Immature Virus Particles Research Articles

Structural and Dynamics Studies of the D54A Mutant of Human T Cell Leukemia Virus-1 Capsid Protein

The human T cell leukemia virus and the human immunodeficiency virus share a highly conserved, predominantly helical two-domain mature capsid (CA) protein structure with an N-terminal beta-hairpin. Despite overall structural similarity, differences exist in the backbone dynamic properties of the CA N-terminal domain. Since studies with other retroviruses suggest that the beta-hairpin is critical for formation of a CA-CA interface, we investigated the functional role of the human T cell leukemia virus beta-hairpin by disrupting the salt bridge between Pro(1) and Asp(54) that stabilizes the beta-hairpin. NMR (15)N relaxation data were used to characterize the backbone dynamics of the D54A mutant in the context of the N-terminal domains, compared with the wild-type counterpart. Moreover, the effect of the mutation on proteolytic processing and release of virus-like particles (VLPs) from human cells in culture was determined. Conformational and dynamic changes resulting from the mutation were detected by NMR spectroscopy. The mutation also altered the conformation of mature CA in cells and VLPs, as reflected by differential antibody recognition of the wild-type and mutated CA proteins. In contrast, the mutation did not detectably affect antibody recognition of the CA protein precursor or release of VLPs assembled by the precursor, consistent with the fact that the hairpin cannot form in the precursor molecule. The particle morphology and size were not detectably affected. The results indicate that the beta-hairpin contributes to the overall structure of the mature CA protein and suggest that differences in the backbone dynamics of the beta-hairpin contribute to mature CA structure, possibly introducing flexibility into interface formation during proteolytic maturation.

Histidine 39 in the dengue virus type 2 M protein has an important role in virus assembly

The mature flavivirus particle comprises a nucleocapsid core surrounded by a lipid bilayer containing the membrane (M) (derived from the precursor prM) and envelope (E) proteins. The formation of intracellular prM/E heterodimers occurs rapidly after translation and is believed to be important for the assembly and secretion of immature virus particles. In this study, the role of the His residue at position 39 in the M protein (M39) of dengue virus type 2 (DENV-2) in the virus life cycle was investigated. Mutations encoding basic (Arg), non-polar (Leu and Pro) and uncharged polar (Asn, Gln and Tyr) amino acids at M39 were introduced into a DENV-2 genomic-length cDNA clone and their effects on virus replication were examined. Substitution of the His residue with non-polar amino acids abolished virus replication, whereas substitution with basic or uncharged polar amino acids decreased virus replication moderately ( approximately 2 log(10) p.f.u. ml(-1) decrease in viral titre for Arg and Asn) or severely (>3.5 log(10) p.f.u. ml(-1) decrease in viral titre for Gln and Tyr). Selected mutations were introduced into a prM-E gene cassette and expressed transiently in COS cells to investigate whether the mutations impaired prM/E association or secretion. None of the mutations was found to disrupt the formation of intracellular prM/E heterodimers. However, the mutations that abolished virus replication prevented secretion of prM/E complexes. The results of this study pinpoint a critical residue in the M protein that potentially plays a role in viral morphogenesis, secretion and entry.

A novel approach for producing lentiviruses that are limited to a single cycle of infection.

We have devised a novel approach for producing simian immunodeficiency virus (SIV) strains and, potentially, human immunodeficiency virus type 1 (HIV-1) strains that are limited to a single cycle of infection. Unlike previous lentiviral vectors, our single-cycle SIV is capable of expressing eight of the nine viral gene products and infected cells release immature virus particles that are unable to complete subsequent rounds of infection. Single-cycle SIV (scSIV) was produced by using a two-plasmid system specifically designed to minimize the possibility of generating replication-competent virus by recombination or nucleotide reversion. One plasmid carried a full-length SIV genome with three nucleotide substitutions in the gag-pol frameshift site to inactivate Pol expression. To ensure inactivation of Pol and to prevent the recovery of wild-type virus by nucleotide reversion, deletions were also introduced into the viral pol gene. In order to provide Gag-Pol in trans, a Gag-Pol-complementing plasmid that included a single nucleotide insertion to permanently place gag and pol in the same reading frame was constructed. We also mutated the frameshift site of this Gag-Pol expression construct so that any recombinants between the two plasmids would remain defective for replication. Cotransfection of both plasmids into 293T cells resulted in the release of Gag-Pol-complemented virus that was capable of one round of infection and one round of viral gene expression but was unable to propagate a spreading infection. The infectivity of scSIV was limited by the amount of Gag-Pol provided in trans and was dependent on the incorporation of a functional integrase. Single-cycle SIV produced by this approach will be useful for addressing questions relating to viral dynamics and viral pathogenesis and for evaluation as an experimental AIDS vaccine in rhesus macaques.

The vaccinia virus G1L putative metalloproteinase is essential for viral replication in vivo.

The function of the putative metalloproteinase encoded by the vaccinia virus G1L gene is unknown. To address this question, we have generated a vaccinia virus strain in which expression of the G1L gene is dependent on the addition of tetracycline (TET) when infection proceeds in a cell line expressing the tetracycline repressor. The vvtetOG1L virus replicated similarly to wild-type Western Reserve (WR) virus in these cells when TET was present but was arrested at a late stage in viral maturation in the absence of TET. This arrest resulted in the accumulation of 98.5% round immature virus particles compared to 6.9% at a similar time point when TET was present. Likewise, the titer of infectious virus progeny decreased by 98.9% +/- 0.97% when the vvtetOG1L virus was propagated in the absence of TET. Mutant virus replication was partially rescued by plasmid-encoded G1L, but not by G1L containing an HXXEH motif mutated to RXXQR. Modeling of G1L revealed a predicted structural similarity to the alpha-subunit of Saccharomyces cerevisiae mitochondrial processing peptidase (alpha-MPP). The HXXEH motif of G1L perfectly overlaps the HXXDR motif of alpha-MPP in this model. These results demonstrate that G1L is essential for virus maturation and suggest that G1L is a metalloproteinase with structural homology to alpha-MPP. However, no obvious effects on the expression and processing of the vaccinia virus major core proteins were observed in the G1L conditional mutant in the absence of TET compared to results for the TET and wild-type WR controls, suggesting that G1L activity is required after this step in viral morphogenesis.

Dimeric Rous Sarcoma Virus Capsid Protein Structure Relevant to Immature Gag Assembly

The structure of the N-terminal domain (NTD) of Rous sarcoma virus (RSV) capsid protein (CA), with an upstream 25 amino acid residue extension corresponding to the C-terminal portion of the Gag p10 protein, has been determined by X-ray crystallography. Purified Gag proteins of retroviruses can assemble in vitro into virus-like particles closely resembling in vivo-assembled immature virus particles, but without a membrane. When the 25 amino acid residues upstream of CA are deleted, Gag assembles into tubular particles. The same phenotype is observed in vivo. Thus, these residues act as a “shape determinant” promoting spherical assembly, when they are present, or tubular assembly, when they are absent. We show that, unlike the NTD on its own, the extended NTD protein has no β-hairpin loop at the N terminus of CA and that the molecule forms a dimer in which the amino-terminal extension forms the interface between monomers. Since dimerization of Gag has been inferred to be a critical step in assembly of spherical, immature Gag particles, the dimer interface may represent a structural feature that is essential in retrovirus assembly.

Two distinct size classes of immature and mature subviral particles from tick-borne encephalitis virus.

Flaviviruses assemble in the endoplasmic reticulum by a mechanism that appears to be driven by lateral interactions between heterodimers of the envelope glycoproteins E and prM. Immature intracellular virus particles are then transported through the secretory pathway and converted to their mature form by cleavage of the prM protein by the cellular protease furin. Earlier studies showed that when the prM and E proteins of tick-borne encephalitis virus are expressed together in mammalian cells, they assemble into membrane-containing, icosahedrally symmetrical recombinant subviral particles (RSPs), which are smaller than whole virions but retain functional properties and undergo cleavage maturation, yielding a mature form in which the E proteins are arranged in a regular T = 1 icosahedral lattice. In this study, we generated immature subviral particles by mutation of the furin recognition site in prM. The mutation resulted in the secretion of two distinct size classes of particles that could be separated by sucrose gradient centrifugation. Electron microscopy showed that the smaller particles were approximately the same size as the previously described mature RSPs, whereas the larger particles were approximately the same size as the virus. Particles of the larger size class were also detected with a wild-type construct that allowed prM cleavage, although in this case the smaller size class was far more prevalent. Subtle differences in endoglycosidase sensitivity patterns suggested that, in contrast to the small particles, the E glycoproteins in the large subviral particles and whole virions might be in nonequivalent structural environments during intracellular transport, with a portion of them inaccessible to cellular glycan processing enzymes. These proteins thus appear to have the intrinsic ability to form alternative assembly products that could provide important clues about the role of lateral envelope protein interactions in flavivirus assembly.

Caprine arthritis encephalitis virus: evidence for a B/D-type assembly pathway in a C-type lentivirus replication.

Lentiviruses, among which is caprine arthritis encephalitis virus (CAEV), are known to concomitantly assemble and bud at the plasma membrane of infected cells, in a C-type defined pathway. Electron microscopy analysis of CAEV-infected cells demonstrated viral particles budding at the plasma membrane and into intracellular membrane-surrounded vesicles. Furthermore, nonenveloped immature virus-like particles, resembling intracytoplasmic type-A particles (ICAPs), accumulated within the cytoplasm of those cells. Fractionation on sucrose density gradients of cytoplasmic lysates from CAEV-infected cells revealed that enveloped immature or mature viral particles had a density of 1.16--1.17 g/ml, whereas ICAPs sedimented at a density of 1.2--1.27 g/ml. Endogenous reverse transcriptase activity was only associated with the 1.16--1.17 g/ml density particles despite the presence of viral RNA in both populations. The intracellular enveloped particles were found to be infectious. The CAEV Gag precursor by itself was shown to direct assembly, budding, and release of immature virus-like particles when expressed in goat primary synovial membrane cells using the same pathways of assembly and budding as observed in CAEV-infected cells. These data suggest that CAEV assembly, driven by the Gag precursor, could unusually proceed via two simultaneous pathways characteristic of type-C and type-B/D retroviruses.

The major core protein P4a (A10L gene) of vaccinia virus is essential for correct assembly of viral DNA into the nucleoprotein complex to form immature viral particles.

The vaccinia virus (VV) A10L gene codes for a major core protein, P4a. This polypeptide is synthesized at late times during viral infection and is proteolytically cleaved during virion assembly. To investigate the role of P4a in the virus life cycle and morphogenesis, we have generated an inducer-dependent conditional mutant (VVindA10L) in which expression of the A10L gene is under the control of the Escherichia coli lacI operator/repressor system. Repression of the A10L gene severely impairs virus growth, as observed by both the inability of the virus to form plaques and the 2-log reduction of viral yields. This defect can be partially overcome by addition of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG). Synthesis of viral proteins other than P4a occurred, although early shutoff of host protein synthesis and expression of viral late polypeptides are clearly delayed, both in the absence and in the presence of IPTG, compared with cells infected with the parental virus. Viral DNA replication and concatemer resolution appeared to proceed normally in the absence of the A10L gene product. In cells infected with VVindA10L in the absence of the inducer virion assembly is blocked, as defined by electron microscopy. Numerous spherical immature viral particles that appear devoid of dense viroplasmic material together with highly electron-dense regular structures are abundant in VVindA10L-infected cells. These regularly spaced structures can be specifically labeled with anti-DNA antibodies as well as with a DNase-gold conjugate, indicating that they contain DNA. Some images suggest that these DNA structures enter into spherical immature viral particles. In this regard, although it has not been firmly established, it has been suggested that DNA uptake occurs after formation of spherical immature particles. Overall, our results showed that P4a and/or its cleaved products are essential for the correct assembly of the nucleoprotein complex within immature viral particles.

Vif is largely absent from human immunodeficiency virus type 1 mature virions and associates mainly with viral particles containing unprocessed gag.

Vif is a human immunodeficiency virus type 1 (HIV-1) protein that is essential for the production of infectious virus. Most of Vif synthesized during HIV infection localizes within cells, and the extent of Vif packaging into virions and its function there remain controversial. Here we show that a small but detectable amount of Vif remains associated with purified virions even after their treatment with the protease subtilisin. However, treatment of these virions with 1% Triton X-100 revealed that most of the virion-associated Vif segregated with detergent-resistant virus particles consisting of unprocessed Gag, indicating that detergent-soluble, mature virions contain very little Vif. To investigate the control of Vif packaging in immature virus particles, we tested its association with Gag-containing virus-like particles (VLPs) in a Vif and Gag coexpression system in human cells. Only a small proportion of Vif molecules synthesized in this system became packaged into VLPs, and the VLP-associated Vif was protected from exogenous protease and detergent treatment, indicating that it is stably incorporated into immature virion-like cores. About 10-fold more Vpr than Vif was packaged into VLPs but most of the VLP-associated Vpr was removed by treatment with detergent. Mutagenesis of the C-terminal sequences in Gag previously shown to be responsible for interaction with Vif did not reduce the extent of Vif packaging into Gag VLPs. Surprisingly, short deletions in the capsid domain (CA) of Gag (amino acid residues 284 to 304 and 350 to 362) increased Vif packaging over 10-fold. The 350 to 363 deletion introduced into CA in HIV provirus also increased Vif incorporation into purified virions. Our results show that Vif can be packaged at low levels into aberrant virus particles or immature virions and that Vif is not present significantly in mature virions. Overall, these results indicate that the Vif content in virions is tightly regulated and also argue against a function of virion-associated Vif.

Membrane Interactions of a Novel Viral Enterotoxin: Rotavirus Nonstructural Glycoprotein NSP4

The rotavirus enterotoxin, NSP4, is a novel secretory agonist that also plays a role in the unique rotavirus morphogenesis that involves a transient budding of newly made immature viral particles into the endoplasmic reticulum. NSP4 and an active peptide corresponding to NSP4 residues 114 to 135 (NSP4(114-135)) mobilize intracellular calcium and induce secretory chloride currents when added exogenously to intestinal cells or mucosa. Membrane-NSP4 interactions may contribute to these alterations; however, details of a lipid-binding domain are unresolved. Therefore, circular dichroism was used to determine (i) the interaction(s) of NSP4 and NSP4(114-135) with model membranes, (ii) the conformational changes elicited in NSP4 upon interacting with membranes, (iii) if NSP4(114-135) is a membrane interacting domain, and (iv) the molar dissociation constant (K(d)) of NSP4(114-135) with defined lipid vesicles. Circular dichroism revealed for the first time that NSP4 and NSP4(114-135) undergo secondary structural changes upon interaction with membrane vesicles. This interaction was highly dependent on both the membrane surface curvature and the lipid composition. NSP4 and NSP4(114-135) preferentially interacted with highly curved, small unilamellar vesicle membranes (SUV), but significantly less with low-curvature, large unilamellar vesicle membranes (LUV). Binding to SUV, but not LUV, was greatly enhanced by negatively charged phospholipids. Increasing the SUV cholesterol content, concomitant with the presence of negatively charged phospholipids, further potentiated the interaction of NSP4(114-135) with the SUV membrane. The K(d) of NSP4(114-135) was determined as well as partitioning of NSP4(114-135) with SUVs in a filtration-binding assay. These data confirmed NSP4 and its active peptide interact with model membranes that mimic caveolae.

Budding of fowlpox and pigeonpox viruses at the surface of infected cells.

Chick embryo fibroblasts and chorioallantoic membranes infected with fowlpox virus (FWPV) or pigeonpox virus (PPV) were examined by transmission and scanning electron microscopy. Immature virus particles were observed in finely granular areas, i.e. virus factories, of the cytoplasm. These particles had various forms depending on their stages of development. Many tubular structures were also seen in these regions. Mature virus particles with ellipsoidal or brick-shaped forms enclosing electron-dense cores were detected throughout the cytoplasm. Notably, there was a high frequency of virus budding at the cell surface, but only occasional virus wrapping in the cytoplasm. Another remarkable feature of the infected cells was accumulation of many virions just beneath the plasma membrane, indicating that this phenomenon is closely related to virus budding. Based on the observed frequency of budding, this mechanism seems to be the predominant way for FWPV and PPV to exit the cell.

The vaccinia virus A9L gene encodes a membrane protein required for an early step in virion morphogenesis.

The A9L open reading frame of vaccinia virus was predicted to encode a membrane-associated protein. A transcriptional analysis of the A9L gene indicated that it was expressed at late times in vaccinia virus-infected cells. Late expression, as well as virion membrane association, was demonstrated by the construction and use of a recombinant vaccinia virus encoding an A9L protein with a C-terminal epitope tag. Immunoelectron microscopy revealed that the A9L protein was associated with both immature and mature virus particles and was oriented in the membrane with its C terminus exposed on the virion surface. To determine whether the A9L protein functions in viral assembly or infectivity, we made a conditional-lethal inducible recombinant vaccinia virus. In the absence of inducer, A9L expression and virus replication were undetectable. Under nonpermissive conditions, viral late protein synthesis occurred, but maturational proteolytic processing was inhibited, and there was an accumulation of membrane-coated electron-dense bodies, crescents, and immature virus particles, many of which appeared abnormal. We concluded that the product of the A9L gene is a viral membrane-associated protein and functions at an early stage in virion morphogenesis.

Assembly of retrovirus capsid-nucleocapsid proteins in the presence of membranes or RNA.

Retrovirus Gag precursor (PrGag) proteins direct the assembly of roughly spherical immature virus particles, while after proteolytic processing events, the Gag capsid (CA) and nucleocapsid (NC) domains condense on viral RNAs to form mature retrovirus core structures. To investigate the process of retroviral morphogenesis, we examined the properties of histidine-tagged (His-tagged) Moloney murine leukemia (M-MuLV) capsid plus nucleocapsid (CANC) (His-MoCANC) proteins in vitro. The His-MoCANC proteins bound RNA, possessed nucleic acid-annealing activities, and assembled into strand, circle (or sphere), and tube forms in the presence of RNA. Image analysis of electron micrographs revealed that tubes were formed by cage-like lattices of CANC proteins surrounding at least two different types of protein-free cage holes. By virtue of a His tag association with nickel-chelating lipids, His-MoCANC proteins also assembled into planar sheets on lipid monolayers, mimicking the membrane-associated immature PrGag protein forms. Membrane-bound His-MoCANC proteins organized into two-dimensional (2D) cage-like lattices that were closely related to the tube forms, and in the presence of both nickel-chelating lipids and RNAs, 2D lattice forms appeared similar to lattices assembled in the absence of RNA. Our observations are consistent with a M-MuLV morphogenesis model in which proteolytic processing of membrane-bound Gag proteins permits CA and NC domains to rearrange from an immature spherical structure to a condensed mature form while maintaining local protein-protein contacts.

Amprenavir: A new human immunodeficiency virus type 1 protease inhibitor

This paper reviews the pharmacologic properties and clinical usefulness of amprenavir, a new human immunodeficiency virus type 1 (HIV-1) protease inhibitor. Amprenavir, the most recent HIV-1 protease inhibitor to receive marketing approval from the US Food and Drug Administration, is a potent competitive inhibitor of HIV-1 protease and a relatively weak inhibitor of HIV-2 protease. Inhibition of the HIV-1 protease enzyme results in immature and noninfectious viral particles. Amprenavir is rapidly absorbed following oral administration. The time to peak concentration (Tmax) in adults is between 1 and 2 hours, the area under the plasma concentration versus time curve is roughly proportional to the dose, the half-life is approximately 8 hours, and the volume of distribution is approximately 430 L. The Tmax in children 4 to 12 years of age is between 1.1 and 1.4 hours. The bioavailability of the solution is 86% relative to the capsule formulation. It is metabolized by the cytochrome P-450 isozyme CYP3A4 and to a lesser extent by CYP2D6 and CYP2C9. We searched MEDLINE (1966 to January 2000), AIDSLINE (1980 to January 2000), International Pharmaceutical Abstracts (1970 to January 2000), PharmaProjects (January 2000 version), and Web sites of major HIV/acquired immunodeficiency syndrome conferences for appropriate published references (1996 to February 2000). Data reported to date indicate that amprenavir is efficacious in the treatment of HIV disease in patients with primary HIV infection, antiretroviral-naïve patients, protease inhibitor-naïve patients, protease inhibitor-experienced patients, and pediatric patients. Adverse effects were usually of early onset (range, 2 to 21 days) and transient (range, 3 to 46 days), although the incidence of metabolic abnormalities such as lipodystrophy, hyperlipidemia, and diabetes mellitus has not yet been defined. Amprenavir should be avoided in patients with a known sulfonamide allergy. Concomitant use of other medications that are CYP3A4 inducers or inhibitors should be done cautiously and only if the potential benefit clearly outweighs potential risk. The dose should be reduced in patients with significant hepatic impairment (Child-Pugh score, > or = 5). Amprenavir probably should not be administered with rifabutin, rifampin, astemizole, midazolam , triazolam, bepridil, dihydroergotamine, ergotamine, or cisapride. The recommended adult dose is 1200 mg twice daily. For patients between 4 and 12 years of age or between 13 and 16 years of age who weigh < 50 kg, the recommended dosage of the capsule form is 20 mg/kg (22.5 mg/kg for oral solution) twice daily or 15 mg/kg (17 mg/kg for oral solution) 3 times a day to a maximum dose of 2400 mg (2800 mg for oral solution). Patients should not take vitamin E supplements because amprenavir is formulated with a large amount of vitamin E (109 IU/capsule and 46 IU/mL oral solution) to improve oral absorption. Amprenavir may be administered with or without food, but a high-fat meal (> 67 g fat) should be avoided. Published clinical data are limited, but amprenavir appears to be efficacious and generally well tolerated in patients with HIV infection. Pharmacoeconomic data are not yet available. The introduction of amprenavir appears to be important, since it provides an additional treatment option as a component of both initial and salvage combination therapies for patients with HIV.

Crosslink Analysis of N-Terminal, C-Terminal, and N/B Determining Regions of the Moloney Murine Leukemia Virus Capsid Protein

To analyze contacts made by Moloney murine leukemia virus (M-MuLV) capsid (CA) proteins in immature and mature virus particles, we have employed a cysteine-specific crosslinking approach that permits the identification of retroviral Gag protein interactions at particular residues. For analysis, single cysteine creation mutations were made in the context of protease-deficient or protease-competent parental constructs. Cysteine creation mutations were chosen near the N- and C-termini of CA and at a site adjacent to the M-MuLV Glu-Ala Fv1 N/B host range determination sequence. Analysis of immature virions showed that PrGag proteins were crosslinked at C-terminal CA residues to form dimers while crosslinking of particle-associated N-terminal and N/B region mutant proteins did not yield dimers, but showed evidence of linking to an unknown 140- to 160-kDa partner. Analysis of mature virions demonstrated that both N- and C-terminal CA residues participated in dimer formation, suggesting that processed CA N- and C-termini are free to establish interprotein associations. Interestingly, N/B region mutant residues in mature virus particles did not crosslink to form dimers, but showed a novel crosslinked band, consistent with an interaction between the N/B tropism determining region and a cellular protein of 45–55 kDa.

Atomic Force Microscopy and Electron Microscopy Analysis of Retrovirus Gag Proteins Assembled in Vitro on Lipid Bilayers

We have used an in vitro system that mimics the assembly of immature Moloney murine leukemia virus (M-MuLV) particles to examine how viral structural (Gag) proteins oligomerize at membrane interfaces. Ordered arrays of histidine-tagged Moloney capsid protein (his-MoCA) were obtained on membrane bilayers composed of phosphatidylcholine (PC) and the nickel-chelating lipid 1,2-di- O-hexadecyl-sn-glycero-3-(1′-2”-R-hydroxy-3′ N-(5-amino-1-carboxypentyl)iminodiacetic acid)propyl ether (DHGN). The membrane-bound arrays were analyzed by electron microscopy (EM) and atomic force microscopy (AFM). Two-dimensional projection images obtained by EM showed that bilayer-bound his-MoCA proteins formed cages surrounding different types of protein-free cage holes with similar cage holes spaced at 81.5-Å distances and distances between dissimilar cage holes of 45.5 Å. AFM images, showing topological features viewed near the membrane-proximal domain of the his-MoCA protein, revealed a cage network of only symmetrical hexamers spaced at 79-Å distances. These results are consistent with a model in which dimers constitute structural building blocks and where membrane-proximal and distal his-MoCA regions interact with different partners in membrane-bound arrays.

Structural maturation of the transmissible gastroenteritis coronavirus.

During the life cycle of the transmissible gastroenteritis coronavirus (TGEV), two types of virus-related particles are detected in infected swine testis cells: large annular viruses and small dense viruses. We have studied the relationships between these two types of particles. Immunoelectron microscopy showed that they are closely related, since both large and small particles reacted equally with polyclonal and monoclonal antibodies specific for TGEV proteins. Monensin, a drug that selectively affects the Golgi complex, caused an accumulation of large annular viral particles in perinuclear elements of the endoplasmic reticulum-Golgi intermediate compartment. A partial reversion of the monensin blockade was obtained in both the absence and presence of cycloheximide, a drug that prevented the formation of new viral particles. After removal of monensin, the Golgi complex recovered its perinuclear location, and a decrease in the number of perinuclear large viral particles was observed. The release of small dense viral particles into secretory vesicles and the extracellular medium was also observed, as was a partial recovery of infectivity in culture supernatants. Small viral particles started to be seen between the third and the fourth Golgi cisternae of normally infected cells. All of these data strongly indicate that the large annular particles are the immature precursors of the small dense viruses, which are the infectious TGEV virions. The immature viral particles need to reach a particular location at the trans side of the Golgi stack to complete their morphological maturation.

In Vitro Assembly of Human Immunodeficiency Virus Type 1 Gag Protein

Retroviral Gag protein is sufficient to produce Gag virus-like particles when expressed in higher eukaryotic cells. Here we describe the in vitro assembly reaction of human immunodeficiency virus Gag protein, which consists of two sequential steps showing the optimal conditions for each reaction. Following expression and purification, Gag protein lacking only the C-terminal p6 domain was present as a monomer (50 kDa) by velocity sedimentation analysis. Initial assembly of the Gag protein to 60 S intermediates occurred by dialysis at 4 degrees C in low salt at neutral to alkaline pH. However, higher order of assembly required incubation at 37 degrees C and was facilitated by the addition of Mg(2+). Prolonged incubation under these conditions produced complete assembly (600 S), equivalent to Gag virus-like particles obtained from Gag-expressing cells. Neither form disassembled by treatment with nonionic detergent, suggesting that correct assembly might occur in vitro. Electron microscopic observation confirmed that the 600 S assembly products were spherical particles similar to authentic immature human immunodeficiency virus particles. The latter assembly stage but not the former was accelerated by the addition of RNA although not inhibited by RNaseA treatment. These results suggest that Gag protein alone assembles in vitro, but that additional RNA facilitates the assembly reaction.

DNA Packaging Mutant: Repression of the Vaccinia Virus A32 Gene Results in Noninfectious, DNA-Deficient, Spherical, Enveloped Particles

The vaccinia virus A32 open reading frame was predicted to encode a protein with a nucleoside triphosphate-binding motif and a mass of 34 kDa. To investigate the role of this protein, we constructed a mutant in which the original A32 gene was replaced by an inducible copy. The recombinant virus, vA32i, has a conditional lethal phenotype: infectious virus formation was dependent on isopropyl-beta-D-thiogalactopyranoside (IPTG). Under nonpermissive conditions, the mutant synthesized early- and late-stage viral proteins, as well as viral DNA that was processed into unit-length genomes. Electron microscopy of cells infected in the absence of IPTG revealed normal-appearing crescents and immature virus particles but very few with nucleoids. Instead of brick-shaped mature particles with defined core structures, there were numerous electron-dense, spherical particles. Some of these spherical particles were wrapped with cisternal membranes, analogous to intracellular and extracellular enveloped virions. Mutant viral particles, purified by sucrose density gradient centrifugation, had low infectivity and transcriptional activity, and the majority were spherical and lacked DNA. Nevertheless, the particle preparation contained representative membrane proteins, cleaved and uncleaved core proteins, the viral RNA polymerase, the early transcription factor and several enzymes, suggesting that incorporation of these components is not strictly coupled to DNA packaging.

Organization of HIV-1 Capsid Proteins on a Lipid Monolayer

In an in vitro system that mimics the assembly of immature human immunodeficiency virus (HIV) particles, ordered arrays of HIV-1 capsid (CA) proteins encoded by the viral gag gene have been obtained by incubation of histidine-tagged capsid proteins (His-HIVCA) beneath lipid monolayers containing the nickel-chelating lipid, 1,2-di-O-hexadecyl-sn-glycero-3-(1'-2"-R-hydroxy-3'-N-(5-amino-1- carboxypentyl)iminodiacetic acid)propyl ether. The membrane-bound His-HIVCA proteins formed small crystalline arrays of primitive (p1) unit cells with dimensions of a = 74.2 A, b = 126.2 A, gamma = 89.3 degrees. The image-analyzed two-dimensional projection of His-HIVCA assemblies shows a cage-like lattice, consisting of hexamer and trimer units, surrounding protein-free cage holes. The hexamer-coordinated cage holes of 26.3-A diameter are spaced at 74. 2-A intervals: these distances, and the hexamer-trimer arrangement, are consistent with previous, lower resolution studies on immature HIV-1 virus particles produced in vivo. Additionally, HIV-1 matrix protein trimer unit structures align to the His-HIVCA trimer units such that residues previously shown to interact with the HIV-1 gp120/gp41 envelope protein complex are oriented toward the hexamer cage holes. Our results form a bridge between results from conventional methods for the analysis of HIV particle structure.