5' SD with heterologous splice sites enabled expression of the gag and pol genes. The incorporation of nonvector RNA into vector particles could be reduced to barely detectable levels with constructs in which the human immunodeficiency virus 5' SD or an unrelated intron sequence was substituted for the FV 5' untranslated region and in which gag expression and pol expression were separated on two different plasmids. By this strategy, efficient vector transfer was achieved with constructs that have minimal genetic overlap.     

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.     

Efficiency and selectivity of RNA packaging by Rous sarcoma virus Gag deletion mutants.

In all retrovirus systems studied, the leader region of the RNA contains a cis-acting sequence called psi that is required for packaging the viral RNA genome. Since the pol and env genes are dispensable for formation of RNA-containing particles, the gag gene product must have an RNA binding domain(s) capable of recognizing psi. To gain information about which portion(s) of Gag is required for RNA packaging in the avian sarcoma and leukemia virus system, we utilized a series of gag deletion mutants that retain the ability to assemble virus-like particles. COS cells were cotransfected with these mutant DNAs plus a tester DNA containing psi, and incorporation of RNA into particles were measured by RNase protection. The efficiency of packaging was determined by normalization of the amount of psi+ RNA to the amount of Gag protein released in virus-like particles. Specificity of packaging was determined by comparisons of psi+ and psi- RNA in particles and in cells. The results indicate that much of the MA domain, much of the p10 domain, half of the CA domain, and the entire PR domain of Gag are unnecessary for efficient packaging. In addition, none of these deleted regions is needed for specific selection of the psi RNA. Deletions within the NC domain, as expected, reduce or eliminate both the efficiency and the specificity of packaging. Among mutants that retain the ability to package, a deletion within the CA domain (which includes the major homology region) is the least efficient. We also examined particles of the well-known packaging mutant SE21Q1b. The data suggest that the random RNA packaging behavior of this mutant is not due to a specific defect but rather is the result of the cumulative effect of many point mutations throughout the gag gene.     

Naturally occurring point mutation in the C terminus of the polymerase gene prevents duck hepatitis B virus RNA packaging.

A duck hepatitis B virus (DHBV) genome cloned from a domestic duck from the People's Republic of China has been sequenced and exhibits no variation in sequences known to be important in viral replication or generation of gene products. Intrahepatic transfection of a dimer of this viral genome into ducklings did not result in viremia or any sign of virus infection, indicating that the genome was defective. Functional analysis of this mutant genome, performed by transfecting the DNA into a chicken hepatoma cell line capable of replicating wild-type virus, indicated that viral RNA is not encapsidated. However, virus core protein is made and can assemble into particles in the absence of encapsidation of viral nucleic acid. Using genetic approaches, it was determined that a change of cysteine to tyrosine in position 711 in the polymerase (P) gene C terminus led to this RNA-packaging defect. By site-directed mutagenesis, it was found that while substitution of Cys-711 with tryptophan also abolished packaging, substitution with methionine did not affect packaging or viral replication. Therefore, Cys-711, which is conserved in all published sequences of DHBV, may not be involved in a disulfide bridge structure essential to viral RNA packaging or replication. Our results, showing that a missense mutation in the region of the DHBV polymerase protein thought to be primarily the RNase H domain results in packaging deficiency, support the previous findings that multiple regions of the complex hepadnaviral polymerase protein may be required for viral RNA packaging.     

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.     

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.     

N-terminal Gag domain required for foamy virus particle assembly and export

Among the Retroviridae, foamy viruses (FVs) exhibit an unusual way of particle assembly and a highly specific incorporation of envelope protein into progeny virions. We have analyzed deletions and point mutants of the prototypic FV gag gene for capsid assembly and egress, envelope protein incorporation, infectivity, and ultrastructure. Deletions introduced at the 3' end of gag revealed the first 297 amino acids (aa) to be sufficient for specific Env incorporation and export of particulate material. Deletions introduced at the 5' end showed the region between aa 6 and 200 to be dispensable for virus capsid assembly but required for the incorporation of Env and particle egress. Point mutations were introduced in the 5' region of gag to target residues conserved among FVs from different species. Alanine substitutions of residues in a region between aa 40 and 60 resulted in severe alterations in particle morphology. Furthermore, at position 50, this region harbors the conserved arginine that is presumably at the center of a signal sequence directing FV Gag proteins to a cytoplasmic assembly site.