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PubMed2003

Do lipid rafts mediate virus assembly and pseudotyping?

Co-infection of a host cell by two unrelated enveloped viruses can lead to the production of pseudotypes: virions containing the genome of one virus but the envelope proteins of both viruses. The selection of components during virus assembly must therefore be flexible enough to allow the incorporation of unrelated viral membrane proteins, yet specific enough to exclude the bulk of host proteins. This apparent contradiction has been termed the pseudotypic paradox. There is mounting evidence that lipid rafts play a role in the assembly pathway of non-icosahedral, enveloped viruses. Viral components are concentrated initially in localized regions of the plasma membrane via their interaction with lipid raft domains. Lateral interactions of viral structural proteins amplify the changes in local lipid composition which in turn enhance the concentration of viral proteins in the rafts. An affinity for lipid rafts may be the common feature of enveloped virus proteins that leads to the formation of pseudotypes.

Animals↗
PubMed1991

Posttranslational modifications within the HIV-1 envelope glycoprotein which restrict virus assembly and CD4-dependent infection.

Alterations in two highly conserved N-linked glycosylation sites within the gp120 envelope glycoprotein of human immunodeficiency virus type I (HIV-1) implicated in the phenotype of a noncytopathic HIV-1 variant were introduced independently and in combination into a cytopathic, infectious HIV-1 clone by site-specific mutagenesis. Neither mutation affected the synthesis of HIV-1 envelope glycoproteins. However, one of the mutations restricted the ability of HIV-1 envelope to localize on the cell membrane and thus markedly impaired virus assembly. The HIV-1 assembly defect could be overcome in trans if site-specific mutants were packaged in HeLa cells constitutively producing wild-type HIV-1 envelope glycoprotein. In addition to inefficient virus assembly, this mutation impaired the ability of the virus to infect CD4+ T cells, but did not affect CD4-independent infection of muscle cells. These results suggest additional functions of posttranslational modification in virus replication (i.e., envelope glycoprotein transport). Given that such modifications can restrict CD4-mediated uptake without affecting CD4-independent uptake, variations in posttranslational env processing between different HIV-1 genotypes may affect virus tropism in vivo.

Amino Acid Sequence↗
PubMed1997

Sindbis virus replicons and Sindbis virus: assembly of chimeras and of particles deficient in virus RNA.

Alphaviruses are a well-characterized group of positive-strand RNA viruses. The identification of cis-acting elements in their genomes and their replication strategy have made them useful as vectors for the expression of heterologous genes. In infected cells, the nonstructural proteins, required for replication and transcription of the viral genes, are translated from the genomic RNA; the structural proteins, the capsid protein that interacts with the RNA to form the nucleocapsid and the proteins embedded in the lipid envelope, are translated from a subgenomic mRNA and can be replaced by heterologous genes. Such modified genomes are self-replicating (replicons); they can be introduced into the cells by transfection and can also be packaged into extracellular particles with defective helper (DH) RNAs. The particular DH RNA determines how well it is replicated and to what extent it is packaged. One potential complication of this system has been that recombination between the replicon genome and the DH RNA may occur. The studies described here were designed to prevent recombination by expressing the capsid protein from one DH RNA and the virus membrane proteins from a second helper RNA. Recombination to yield a nonsegmented infectious virus genome would then require several independent crossover events. There is a translational enhancer located downstream of the initiating AUG in the RNA of the capsid gene that had to be conserved in the second helper to achieve high-level expression of the viral glycoproteins. For this reason, we modified the capsid protein gene in two ways: the first was to use the capsid protein gene from a different alphavirus, Ross River virus, and the second was to make deletions in that gene to maintain the translational enhancer in the RNA but to eliminate the positively charged region in the protein that should be essential for the specific and nonspecific interactions with RNA. Transfections with replicon RNA and the deleted chimeric DH RNA as the only helper resulted in the high-level production of particles that were almost completely devoid of RNA. The inclusion of a helper expressing an intact Sindbis virus capsid protein gene led to the production of high levels of packaged replicons. Recombinants were not detected even after several undiluted passages.

Amino Acid Sequence↗
PubMed1996

Do light-induced pH changes within the chloroplast drive turnip yellow mosaic virus assembly?

Turnip yellow mosaic virus (TYMV) induces gross morphological and biochemical changes in the chloroplasts of infected cells. Viral RNA is synthesized in vesicles formed by invagination of the outer chloroplast bilayer. Virion assembly occurs at the neck of these vesicles and requires illumination. Data collected over the last three decades are consistent with the hypothesis that light-induced generation of a low pH drives TYMV assembly within the intermembrane space of chloroplasts. In a low-pH environment, poly(C) regions within the genomic RNA of TYMV may interact to form tertiary structures, and the recognition of these structures by TYMV coat protein initiates virion assembly.

Chloroplasts↗
PubMed1998

Identification and characterization of virus assembly intermediate complexes in HIV-1-infected CD4+ T cells.

For type-C and lentiviruses, including human immunodeficiency virus type 1 (HIV-1), the pathway of virus assembly remains poorly defined, and the assembly and budding of capsids are believed to occur simultaneously at the plasma membrane of the infected cell. We have now identified two putative HIV-1 assembly intermediate complexes in infected CD4+ T cells. The first of these intermediates, a detergent-resistant complex (DRC), was identified as a large oligomer that had a density of 1.10-1.13 g/ml and was primarily composed of Pr55Gag and Pr160Gag-Pol precursors. The other putative intermediate was a detergent-sensitive complex (DSC) with a density of 1.15-1.17 g/ml, which apparently represented the products of extensive proteolytic processing of both the Pr55Gag and Pr160Gag-Pol precursors. Both complexes could be distinguished from released mature virions as well as immature viral particles. Surprisingly, the formation of DRC was not dependent upon the myristylation at the N-terminus of the Gag proteins, a signal required for plasma membrane targeting and virus production. However, the myristic acid modification was essential for the formation of DSC. These data suggest that interactions between individual Gag molecules and between Gag and Gag-Pol precursors may occur before their targeting to the plasma membrane during HIV-1 assembly. However, formation of the late virus assembly complex and productive processing of Pr55Gag and Pr160Gag-Pol precursors apparently do not occur until these precursors are targeted to the plasma membrane.

CD4-Positive T-Lymphocytes↗
PubMed2006

A domain directly C-terminal to the major homology region of human immunodeficiency type 1 capsid protein plays a crucial role in directing both virus assembly and incorporation of Gag-Pol.

We demonstrate here that a deletion of 14 amino acid residues directly C-terminal to the major homology region (MHR) of the HIV-1 capsid (CA) in Gag-Pol markedly affects the incorporation of Gag-Pol into virions. The 14-amino acid deletion also significantly impaired virus assembly. In agreement with previous reports, mutations at the very C-terminus of CA resulted in a remarkable reduction in virus production. However, HIV-1 Gag-Pol precursors containing the C-terminal CA mutation were still capable of being incorporated into virions at a level of about 50% that of the wild-type. These results suggest that the domain immediately C-terminal to the MHR is functionally involved in Gag-Gag and Gag/Gag-Pol interaction, and this supports the notion that Gag or Gag-Pol mutants blocked in assembly into virus particles can be rescued into virions provided they retain the domains that are able to interact with the Gag precursor. An HIV-1 Gag-Pol deletion mutant retaining a minimal sequence consisting of the MHR and the adjacent CA-SP1 was efficiently incorporated into virions. Analysis by immunofluorescence staining indicated that the subcellular localization patterns shown by the Gag-Pol mutants were not fully compatible with their efficiency in being incorporated into virions, suggesting that the ability of Gag-Pol mutants to be incorporated into virions largely depends on their interactions with the Gag precursor.

Blotting, Western↗
PubMed2006

Physical controls on directed virus assembly at nanoscale chemical templates.

Viruses are attractive building blocks for nanoscale heterostructures, but little is understood about the physical principles governing their directed assembly. In situ force microscopy was used to investigate organization of Cowpea Mosaic Virus engineered to bind specifically and reversibly at nanoscale chemical templates with sub-30 nm features. Morphological evolution and assembly kinetics were measured as virus flux and inter-viral potential were varied. The resulting morphologies were similar to those of atomic-scale epitaxial systems, but the underlying thermodynamics was analogous to that of colloidal systems in confined geometries. The 1D templates biased the location of initial cluster formation, introduced asymmetric sticking probabilities, and drove 1D and 2D condensation at sub-critical volume fractions. The growth kinetics followed a t(1/2) law controlled by the slow diffusion of viruses. The ability of poly(ethylene glycol) (PEG) to induce the lateral expansion of virus clusters away from the 1D templates suggests a significant role for weak interactions.

Comovirus↗
PubMed1999

Virus assembly: Imaging a molecular machine.

A recent structural study of a double-stranded DNA bacteriophage has provided remarkable new insights into the assembly of a complex virus particle and ushers in a new era in the imaging of non-icosahedral viruses.

Bacillus Phages↗
PubMed1991

Fate of the 6K membrane protein of Semliki Forest virus during virus assembly.

The Semliki Forest virus directs the synthesis of three virus-specific transmembrane proteins p62, 6K, and E1, which all are made in equimolar amounts from a polyprotein precursor molecule. The p62 and E1 spike proteins form heterodimeric complexes in the endoplasmic reticulum before being transported to the cell surface where virus budding occurs. In this study we show that the 6K protein becomes associated to the p62E1 complex in the endoplasmic reticulum and transported with the complex to the cell surface. During virus budding, E1 and p62 (which has matured into the E2 protein) are incorporated into new virions whereas the 6K is mostly excluded. Virus particles released from infected BHK cells contain only about 3% of 6K in their membrane as compared to the spike protein content. The relevance of these findings for the mechanism of SFV assembly is discussed.

Animals↗
PubMed2002

Pressure induces folding intermediates that are crucial for protein-DNA recognition and virus assembly.

Protein-nucleic acid interactions are crucial for a variety of fundamental biological processes such as replication, transcription, restriction, translation and virus assembly. The molecular basis of protein-DNA and protein-RNA recognition is deeply related to the thermodynamics of the systems. We review here how protein-nucleic acid interactions can be approached in the same way as protein-protein interactions involved in protein folding and protein assembly, using hydrostatic pressure as the primary tool and employing several spectroscopic techniques, especially fluorescence, circular dichroism and high-resolution nuclear magnetic resonance. High pressure has the unique property of stabilizing partially folded states or molten-globule states of a protein. The competition between correct folding and misfolding, which in many proteins leads to formation of insoluble aggregates is an important problem in the biotechnology industry and in human diseases such as amyloidosis, Alzheimer's, prion and tumor diseases. The pressure studies reveal that a gradient of partially folded (molten globule) conformations is present between the unfolded and fully folded structure of several bacteria, plant and mammalian viruses. Using pressure, we have detected the presence of a ribonucleoprotein intermediate, where the coat protein is partially unfolded but bound to RNA. These intermediates are potential targets for antiviral compounds. Pressure studies on viruses have direct biotechnological applications. The ability of pressure to inactivate viruses has been evaluated with a view toward the applications of vaccine development and virus sterilization. Recent studies demonstrate that pressure causes virus inactivation while preserving the immunogenic properties. There is substantial evidence that a high-pressure cycle traps a virus in the 'fusion intermediate state', not infectious but highly immunogenic.

Amyloid↗
PubMed2000

Accumulation of virion tegument and envelope proteins in a stable cytoplasmic compartment during human cytomegalovirus replication: characterization of a potential site of virus assembly.

The assembly of human cytomegalovirus (HCMV) is thought to be similar to that which has been proposed for alphaherpesviruses and involve envelopment of tegumented subviral particles at the nuclear membrane followed by export from the cell by a poorly defined pathway. However, several studies have shown that at least two tegument virion proteins remain in the cytoplasm during the HCMV replicative cycle, thereby suggesting that HCMV cannot acquire its final envelope at the nuclear envelope. We investigated the assembly of HCMV by determining the intracellular trafficking of the abundant tegument protein pp150 (UL32) in productively infected human fibroblasts. Our results indicated that pp150 remained within the cytoplasm throughout the replicative cycle of HCMV and accumulated in a stable, juxtanuclear structure late in infection. Image analysis using a variety of cell protein-specific antibodies indicated that the pp150-containing structure was not a component of the endoplasmic reticulum, (ER), ER-Golgi intermediate compartment, cis or medial Golgi, or lysosomes. Partial colocalization of the structure was noted with the trans-Golgi network, and it appeared to lie in close proximity to the microtubule organizing center. Two additional tegument proteins (pp28 and pp65) and three envelope glycoproteins (gB, gH, and gp65) localized in this same structure late infection. This compartment appeared to be relatively stable since pp150, pp65, and the processed form of gB could be coisolated following cell fractionation. Our findings indicated that pp150 was expressed exclusively within the cytoplasm throughout the infectious cycle of HCMV and that the accumulation of the pp150 in this cytoplasmic structure was accompanied by at least five other virion proteins. These results suggested the possibility that this virus-induced structure represented a cytoplasmic site of virus assembly.

Animals↗
PubMed1981

Effect of polymerase mutations on packaging of primer tRNAPro during murine leukemia virus assembly.

The role of reverse transcriptase in selective encapsidation of the murine leukemia virus (MuLV) tRNA primer, tRNAPro, was investigated by examining the tRNA composition of several nonconditional pol mutants. One mutant, clone 23, which contains an altered polymerase about 40% smaller than the wild-type enzyme (B. I. Gerwin et al., J. Virol. 31:741-751, 1979) had a typical viral tRNA pattern, including normal levels of tRNAPro in free and 70S-associated 4S RNA. Another class of mutants, produced by Moloney murine leukemia virus-infected cell clone M13 and subclone M13/1, does not contain any detectable polymerase protein (A. Shields et al., Cell 14:601-609, 1978) and was found to have reduced amounts of tRNAPro in free 4S RNA. However, the level of tRNAPro associated with the genome was normal in the mutant virions. These results suggest that the reverse transcriptase protein is involved in the initial selection of tRNA primer during virus assembly, but not in the subsequent association of this tRNA with genomic RNA.

Leukemia Virus, Murine↗
PubMed1995

Cold denaturation of an icosahedral virus. The role of entropy in virus assembly.

Assembly of icosahedral viruses is not completely understood at the molecular level. The main puzzle is to answer how chemically identical protein subunits take up unique positionally dependent conformations during the process of assembly. The stability of the ribonucleoprotein particles of cowpea mosaic virus (CPMV) to pressures and subzero temperatures has been studied. At room temperature, reversible pressure denaturation of CPMV is obtained only in the presence of 5.0 M urea. On the other hand, when the temperature is decreased to -15 degrees C, the ribonucleoprotein components denature, at 2.5 kbar, in the presence of 1.0 M urea. At temperatures close to -20 degrees C, denaturation is obtained even in the absence of urea. Whereas the denaturation promoted by pressure and urea at room temperature is reversible, virus particles denatured when the temperature is decreased under pressure cannot reassemble. Bis-ANS binding data suggest that this irreversibility may be related to protein release from RNA, which probably does not occur under denaturating conditions at room temperature. The contributions of enthalpy (delta H*) and entropy (delta S*) for the free energy of association of CPMV are calculated from the cold denaturation curves under pressure. The entropy change is positive and large, making the assembly of ribonucleoprotein components an entropy-driven process, suggesting that the burial of nonpolar side chains during the process of assembly is the structural foundation for CPMV assembly.

Cold Temperature↗
PubMed1992

Synthetic peptides and anti-peptide antibodies as probes to study interdomain interactions involved in virus assembly: the envelope of the human immunodeficiency virus (HIV-1).

Synthetic peptides and anti-peptide antibodies have been widely used as probes to map B- and T-cell epitopes on proteins. Such probes also have the potential to delineate contact sites involved generally in protein-protein interactions or in association of domains within a protein. We applied peptide/anti-peptide probes to define: (1) regions on the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins gp120 and gp41 involved in the association between these two glycoproteins; and (2) sites on gp120/gp41, essential for the association of HIV-1 with the CD4 cell receptor. Results of this examination suggested the following: (1) two segments on gp120, encompassing residues (102-126) and (425-452), contribute to the binding site for CD4 and are expected to be juxtaposed in the folded gp120 chain; (2) portions of immunodominant gp120 and gp41 epitopes, encompassing residues (303-338) and (579-611), respectively, appeared to be involved in the gp120-gp41 association, as suggested by direct binding studies and by the limited accessibility of these epitopes on HIV-1 virions: other portions of gp120 also appeared to contribute to the association between these two glycoproteins; (3) there is a partial overlap between gp41 and CD4 binding sites on gp120; (4) the fusion domain and a segment (637-666) of gp41 are not accessible to antibodies after oligomerization of gp41; and 5) the gp120-gp41 association was blocked by aurintricarboxylic acid, suggesting the possibility of developing antiviral compounds interfering with HIV-1 assembly.

Amino Acid Sequence↗
PubMed2003

Intra- and intermolecular disulfide bonds of the GP2b glycoprotein of equine arteritis virus: relevance for virus assembly and infectivity.

Equine arteritis virus (EAV) is an enveloped, positive-strand RNA virus belonging to the family Arteriviridae of the order NIDOVIRALES: EAV virions contain six different envelope proteins. The glycoprotein GP(5) (previously named G(L)) and the unglycosylated membrane protein M are the major envelope proteins, while the glycoproteins GP(2b) (previously named G(S)), GP(3), and GP(4) are minor structural proteins. The unglycosylated small hydrophobic envelope protein E is present in virus particles in intermediate molar amounts compared to the other transmembrane proteins. The GP(5) and M proteins are both essential for particle assembly. They occur as covalently linked heterodimers that constitute the basic protein matrix of the envelope. The GP(2b), GP(3), and GP(4) proteins occur as a heterotrimeric complex in which disulfide bonds play an important role. The function of this complex has not been established yet, but the available data suggest it to be involved in the viral entry process. Here we investigated the role of the four cysteine residues of the mature GP(2b) protein in the assembly of the GP(2b)/GP(3)/GP(4) complex. Open reading frames encoding cysteine-to-serine mutants of the GP(2b) protein were expressed independently or from a full-length infectious EAV cDNA clone. The results of these experiments support a model in which the cysteine residue at position 102 of GP(2b) forms an intermolecular cystine bridge with one of the cysteines of the GP(4) protein, while the cysteine residues at positions 48 and 137 of GP(2b) are linked by an intrachain disulfide bond. In this model, another cysteine residue in the GP(4) protein is responsible for the covalent association of GP(3) with the disulfide-linked GP(2b)/GP(4) heterodimer. In addition, our data highlight the importance of the correct association of the minor EAV envelope glycoproteins for their efficient incorporation into viral particles and for virus infectivity.

Animals↗
PubMed2000

Measles virus assembly within membrane rafts.

During measles virus (MV) replication, approximately half of the internal M and N proteins, together with envelope H and F glycoproteins, are selectively enriched in microdomains rich in cholesterol and sphingolipids called membrane rafts. Rafts isolated from MV-infected cells after cold Triton X-100 solubilization and flotation in a sucrose gradient contain all MV components and are infectious. Furthermore, the H and F glycoproteins from released virus are also partly in membrane rafts (S. N. Manié et al., J. Virol. 74:305-311, 2000). When expressed alone, the M but not N protein shows a low partitioning (around 10%) into rafts; this distribution is unchanged when all of the internal proteins, M, N, P, and L, are coexpressed. After infection with MGV, a chimeric MV where both H and F proteins have been replaced by vesicular stomatitis virus G protein, both the M and N proteins were found enriched in membrane rafts, whereas the G protein was not. These data suggest that assembly of internal MV proteins into rafts requires the presence of the MV genome. The F but not H glycoprotein has the intrinsic ability to be localized in rafts. When coexpressed with F, the H glycoprotein is dragged into the rafts. This is not observed following coexpression of either the M or N protein. We propose a model for MV assembly into membrane rafts where the virus envelope and the ribonucleoparticle colocalize and associate.

HeLa Cells↗
PubMed1998

Molecular Assembly of Virus.

This short review article describes the recent progress in the research field of virus assembly. Two fundamental types of macromolecular interactions: the protein-protein and protein-nucleic acid are involved in the process of virus assembly. Virus assembly at least includes the following three major questions: 1. How the capsid subunits recognize and interact with each other? 2. What protein-protein and protein-nucleic acid interactions are involved in this process? 3. How is the specific selection of viral nucleic acid to be packaged into the virions? Virus assembly is an essential event in virus multiplication and maturation. Elucidation of virus assembly is not only important for better understanding of the viral maturation process, also will play the critical role in practical application, such as the development of vaccine against virus infection, the antiviral drug design and so on.

Journal Article↗
PubMed1976

Protein cleavage during virus assembly: a novel specificity of assembly dependent cleavage in bacteriophage T4.

Cleavage of precursor proteins occurs during assembly of numerous viruses. Seven bacteriophage T4 head-related proteins areknown to be cleaved during morphogenesis. Sequences surrounding the cleavage sites in T4 head precursors P23 and IPIII are reported here. We previously determined the sequences of precursor and processed forms of IPII and IPI. Cleavage occurs at a glutamyl-alanyl bond in each protein. By comparison of sequences around five cleaved and four uncleaved Glu-Ala bonds in head precursors, it appears that cleavage is limited to the Thr or Ala, and X2 to hydrophilic residues. The results suggest the viral-induced assembly protease recognizes and cleaves an extended primary structure in the structurally dissimilar precursors.

Amino Acid Sequence↗