Solution structure and backbone dynamics of Mason-Pfizer monkey virus (MPMV) nucleocapsid protein.

Retroviral nucleocapsid proteins (NCPs) are CCHC-type zinc finger proteins that mediate virion RNA binding activities associated with retrovirus assembly and genomic RNA encapsidation. Mason-Pfizer monkey virus (MPMV), a type D retrovirus, encodes a 96-amino acid nucleocapsid protein, which contains two Cys-X2-Cys-X4-His-X4-Cys (CCHC) zinc fingers connected by an unusually long 15-amino acid linker. Homonuclear, two-dimensional sensitivity-enhanced 15N-1H, three-dimensional 15N-1H, and triple resonance NMR spectroscopy have been used to determine the solution structure and residue-specific backbone dynamics of the structured core domain of MPMV NCP containing residues 21-80. Structure calculations and spectral density mapping of N-H bond vector mobility reveal that MPMV NCP 21-80 is best described as two independently folded, rotationally uncorrelated globular domains connected by a seven-residue flexible linker consisting of residues 42-48. The N-terminal CCHC zinc finger domain (residues 24-37) appears to adopt a fold like that described previously for HIV-1 NCP; however, residues within this domain and the immediately adjacent linker region (residues 38-41) are characterized by extensive conformational averaging on the micros-ms time scale at 25 degrees C. In contrast to other NCPs, residues 49-77, which includes the C-terminal CCHC zinc-finger (residues 53-66), comprise a well-folded globular domain with the Val49-Pro-Gly-Leu52 sequence and C-terminal tail residues 67-77 characterized by amide proton exchange properties and 15N R1, R2, and (1H-15N) NOE values indistinguishable to residues in the core C-terminal finger. Twelve refined structural models of MPMV NCP residues 49-80 (pairwise backbone RMSD of 0.77 A) reveal that the side chains of the conserved Pro50 and Trp62 are in van der Waals contact with one another. Residues 70-73 in the C-terminal tail adopt a reverse turn-like structure. Ile77 is involved in extensive van der Waals contact with the core finger domain, while the side chains of Ser68 and Asn75 appear to form hydrogen bonds that stabilize the overall fold of this domain. These residues outside of the core finger structure are conserved in D-type and related retroviral NCPs, e.g., MMTV NCP, suggesting that the structure of MPMV NCP may be representative of this subclass of retroviral NCPs.     

Mason-Pfizer monkey virus: analysis and localization of virion proteins and glycoproteins.

The polypeptide composition of Mason-Pfizer monkey virus was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Six major polypeptides of molecular weights 68,000, 27,000, 20,000, 14,000, 12,000, and 10,000 were resolved regardless of the cell type (i.e., two human and two rhesus) in which the virus was grown. Protein gp68 (68,000) represented the major virus glycoprotein and protein gp20 (20,000) represented a minor glycoprotein of the virion, again regardless of the cell type of origin of the virus. Protein gp68 appears to be located on the outer surface of the viral envelope, as demonstrated by lactoperoxidase catalyzed iodination of intact virions. Additional glycoproteins were shown to be virion associated; their presence depended, however, on the cell type in which the virus was propagated.     

Activity of the Mason-Pfizer monkey virus fusion protein is modulated by single amino acids in the cytoplasmic tail

Mason-Pfizer monkey virus (M-PMV) encodes a transmembrane glycoprotein with a 38-amino-acid-long cytoplasmic tail. After the release of the immature virus, a viral protease-mediated cleavage of the cytoplasmic tail (CT) results in the loss of 17 amino acids from the carboxy terminus and renders the envelope protein fusion competent. To investigate the role of individual amino acid residues in the CT in fusion, a series of mutations was introduced, and the effects of these mutations on glycoprotein biosynthesis and fusion were examined. Most of the alanine-scanning mutations in the CT had little effect on fusion activity. However, four amino acid substitutions (threonine 4, lysine 7, glutamine 9, and isoleucine 10) resulted in substantially increased fusogenicity, while six (leucine 2, phenylalanine 5, isoleucine 13, lysine 16, proline 17, and glycine 31) resulted in much-reduced fusion. Interestingly, the bulk of these mutations are located upstream of the CT cleavage site in a region that has the potential to form a coiled-coil in the Env trimer. Substitutions at glutamine 9 and isoleucine 10 with alanine had the most dramatic positive effect and resulted in the formation of large syncytia. Taken together, these data demonstrate that individual residues within the cytoplasmic domain of M-PMV Env can modulate, in both a positive and negative manner, biological functions that are associated with the extracellular domains of the glycoprotein complex.     

Molecular organization of Mason-Pfizer monkey virus capsids assembled from Gag polyprotein in Escherichia coli

We describe the results of a study by electron microscopy and image processing of Gag protein shells-immature capsids--of Mason-Pfizer monkey virus assembled in Escherichia coli from two truncated forms of the Gag precursor: Deltap4Gag, in which the C-terminal p4Gag was deleted, and Pro(-)CA.NC, in which the N-terminal peptides and proline 1 of the CA domain were deleted. Negative staining of capsids revealed small patches of holes forming a trigonal or hexagonal pattern most clearly visible on occasional tubular forms. The center-to-center spacing of holes in the network was 7.1 nm in Deltap4Gag capsids and 7.4 nm in Pro(-)CA.NC capsids. Image processing of Deltap4Gag tubes revealed a hexagonal network of holes formed by six subunits with a single subunit shared between rings. This organization suggests that the six subunits are contributed by three trimers of the truncated Gag precursor. Similar molecular organization was observed in negatively stained Pro(-)CA.NC capsids. Shadowed replicas of freeze-etched capsids produced by either construct confirmed the presence of a hexagonal network of holes with a similar center-to-center spacing. We conclude that the basic building block of the cage-like network is a trimer of the Deltap4Gag or Pro(-)CA.NC domains. In addition, our results point to a key role of structurally constrained CA domain in the trimeric interaction of the Gag polyprotein.     

Importance of p12 protein in Mason-Pfizer monkey virus assembly and infectivity.

Mason-Pfizer monkey virus (M-PMV) represents the prototype type D retrovirus, characterized by the assembly of intracytoplasmic A-type particles within the infected-cell cytoplasm. These immature particles migrate to the plasma membrane, where they are released by budding. The gag gene of M-PMV encodes a novel protein, p12, just 5' of the major capsid protein (CA) p27 on the polyprotein precursor. The function of p12 is not known, but an equivalent protein is found in mouse mammary tumor virus and is absent from the type C retroviruses. In order to determine whether the p12 protein plays a role in the intracytoplasmic assembly of capsids, a series of in-frame deletion mutations were constructed in the p12 coding domain. The mutant gag genes were expressed by a recombinant vaccinia virus-T7 polymerase-based system in CV-1 cells or in the context of the viral genome in COS-1 cells. In both of these high-level expression systems, mutant Gag precursors were competent to assemble but were not infectious. In contrast, when stable transfectant HeLa cell lines were established, assembly of the mutant precursors into capsids was drastically reduced. Instead, the polyprotein precursors remained predominantly soluble in the cytoplasm. These results show that while p12 is not required for the intracytoplasmic assembly of M-PMV capsids, under the conditions of low-level protein biosynthesis seen in virus-infected cells, it may assist in the stable association of polyprotein precursors for capsid assembly. Moreover, the presence of the p12 coding domain is absolutely required for the infectivity of M-PMV virions.     

Variable sensitivity to substitutions in the N-terminal heptad repeat of Mason-Pfizer monkey virus transmembrane protein

The transmembrane protein of Mason-Pfizer monkey virus contains two heptad repeats that are predicted to form amphipathic alpha-helices that mediate the conformational change necessary for membrane fusion. To analyze the relative sensitivity of the predicted hydrophobic face of the N-terminal heptad repeat to the insertion of uncharged, polar, and charged substitutions, mutations that introduced alanine, serine, or glutamic acid into positions 436, 443, 450, and 457 of the envelope protein were examined. Novel systems using Tat protein and the GHOST cell line were developed to test and quantitate the effects of the mutations on Env-mediated fusion and infectivity of the virus. While no single amino acid change at any of the positions interfered significantly with the synthesis, processing, or transport to the plasma membrane of glycoprotein complexes, 9 of the 12 nonconservative mutations in these residues completely abolished fusion activity and virus infectivity. Mutations in the central positions (443 and 450) of the heptad repeat region were the most detrimental to Env function, and even single alanine substitutions in these positions dramatically altered the fusogenicity of the protein. These results demonstrate that this N-terminal heptad repeat plays a critical role in Env-mediated membrane fusion and highlight the key function of central hydrophobic residues in this process and the sensitivity of all positions to charge substitutions.     

Herpes simplex virus phosphoproteins. II. Characterization of the virion protein kinase and of the polypeptides phosphorylated in the virion.

The protein kinase associated with purified herpes simplex virus 1 and 2 virions partitioned with the capsid-tegument structures and was not solubilized by non-ionic detergents and low, non-inhibitory concentrations of urea. The enzyme required Mg2+ or Mn2+ and utilized ATP or GTP. The activity was enhanced by non-ionic detergents and by Na+ even in the presence of high concentrations of of Mg2+, but not by cyclic nucleotides. The enzyme associated with capsid-tegument structures phosphorylated virion polypeptides only; exogenously added substrates (acidic and basic histones, casein, phosphovitin, protamine, and bovine serum albumin) were not phosphorylated. The major phosphorylated species were virion polypeptides (VP) 1-2, 4, 11-12, 13-14, 18.7, 18.8 and 23. VP 18.7 and VP 18.8 have not been previously detected, but may be phosphorylated forms of polypeptides co-migrating with VP 19. Of the remainder, only VP 23 has been previously identified as a capsid protein; the others are constituents of the tegument or of the under surface of the virion envelope. The distribution of the phosphate bound to viral polypeptides varied depending on the Mg2+ concentration and pH. In the absence of dithiothreitol, in vitro phosphate exchange was demonstrable in VP 23 and to a lesser extent in two other polypeptides on sequential phosphorylation frist with saturating amounts off unlabeled ATP and then with [gamma-32P]ATP. Analysis of the virion polypeptides specified by herpes simplex virus 1 X herpes simplex virus 2 recombinants indicates that the genes specifying the polypeptides which serve as a substrate for the protein kinase map in the unique sequences near the left and right reinterated DNA sequences of the L component.     

Expression and purification of myristoylated matrix protein of Mason-Pfizer monkey virus for NMR and MS measurements

Matrix proteins play multiple roles both in early and late stages of the viral replication cycle. Their N-terminal myristoylation is important for interaction with the host cell membrane during virus budding. We used Escherichia coli, carrying N-myristoyltransferase gene, for the expression of the myristoylated His-tagged matrix protein of Mason-Pfizer monkey virus. An efficient, single-step purification procedure eliminating all contaminating proteins including, importantly, the non-myristoylated matrix protein was designed. The comparison of NMR spectra of matrix protein with its myristoylated form revealed substantial structural changes induced by this fatty acid modification.     

Mutations within the env gene of Mason-Pfizer monkey virus: effects on protein transport and SU-TM association.

By deletion mutagenesis analyses, we have examined the contribution of the immunosuppressive peptide (ISP) region within the transmembrane (TM) protein of Mason-Pfizer monkey virus to viral maturation and infectivity. Deletion of the entire region (mutant D105) results in the production of an Env precursor that is transport defective and therefore unable to be processed to mature glycoproteins. This mutation results in the release of noninfectious virions devoid of surface glycoproteins. A second deletion that removes the most highly conserved 11 amino acids of the ISP (mutant D33) does not affect the production, transport, or processing of the Env precursor yet produces virions that are noninfectious. The mutation was shown to cause the loss of interaction between the surface (SU) and TM proteins and result in the efficient shedding of gp70 into the culture medium. The released gp70 protein was biologically active and could still bind with high specificity to susceptible target cells. Since the ISP domain may represent an area of contact between SU and TM, it could provide an additional explanation for the amino acid sequence homology observed within this region of a variety of retroviruses.     

Membrane and protein interactions of a soluble form of the Semliki Forest virus fusion protein.

Semliki Forest virus is an enveloped alphavirus that infects cells by a membrane fusion reaction triggered by the low pH present in endocytic vacuoles. Fusion is mediated by the E1 spike protein subunit. During fusion, several conformational changes occur in E1 and E2, the two transmembrane subunits of the spike protein. These changes include dissociation of the E1-E2 dimer, alteration of the trypsin sensitivity and monoclonal antibody binding patterns of E1, and formation of a sodium dodecyl sulfate (SDS)-resistant E1 homotrimer. A critical characteristic of Semliki Forest virus fusion is also its dependence on the presence of both cholesterol and sphingomyelin in the target membrane. We have here examined the conformational changes induced by low pH treatment of E1*, the water-soluble, proteolytically truncated ectodomain of the E1 subunit. Following low pH treatment, E1* was shown to bind efficiently to artificial liposomes. Similar to virus fusion, optimal E1*-liposome binding required low pH, cholesterol, and sphingomyelin. The E1 ectodomain, although monomeric in its neutral pH form, assembled into an SDS-resistant oligomer following treatment at low pH. This low pH-induced oligomerization required target membranes containing both cholesterol and sphingomyelin. Our results demonstrate that the E1 ectodomain responds to low pH similarly to the full-length E1 subunit. The ectodomain facilitates the characterization of conformational changes and membrane binding in the absence of virus fusion or other virus components.     

Role of the virion host shutoff protein in neurovirulence of monkey B virus (Macacine herpesvirus 1)

Monkey B virus(Macacine herpesvirus 1; BV) is noted for its extreme neurovirulence in humans. Since the vhs protein encoded by the UL41 gene has been shown to be a neurovirulence factor in the related human herpes simplex viruses, the role of the UL41 gene in BV neurovirulence was investigated. BV mutants were constructed that lacked the entire UL41 ORF(Δ41) or had the RNase active site mutated(Δ41A). Neither mutant shut off host protein synthesis, degraded β-actin mRNA, or prevented an IFN-β response, indicating that the vhs protein and its RNase activity are both necessary for these activities. Replication of both mutants in primary mouse cells was impaired and they exhibited a prolonged disease course in mice. Whereas Δ41 infected mice were euthanized for symptoms related to central nervous system(CNS) infection, Δ41A infected mice were euthanized primarily for symptoms of autonomic nervous system dysfunction. While neuroinvasiveness was not affected, lesions in the CNS were more limited in size, anatomical distribution, and severity than for wild-type virus. These results indicate that the vhs protein affects the general replicative efficiency of BV in vivo rather than being a specific neurovirulence factor critical for invasion of or preferential replication in the CNS.     

Role of the virion host shutoff protein in neurovirulence of monkey B virus(Macacine herpesvirus 1)

Monkey B virus(Macacine herpesvirus 1; BV) is noted for its extreme neurovirulence in humans. Since the vhs protein encoded by the UL41 gene has been shown to be a neurovirulence factor in the related human herpes simplex viruses, the role of the UL41 gene in BV neurovirulence was investigated. BV mutants were constructed that lacked the entire UL41 ORF(Δ41) or had the RNase active site mutated(Δ41A). Neither mutant shut off host protein synthesis, degraded β-actin mRNA, or prevented an IFN-β response, indicating that the vhs protein and its RNase activity are both necessary for these activities. Replication of both mutants in primary mouse cells was impaired and they exhibited a prolonged disease course in mice. Whereas Δ41 infected mice were euthanized for symptoms related to central nervous system(CNS) infection, Δ41A infected mice were euthanized primarily for symptoms of autonomic nervous system dysfunction. While neuroinvasiveness was not affected, lesions in the CNS were more limited in size, anatomical distribution, and severity than for wild-type virus. These results indicate that the vhs protein affects the general replicative efficiency of BV in vivo rather than being a specific neurovirulence factor critical for invasion of or preferential replication in the CNS.     

Basic residues in the Mason-Pfizer monkey virus gag matrix domain regulate intracellular trafficking and capsid-membrane interactions

Mason-Pfizer monkey virus (M-PMV) capsids that have assembled in the cytoplasm must be transported to and associate with the plasma membrane prior to being enveloped by a lipid bilayer during viral release. Structural studies have identified a positive-charge density on the membrane-proximal surface of the matrix (MA) protein component of the Gag polyprotein. To investigate if basic amino acids in MA play a role in intracellular transport and capsid-membrane interactions, mutants were constructed in which lysine and arginine residues (R10, K16, K20, R22, K25, K27, K33, and K39) potentially exposed on the capsid surface were replaced singly and in pairs by alanine. A majority of the charge substitution mutants were released less efficiently than the wild type. Electron microscopy of mutant Gag-expressing cells revealed four distinct phenotypes: K16A and K20A immature capsids accumulated on and budded into intracellular vesicles; R10A, K27A, and R22A capsid transport was arrested at the cellular cortical actin network, while K25A immature capsids were dispersed throughout the cytoplasm and appeared to be defective at an earlier stage of intracellular transport; and the remaining mutant (K33A and K39A) capsids accumulated at the inner surface of the plasma membrane. All mutants that released virions exhibited near-wild-type infectivity in a single-round assay. Thus, basic amino acids in the M-PMV MA define both cellular location and efficiency of virus release.     

Secondary structure and mutational analysis of the Mason-Pfizer monkey virus RNA constitutive transport element.

The Mason-Pfizer monkey virus (MPMV) genome contains a cis-acting element that serves to facilitate nucleocytoplasmic export of intron-containing RNA. This element, known as the constitutive transport element (CTE), has been mapped to a 154-nt region close to the 3' end of the MPMV genome. The CTE contains a degenerate direct repeat of approximately 70 nt. We have probed the secondary structure of the CTE using double-strand- and single-strand-specific ribonucleases and chemical modification agents. A mutational analysis was also performed to confirm critical features of the CTE structure, as well as to identify regions that contain sequence-specific information required for function. Our results indicate that the CTE forms a long stem structure that contains a 9-nt terminal hairpin loop as well as two identical 16-nt inner loops. The inner loop sequences are rotated 180 degrees relative to each other within the structure. The mutational analysis shows that primary sequences in the loop regions are important for function, suggesting that they may contain binding sites for cellular proteins involved in RNA export. Interestingly, sequences with significant homology to the inner loop regions are found in the genomes of spumaviruses and mouse intracisternal A particles.     

In vivo generation and characterization of a soluble form of the Semliki forest virus fusion protein

During infection of host cells, a number of enveloped animal viruses are known to produce soluble forms of viral membrane glycoproteins lacking the transmembrane domain. The roles of such soluble glycoproteins in viral life cycles are incompletely understood, but in several cases they are believed to modulate host immune response and viral pathogenesis. Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells through low-pH-dependent fusion and buds from the plasma membrane. Fusion is mediated by the E1 subunit of the SFV spike protein. Previous studies described the in vivo generation of E1s, a truncated soluble form of E1, under conditions in which budding is inhibited in mammalian host cells. We have here examined the properties of E1s generation and the biological activity of E1s. E1s cleavage required spike protein transport out of the endoplasmic reticulum and was independent of virus infection. Cell surface E1 efficiently acted as a precursor for E1s. E1s generation was strongly pH dependent in BHK cells, with optimal cleavage at a pH of < or =7.0, conditions that inhibited the budding of SFV but not the budding of the rhabdovirus vesicular stomatitis virus. The pH dependence of E1s production and SFV budding was unaffected by the stability of the spike protein dimer but was a function of the host cell. Similar to the intact virus and in vitro-generated E1 ectodomain, treatment of E1s at low pH in the presence of target membranes triggered specific acid-dependent conformational changes. Thus, under a variety of conditions, SFV-infected cells can produce a soluble form of E1 that is biologically active.     

Comparison of classical and affinity purification techniques of Mason-Pfizer monkey virus capsid protein: the alteration of the product by an affinity tag

The efficiencies of different procedures for purification of the capsid protein (CA) of Mason-Pfizer monkey virus are compared. Plasmids encoding both wild-type CA and two C-terminally modified sequences of CA suitable for affinity chromatography purification were prepared. CA was expressed in Escherichia coli (i) as a wild-type protein, (ii) C-terminally extended with a six-histidine tag (CA 6His), and (iii) as a protein containing a C-terminal fusion to a viral protease cleavage site followed by a six-histidine tag (CA 6aa6His). Electron microscopy was used for comparison of the resulting proteins, as CA is a structural protein with no enzymatic activity. We have found that these C-terminal fusions dramatically influenced the properties and morphology of structures formed by CA protein in E. coli. The formation of amorphous aggregates of CA was abolished and CA 6His and CA 6aa6His proteins formed organized structures. CA and CA 6aa6His accumulated in bacteria in inclusion bodies as insoluble proteins, CA 6His was found in a soluble form. Both six-histidine-tagged proteins were purified using affinity chromatography under either native (CA 6His) or denaturing (CA 6aa6His) conditions. CA protein was purified under denaturing conditions using gel-filtration chromatography followed by refolding. All proteins were obtained at a purity >98%. Both aforementioned C-terminal extensions led to dramatic changes in behavior of the products and they also affected the tendency to form organized structures within E. coli. We show here that the widely used histidine anchor may significantly alter the properties of the protein of interest.     

Mutated form of the Newcastle disease virus hemagglutinin-neuraminidase interacts with the homologous fusion protein despite deficiencies in both receptor recognition and fusion promotion

The Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) protein mediates attachment to cellular receptors. The fusion (F) protein promotes viral entry and spread. However, fusion is dependent on a virus-specific interaction between the two proteins that can be detected at the cell surface by a coimmunoprecipitation assay. A point mutation of I175E in the neuraminidase (NA) active site converts the HN of the Australia-Victoria isolate of the virus to a form that can interact with the F protein despite negligible receptor recognition and fusion-promoting activities. Thus, I175E-HN could represent a fusion intermediate in which HN and F are associated and primed for the promotion of fusion. Both the attachment and fusion-promoting activities of this mutant HN protein can be rescued either by NA activity contributed by another HN protein or by a set of four substitutions at the dimer interface. These substitutions were identified by the evaluation of chimeras composed of segments from HN proteins derived from two different NDV strains. These findings suggest that the I175E substitution converts HN to an F-interactive form, but it is one for which receptor binding is still required for fusion promotion. The data also indicate that the integrity of the HN dimer interface is critical to its receptor recognition activity.     

Identification of the nucleocapsid, tegument, and envelope proteins of the shrimp white spot syndrome virus virion

The protein components of the white spot syndrome virus (WSSV) virion have been well established by proteomic methods, and at least 39 structural proteins are currently known. However, several details of the virus structure and assembly remain controversial, including the role of one of the major structural proteins, VP26. In this study, Triton X-100 was used in combination with various concentrations of NaCl to separate intact WSSV virions into distinct fractions such that each fraction contained envelope and tegument proteins, tegument and nucleocapsid proteins, or nucleocapsid proteins only. From the protein profiles and Western blotting results, VP26, VP36A, VP39A, and VP95 were all identified as tegument proteins distinct from the envelope proteins (VP19, VP28, VP31, VP36B, VP38A, VP51B, VP53A) and nucleocapsid proteins (VP664, VP51C, VP60B, VP15). We also found that VP15 dissociated from the nucleocapsid at high salt concentrations, even though DNA was still present. These results were confirmed by CsCl isopycnic centrifugation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry, by a trypsin sensitivity assay, and by an immunogold assay. Finally, we propose an assembly process for the WSSV virion.