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PubMed1993

Overexpression of the HIV-1 gag-pol polyprotein results in intracellular activation of HIV-1 protease and inhibition of assembly and budding of virus-like particles.

Some retroviruses, including HIV-1, regulate the relative amounts of gag and pol gene products by a translational frameshift mechanism. The consequences of altering the ratios of the Gag and Pol proteins were tested using vaccinia virus expression vectors, in which the gag and pol genes were fused by placing them in the same open reading frame. Immunoblotting of cell lysates indicated that a protein of approximately 160 kDa, the expected translation product of the fused gag-pol gene, was the dominant species detected with HIV-specific antiserum during the first several hours of infection with this recombinant virus. Subsequently, the full-length polyprotein diminished in amount and a series of Gag-related intermediate size proteins appeared. Later in infection, p24 and myristoylated p17 Gag proteins predominated and larger amounts of intracellularly processed reverse transcriptase, integrase, and protease were detected compared to the amounts formed with the wild-type gag-pol gene. Large numbers of budding, immature, and mature retrovirus-like particles were visualized by electron microscopy when the wild-type gag-pol gene was expressed, whereas no particles were detected in cells that expressed the fused gag-pol gene. The block to virus assembly was partially overcome by (i) inhibition of the HIV-1 protease with a peptidomimetic inhibitor, (ii) mutagenesis of the active site of the protease, or (iii) shortening of the Gag-Pol polyprotein by deletion of most of the reverse transcriptase gene. Nevertheless, budding was inefficient and the structures appeared immature and frequently aberrant. These results indicated that overproduction of the full-length Gag-Pol polyprotein and increased intracellular protease activity were both detrimental to viral assembly. Further experiments indicated that intracellular processing of Gag and Gag-Pol polyproteins occurred in the absence of particle formation when myristoylation was prevented.

Animals↗
PubMed1990

Hepatitis B pre-S1 and pre-S2 proteins: clinical significance and relation to hepatitis B virus DNA.

Pre-S proteins may have an important role in virus assembly and virus entry into the host cell. The presence of pre-S proteins in serum has also been thought to correlate with active viral replication. To investigate whether pre-S proteins in serum might have additional diagnostic and/or predictive value for liver sequelae in HBV infection, sera from six different serological groups of patients with HBV markers (total number 363) and different manifestations of liver histology were examined for the presence of pre-S1 and pre-S2 proteins using micro-ELISAs. Pre-S1 and pre-S2 proteins were detected significantly more often in HBV-DNA-positive than in HBV-DNA-negative sera from HBsAg carriers. However, pre-S1 and pre-S2 proteins were also found in HBV-DNA-negative HBsAg carriers irrespective of serum HBeAg/anti-HBe or liver histologic findings. These results suggest that the presence of the pre-S1 and or pre-S2 proteins in serum either does not seem to reflect the presence of active viral replication and active liver disease or pre-S proteins are more readily detectable than HBeAg and HB-DNA as measured by a dot-blot technique. Furthermore, the presence of pre-S proteins in serum is strongly correlated with that of HBsAg.

Chronic Disease↗
PubMed2000

Design and evolution of artificial M13 coat proteins.

Using simple design and selective pressure, we have evolved an artificial M13 bacteriophage coat protein. M13 coat proteins first reside in the bacterial inner membrane and subsequently surround the DNA core of the assembled virus. The artificial coat protein (ACP) was designed and evolved to mimic both functions of the natural M13 coat proteins, but with an inverted orientation. ACP is a non-functional coat protein because it is not required for the production of phage particles. Instead, it incorporates into a phage coat which still requires all the natural coat proteins for structural integrity. In contrast with other M13 coat proteins, which can display polypeptides as amino-terminal fusions, ACP permits the carboxy-terminal display of large polypeptides. The results suggest that viruses can co-opt host membrane proteins to acquire new coat proteins and thus new functions. In particular, M13 bacteriophage can be engineered for new functions, such as carboxy-terminal phage display.

Amino Acid Sequence↗
PubMed1985

Effect of tunicamycin on the development of the cytopathic effect in Sindbis virus-infected avian fibroblasts.

In Sindbis virus-infected avian cells the development of the cytopathic effect is correlated with the disruption of plasma membrane function. Sindbis virus inhibits the activity of the Na+K+ATPase, a membrane-associated enzyme complex which regulates intracellular monovalent cation levels. Tunicamycin, which blocks envelope protein glycosylation, prevents inhibition of Na+K+ATPase activity and the development of morphological changes in Sindbis virus-infected cells. Although inhibition of Na+K+ATPase activity is not essential for the termination of host protein synthesis, membrane-mediated events may favor the selective translation of viral proteins. The termination of host protein synthesis does not contribute to the development of these cytopathic changes in the time frame examined. In tunicamycin-treated, Sindbis virus-infected cells, unglycosylated E1 is inserted into the plasma membrane but virus release is prevented. In productively infected cells, therefore, the inhibition of Na+K+ATPase activity and the development of the cytopathic effect may result from terminal events in virus assembly and/or virus release.

Animals↗
PubMed1986

A morphological study of the replication of Breda virus (proposed family Toroviridae) in bovine intestinal cells.

The morphological aspects of Breda virus serotype 2 replication in intestinal cells of gnotobiotic calves were investigated by electron microscopy. Ultrastructural findings suggest a morphogenetic pathway involving cytoplasmic vesicles, the Golgi apparatus and the cell nucleus. Virus uptake probably occurs via a receptor-mediated endocytosis-like mechanism. Endocytotic vesicles then carry virus to an as yet undetermined site of uncoating. Masses of tubules having the same diameters as Breda virion cores are found in nuclei, suggesting a role for the cell nucleus in replication of nucleocapsids. Similar tubules, as well as complete virions, were found in the Golgi region, the apparent site of virus assembly. Virus-containing Golgi vesicles then presumably move to cell surfaces where they fuse with apical and baso-lateral cell membranes to release virions in a way that permits more than one viral replicative cycle to occur without damage to host cell integrity. Virions are elongated with rounded ends and measure 42 X 100.5 nm. The morphogenesis and replication of Breda virus most closely resembles that of Berne virus of the proposed family Toroviridae.

Animals↗
PubMed2005

From assembly to virus particle budding: pertinence of the detergent resistant membranes.

Detergent resistant membranes (DRMs) are the site of assembly for a variety of viruses. Here, we make use of Sendai virus mutant proteins that are not packaged into virus particles to determine the involvement of this assembly for the virus particle production. We found that, in the context of an infection, (1) all the Sendai virus proteins associated in part with DRMs, (2) mutant HN and M proteins not packaged into virus particles were similarly part of this association, (3) after M protein suppression resulting in a significant reduction of virus production, the floatation profile of the other viral proteins was not altered and finally (4) cellular cholesterol depletion did not decrease the virus particle production, although it somehow reduced their virus infectivity. These results led us to conclude that the assembly complex found in DRM fractions does not constitute a direct precursor of virus particle budding.

Cell Line↗
PubMed2004

Sindbis virus nucleocapsid assembly: RNA folding promotes capsid protein dimerization.

In Sindbis virus, initiation of nucleocapsid core assembly begins with recognition of the encapsidation signal of the viral RNA genome by capsid protein. This nucleation event drives the recruitment of additional capsid proteins to fully encapsidate the genome, generating an icosahedral nucleocapsid core. The encapsidation signal of the Sindbis virus genomic RNA has previously been localized to a 132-nucleotide region of the genome within the coding region of the nsP1 protein, and the RNA-binding activity of the capsid was previously mapped to a central region of the capsid protein. It is unknown how capsid protein binding to encapsidation signal leads to ordered oligomerization of capsid protein and nucleocapsid core assembly. To address this question, we have developed a mobility shift assay to study this interaction. We have characterized a 32 amino acid peptide capable of recognizing the Sindbis virus encapsidation signal RNA. Using this peptide, we were able to observe a conformational change in the RNA induced by capsid protein binding. Binding is tight (K(d)(app) = 12 nM), and results in dimerization of the capsid peptide. Mutational analysis reveals that although almost every predicted secondary structure within the encapsidation signal is required for efficient protein binding, the identities of the bases within the helices and hairpin turns of the RNA do not need to be maintained. In contrast, two purine-rich loops are essential for binding. From these data, we have developed a model in which the encapsidation signal RNA adopts a highly folded structure and this folding process directs early events in nucleocapsid assembly.

Base Pairing↗
PubMed1996

Involvement of the endoplasmic reticulum in the assembly and envelopment of African swine fever virus.

African swine fever (ASF) virus is a large enveloped DNA virus assembled in the cytoplasm of cells. In this study, the membrane compartments involved in the envelopment of ASF virus were investigated. A monoclonal antibody recognizing p73, the major structural protein of ASF virus, was generated to analyze the binding of p73 to membranes during the assembly of the virus. Approximately 50% of the intracellular pool of p73 associated with membranes as a peripheral membrane protein. Binding was rapid and complete within 15 min of synthesis. Subcellular membrane fractionation showed that newly synthesized p73 molecules cosedimented with endoplasmic reticulum (ER) membranes and remained associated with the ER during a 2-h chase. A similar distribution on gradients was recorded for p17, a structural membrane protein of ASF virus. The results suggested that the ER was involved in the assembly of ASF virus. A protease protection assay demonstrated a time-dependent envelopment of the membrane bound, but not cytosolic, pool of p73. Envelopment of p73 took place 1 h after binding to membranes and was completed 1 h before the first detection of p73 in virions secreted from cells. Envelopment was unaffected by brefeldin A and monensin, drugs that block membrane transport between the ER and Golgi. Taken together the results provide evidence for the binding of ASF virus structural proteins to a specific membrane compartment and implicate a role for the ER in the assembly and envelopment of ASF virus.

African Swine Fever Virus↗
PubMed1990

Hepatitis B virus nucleocapsid assembly: primary structure requirements in the core protein.

As a step toward understanding the assembly of the hepatitis B virus (HBV) nucleocapsid at a molecular level, we sought to define the primary sequence requirements for assembly of the HBV core protein. This protein can self assemble upon expression in Escherichia coli. Applying this system to a series of C-terminally truncated core protein variants, we mapped the C-terminal limit for assembly to the region between amino acid residues 139 and 144. The size of this domain agrees well with the minimum length of RNA virus capsid proteins that fold into an eight-stranded beta-barrel structure. The entire Arg-rich C-terminal domain of the HBV core protein is not necessary for assembly. However, the nucleic acid content of particles formed by assembly-competent core protein variants correlates with the presence or absence of this region, as does particle stability. The nucleic acid found in the particles is RNA, between about 100 to some 3,000 nucleotides in length. In particles formed by the full-length protein, the core protein mRNA appears to be enriched over other, cellular RNAs. These data indicate that protein-protein interactions provided by the core protein domain from the N terminus to the region around amino acid 144 are the major factor in HBV capsid assembly, which proceeds without the need for substantial amounts of nucleic acid. The presence of the basic C terminus, however, greatly enhances encapsidation of nucleic acid and appears to make an important contribution to capsid stability via protein-nucleic acid interactions. The observation of low but detectable levels of nucleic acid in particles formed by core protein variants lacking the Arg-rich C terminus suggests the presence of a second nucleic acid-binding motif in the first 144 amino acids of the core protein. Based on these findings, the potential importance of the C-terminal core protein region during assembly in vivo into authentic, replication-competent nucleocapsids is discussed.

Amino Acid Sequence↗
PubMed1986

A block in initiation of simian virus 40 assembly results in the accumulation of minichromosomes containing an exposed regulatory region.

The initiation of simian virus 40 assembly is blocked at the nonpermissive temperature in cells infected with the viral capsid protein VP1 mutant tsC219. Greater than 95% of the minichromosomes isolated from these cells are accessible to cleavage by Bgl I and Sph I, which recognize the sequences near the viral replication origin and in the transcription enhancer elements, respectively. The accessibility of the Ori region to Bgl I is considerably reduced when virion assembly is allowed to proceed in tsC219-infected cells at the permissive temperature. A reduced accessibility to Bgl I is also observed for chromatin isolated from cells infected with wt776, the wild-type parental strain of tsC219. For wt776 chromatin, variability to Bgl I sensitivity is observed and this can be correlated to the relative virion-to-chromatin yield. A similar correlation is not apparent for restriction endonucleases that recognize sequences within the coding region of simian virus 40 chromatin. These results, considered together, indicate that, when virion assembly initiation is blocked, nucleosomes are nonrandomly arranged with respect to the viral regulatory sequences. It appears that the open regulatory region in minichromosomes is established during replication and that a protected regulatory region is generated with the onset of virion assembly.

Animals↗
PubMed2003

Phosphorylation of rubella virus capsid regulates its RNA binding activity and virus replication.

Rubella virus is an enveloped positive-strand RNA virus of the family TOGAVIRIDAE: Virions are composed of three structural proteins: a capsid and two membrane-spanning glycoproteins, E2 and E1. During virus assembly, the capsid interacts with genomic RNA to form nucleocapsids. In the present study, we have investigated the role of capsid phosphorylation in virus replication. We have identified a single serine residue within the RNA binding region that is required for normal phosphorylation of this protein. The importance of capsid phosphorylation in virus replication was demonstrated by the fact that recombinant viruses encoding hypophosphorylated capsids replicated at much lower titers and were less cytopathic than wild-type virus. Nonphosphorylated mutant capsid proteins exhibited higher affinities for viral RNA than wild-type phosphorylated capsids. Capsid protein isolated from wild-type strain virions bound viral RNA more efficiently than cell-associated capsid. However, the RNA-binding activity of cell-associated capsids increased dramatically after treatment with phosphatase, suggesting that the capsid is dephosphorylated during virus assembly. In vitro assays indicate that the capsid may be a substrate for protein phosphatase 1A. As capsid is heavily phosphorylated under conditions where virus assembly does not occur, we propose that phosphorylation serves to negatively regulate binding of viral genomic RNA. This may delay the initiation of nucleocapsid assembly until sufficient amounts of virus glycoproteins accumulate at the budding site and/or prevent nonspecific binding to cellular RNA when levels of genomic RNA are low. It follows that at a late stage in replication, the capsid may undergo dephosphorylation before nucleocapsid assembly occurs.

Amino Acid Sequence↗
PubMed2003

Characterization of self-assembled virus-like particles of human polyomavirus BK generated by recombinant baculoviruses.

The major structural protein of the human polyomavirus BK (BKV), VP1, was expressed by using recombinant baculoviruses. A large amount of protein with a molecular mass of about 42 kDa was synthesized and identified by Western blotting. The protein was detected exclusively in the nuclei by immunofluorescent analysis and it was released into culture medium. The expressed BKV VP1 protein was self-assembled into virus-like particles (BK-VLPs) with two different sizes (50 and 26 nm in diameter), which migrated into four different bands in CsCl gradient with buoyant densities of 1.29, 1.30, 1.33, and 1.35 g/cm(3). The immunological studies on the BK-VLPs suggested that they have similar antigenicity with those of authentic BKV particles. Cryoelectron microscopy and 3D image analysis further revealed that the larger BK-VLPs were composed of 72 capsomers which all were pentamers arranged in a T = 7 surface lattice. This system provides useful information for detailed studies of viral morphogenesis and the structural basis for the antigenicity of BKV.

Amino Acid Sequence↗
PubMed1992

Viral antigen presentation and MHC assembly.

Viruses and other intracellular pathogens represent a unique challenge to the vertebrate immune system. To deal with this problem of intracellular invasion the immune system has developed an elegant strategy to mark virally infected cells. This review discusses current issues relating to the cell biology of MHC molecule assembly and antigen presentation events from the standpoint of viruses and anti-viral immunity.

Animals↗
PubMed2004

Insertion of a classical nuclear import signal into the matrix domain of the Rous sarcoma virus Gag protein interferes with virus replication.

The Rous sarcoma virus Gag protein undergoes transient nuclear trafficking during virus assembly. Nuclear import is mediated by a nuclear targeting sequence within the MA domain. To gain insight into the role of nuclear transport in replication, we investigated whether addition of a "classical " nuclear localization signal (NLS) in Gag would affect virus assembly or infectivity. A bipartite NLS derived from nucleoplasmin was inserted into a region of the MA domain of Gag that is dispensable for budding and infectivity. Gag proteins bearing the nucleoplasmin NLS insertion displayed an assembly defect. Mutant virus particles (RC.V8.NLS) were not infectious, although they were indistinguishable from wild-type virions in Gag, Gag-Pol, Env, and genomic RNA incorporation and Gag protein processing. Unexpectedly, postinfection viral DNA synthesis was also normal, as similar amounts of two-long-terminal-repeat junction molecules were detected for RC.V8.NLS and wild type, suggesting that the replication block occurred after nuclear entry of proviral DNA. Phenotypically revertant viruses arose after continued passage in culture, and sequence analysis revealed that the nucleoplasmin NLS coding sequence was deleted from the gag gene. To determine whether the nuclear targeting activity of the nucleoplasmin sequence was responsible for the infectivity defect, two critical basic amino acids in the NLS were altered. This virus (RC.V8.KR/AA) had restored infectivity, and the MA.KR/AA protein showed reduced nuclear localization, comparable to the wild-type MA protein. These data demonstrate that addition of a second NLS, which might direct MA and/or Gag into the nucleus by an alternate import pathway, is not compatible with productive virus infection.

Active Transport, Cell Nucleus↗
PubMed2000

Role of the Gag matrix domain in targeting human immunodeficiency virus type 1 assembly.

Human immunodeficiency virus type 1 (HIV-1) particle formation and the subsequent initiation of protease-mediated maturation occur predominantly on the plasma membrane. However, the mechanism by which HIV-1 assembly is targeted specifically to the plasma membrane versus intracellular membranes is largely unknown. Previously, we observed that mutations between residues 84 and 88 of the matrix (MA) domain of HIV-1 Gag cause a retargeting of virus particle formation to an intracellular site. In this study, we demonstrate that the mutant virus assembly occurs in the Golgi or in post-Golgi vesicles. These particles undergo core condensation in a protease-dependent manner, indicating that virus maturation can occur not only on the plasma membrane but also in the Golgi or post-Golgi vesicles. The intracellular assembly of mutant particles is dependent on Gag myristylation but is not influenced by p6(Gag) or envelope glycoprotein expression. Previous characterization of viral revertants suggested a functional relationship between the highly basic domain of MA (amino acids 17 to 31) and residues 84 to 88. We now demonstrate that mutations in the highly basic domain also retarget virus particle formation to the Golgi or post-Golgi vesicles. Although the basic domain has been implicated in Gag membrane binding, no correlation was observed between the impact of mutations on membrane binding and Gag targeting, indicating that these two functions of MA are genetically separable. Plasma membrane targeting of Gag proteins with mutations in either the basic domain or between residues 84 and 88 was rescued by coexpression with wild-type Gag; however, the two groups of MA mutants could not rescue each other. We propose that the highly basic domain of MA contains a major determinant of HIV-1 Gag plasma membrane targeting and that mutations between residues 84 and 88 disrupt plasma membrane targeting through an effect on the basic domain.

Binding Sites↗
PubMedDate not supplied

BAY 41-4109 has multiple effects on Hepatitis B virus capsid assembly.

Here we report the effect of a heteroaryldihydropyrimidine (HAP) antiviral compound, BAY 41-4109, on Hepatitis B virus (HBV) capsid assembly and on preformed HBV capsids. The HBV capsid is an icosahedral complex of 120 capsid protein dimers. BAY41-4109 inhibits virus production in vivo by a mechanism that targets the viral capsid. We found that BAY 41-4109 was able to both accelerate and misdirect capsid assembly in vitro. As little as one HAP molecule for every five HBV dimers was sufficient to induce formation of non-capsid polymers. Unlike the related molecule HAP-1 (Stray et al., Proc. Natl. Acad. Sci. USA 102:8138-43, 2005), no stable assembly intermediates were observed in assembly reactions with BAY 41-4109, indicating that accelerated assembly by BAY 41-4109 was still kinetically regulated by the nucleation rate. Preformed capsids were stabilized by BAY 41-4109, up to a ratio of one inhibitor molecule per two dimers. However, at BAY 41-4109:dimer ratios of 1:1 and greater, capsids were destabilized to yield very large non-capsid polymers. These data suggest the existence of two functionally distinguishable classes of drug-binding sites on HBV capsids. Occupation of the first class of site stabilizes capsid, while binding at the second class requires or induces structural changes that cannot be tolerated without destabilizing the capsid. Our data suggest that HAP compounds may inhibit virus replication by inducing assembly inappropriately and, when in excess, by misdirecting assembly decreasing the stability of normal capsids.

Antiviral Agents↗
PubMed2006

Virion tails of Beet yellows virus: Coordinated assembly by three structural proteins.

Filamentous virions of Beet yellows virus contain a long body formed by a major capsid protein and a short tail that is assembled by a minor capsid protein (CPm), an Hsp70-homolog (Hsp70h), a 64-kDa protein (p64), and a 20-kDa protein (p20). Using mutation analysis and newly developed in planta assays, here we investigate the genetic requirements for the tail assembly. We show that the inactivation of CPm dramatically reduces incorporation of both Hsp70h and p64. Furthermore, inactivation of Hsp70h prevents incorporation of p64 into virions and vice versa. Hsp70h and p64 are each required for efficient incorporation of CPm. We also show that the tails possessing normal relative amounts of CPm, Hsp70h, and p64 can be formed in the absence of the major capsid protein and p20. Similar to the tails isolated from the wild-type virions, these mutant tails encapsidate the approximately 700 nt-long, 5'-terminal segments of the viral RNA. Taken together, our results imply that CPm, Hsp70h and p64 act cooperatively to encapsidate a defined region of the closterovirus genome.

Base Sequence↗