Isolation and characterization of the Mason-Pfizer monkey virus p12 protein

The Mason-Pfizer monkey virus (M-PMV) Gag protein, precursor to the structural proteins of the infectious virion, assembles into immature capsid-like particles when expressed at high levels in bacterial cells. Similar capsid-like particles can be obtained by in vitro assembly using a high concentration of isolated Gag. M-PMV Gag contains a p12 protein that has no corresponding analogues in most other retroviruses and has been suggested to contain an internal scaffold domain (ISD). We have expressed and purified p12 and the N- and C-terminal halves (Np12 and Cp12) that are predicted to be structurally independent domains. The behavior of these proteins was analyzed using chemical cross-linking, CD spectroscopy, and electron microscopy. The N-terminal half of p12 is largely alpha-helical although the C-terminal portion lacks any apparent ordered structure. Both p12 and Np12 form high-order oligomers in vitro and when expressed in E. coli produce organized structures that are visible by electron microscopy. Interestingly, Cp12, as well as the whole protein, can form dimers in the presence of SDS. The data show that both domains of p12 contribute to its ability to multimerize with much of this potential residing in its N-terminal part, most probably within the leucine zipper-like (LZL) sequence.     

The effect of point mutations within the N-terminal domain of Mason-Pfizer monkey virus capsid protein on virus core assembly and infectivity

Retroviral capsid protein (CA) mediates protein interactions driving the assembly of both immature viral particles and the core of the mature virions. Structurally conserved N-terminal domains of several retroviruses refold after proteolytic cleavage into a β-hairpin, stabilized by a salt bridge between conserved N-terminal Pro and Asp residues. Based on comparison with other retroviral CA, we identified Asp50 and Asp57 as putative interacting partners for Pro1 in Mason-Pfizer monkey virus (M-PMV) CA. To investigate the importance of CA Pro1 and its interacting Asp in M-PMV core assembly and infectivity, P1A, P1Y, D50A, T54A and D57A mutations were introduced into M-PMV. The P1A and D57A mutations partially blocked Gag processing and the released viral particles exhibited aberrant cores and were non-infectious. These data indicate that the region spanning residues Asp50-Asp57 plays an important role in stabilization of the β-hairpin and that Asp57 likely forms a salt-bridge with P1 in M-PMV CA.     

Rescue of internal scaffold-deleted Mason-Pfizer monkey virus particle production by plasma membrane targeting

The Mason-Pfizer monkey virus (M-PMV) Gag protein follows a morphogenesis pathway in which immature capsids are preassembled within the cytoplasm before interaction with and budding through the plasma membrane. Intracytoplasmic assembly is facilitated by sequences within the p12 domain of Gag that we have termed the Internal Scaffold Domain (ISD). If M-PMV utilizes an ISD then what provides the equivalent function for most other retroviruses that assemble at the plasma membrane? To investigate the possibility that the membrane itself fulfills this role, we have combined functional deletion of the ISD with a mutation that disrupts intracellular targeting or with a plasma membrane targeting signal. By either modification, targeting of ISD-deleted Gag to the plasma membrane restores particle production. These results provide support for a model in which the plasma membrane and the D-type ISD provide an interchangeable scaffold-like function in retrovirus assembly.     

Specific in vitro cleavage of Mason-Pfizer monkey virus capsid protein: evidence for a potential role of retroviral protease in early stages of infection

Processing of Gag polyproteins by viral protease (PR) leads to reorganization of immature retroviral particles and formation of a ribonucleoprotein core. In some retroviruses, such as HIV and RSV, cleavage of a spacer peptide separating capsid and nucleocapsid proteins is essential for the core formation. We show here that no similar spacer peptide is present in the capsid-nucleocapsid (CA-NC) region of Mason-Pfizer monkey virus (M-PMV) and that the CA protein is cleaved in vitro by the PR within the major homology region (MHR) and the NC protein in several sites at the N-terminus. The CA cleavage product was also identified shortly after penetration of M-PMV into COS cells, suggesting that the protease-catalyzed cleavage is involved in core disintegration.     

The impact of altered polyprotein ratios on the assembly and infectivity of Mason-Pfizer monkey virus

Most retroviruses employ a frameshift mechanism during polyprotein synthesis to balance appropriate ratios of structural proteins and enzymes. To investigate the requirements for individual precursors in retrovirus assembly, we modified the polyprotein repertoire of Mason-Pfizer monkey virus (M-PMV) by mutating the frameshift sites to imitate the polyprotein organization of Rous sarcoma virus (Gag-Pro and Gag-Pro-Pol) or Human immunodeficiency virus (Gag and Gag-Pro-Pol). For the “Rous-like” virus, assembly was impaired with no incorporation of Gag-Pro-Pol into particles and for the “HIV-like” virus an altered morphogenesis was observed. A mutant expressing Gag and Gag-Pro polyproteins and lacking Gag-Pro-Pol assembled intracellular particles at a level similar to the wild-type. Gag-Pro-Pol polyprotein alone neither formed immature particles nor processed the precursor. All the mutants were non-infectious except the “HIV-like”, which retained fractional infectivity.     

Atomic force microscopy investigation of Mason-Pfizer monkey virus and human immunodeficiency virus type 1 reassembled particles

Particles of DeltaProCANC, a fusion of capsid (CA) and nucleocapsid (NC) protein of Mason-Pfizer monkey virus (M-PMV), which lacks the amino terminal proline, were reassembled in vitro and visualized by atomic force microscopy (AFM). The particles, of 83-84 nm diameter, exhibited ordered domains based on trigonal arrays of prominent rings with center to center distances of 8.7 nm. Imperfect closure of the lattice on the spherical surface was affected by formation of discontinuities. The lattice is consistent only with plane group p3 where one molecule is shared between contiguous rings. There are no pentameric clusters nor evidence that the particles are icosahedral. Tubular structures were also reassembled, in vitro, from two HIV fusion proteins, DeltaProCANC and CANC. The tubes were uniform in diameter, 40 nm, but varied in length to a maximum of 600 nm. They exhibited left handed helical symmetry based on a p6 hexagonal net. The organization of HIV fusion proteins in the tubes is significantly different than for the protein units in the particles of M-PMV DeltaProCANC.     

Mason-Pfizer monkey virus Gag proteins interact with the human sumo conjugating enzyme, hUbc9

Retroviral Gag proteins function during early and late stages of the viral life cycle. To gain additional insight into the cellular requirements for viral replication, a two-hybrid screen was used to identify cellular proteins that interact with the Mason-Pfizer monkey virus Gag protein. One of the cellular proteins found was identified as hUbc9, a nuclear pore-associated E2 SUMO conjugating enzyme. In vitro protein interaction assays verified the association and mapped the interaction domain to the CA protein. In vivo, hUbc9 and Gag colocalized in the cytoplasm as discrete foci near the nuclear membrane. In addition, overexpression of hUbc9 in cells caused a fraction of Gag to colocalize with hUbc9 in the nucleus. These experiments demonstrate that hUbc9 and Gag interact in cells, strengthen the hypothesis that Gag proteins transiently associate with the nuclear compartment during viral replication, and suggest that hUbc9 plays a role in this process.     

Varicella-zoster virus assembly protein p32/p36 is present in DNA-containing as well as immature capsids

Varicella-zoster virus (VZV) produces a group of nucleocapsid proteins (the p32/p36 nucleoprotein complex) which are the VZV analogues of the herpes simplex virus type 1 (HSV-1) and cy0tomegalovirus (CMV) assembly proteins. There are multiple components in the VZV p32/p36 complex, with major proteins of 32 and 36 kDa and minor proteins of 34 and 38 kDa. In HSV-1 the assembly proteins have been shown to be present in immature (B) capsids, but are removed prior to the formation of mature (C) capsids containing the viral DNA genome. Our work has shown that VZV produces capsids corresponding to the B and C forms. However, in contrast to HSV-1, VZV also produces “B/C” capsids that appear to contain both the assembly proteins and the viral DNA genome. Possible mechanisms for this are discussed. In addition, it was shown that VZV capsids appear to lack the 36 and 38 kDa proteins, and based on this observation we suggest that these may represent unprocessed forms of the assembly protein. In both HSV and CMV, a much larger, crossreactive protein has been identified as the fulllength product of the gene coding for the assembly protein. The homologous VZV gene (ORF 33) theoretically has the capacity to produce a 66 kDa protein. However, no such protein is readily apparent in VZV-infected cells. The presence of an immunoreactive 64 kDa protein was demonstrated in purified VZV capsids which may represent the full-length ORF 33 protein. © 1995 Wiley-Liss, Inc.     

Inhibition of viral assembly in murine cells by HIV-1 matrix

In human cells, the N-terminal matrix (MA) domain of the human immunodeficiency virus type 1 (HIV-1) Gag targets assembly to specific membrane compartments. In murine fibroblasts, membrane targeting of Gag and assembly of HIV-1 are inefficient. These deficiencies are relieved by replacement of HIV-1 MA with murine leukemia virus (MLV) MA in chimeric proviruses. In this study, we examined chimeric HIV-1 carrying tandem MLV and HIV-1 MA domains and found that the addition of MLV MA to the N-terminus of HIV-1 Gag enhanced membrane binding in murine cells, but was not sufficient to stimulate virus production. Removal of HIV MA was required to observe more efficient Gag processing and increased virus production in murine cells. Deletion of the globular head of MA also alleviated the blocks to membrane binding and Gag processing in murine cells, yet did not lead to increased virus production. These MA-dependent, cell-type-specific phenotypes suggest that host factors interact with the globular head of MA to regulate membrane binding and additional membrane-independent step(s) required for assembly.     

Self-interaction of ORF II protein through the leucine zipper is essential for Soybean chlorotic mottle virus infectivity

The ORF II protein (PII) of Soybean chlorotic mottle virus (SbCMV) is essential for the virus life cycle. We investigated the interactions of SbCMV PII with itself and with other essential virus proteins using a Gal4-based yeast two-hybrid system. PII interacted only with itself and not with any other virus proteins. The PII-PII interaction was confirmed by a Sos-based yeast two-hybrid system and a far-western analysis. Deletion mutagenesis mapped the self-interacting domain to the C-terminal 48 amino acids (amino acids 154-201), which contain two putative leucine zipper motifs. Introduction of amino acid substitutions to leucine/isoleucine in zipper sequences prevented the PII-PII interaction and abolished the infectivity of SbCMV. These results revealed that the self-interaction of PII through a leucine zipper is necessary for virus infection.     

Prenatal diagnosis and clinical findings in a case of hexasomy 12p

We report the first case of hexasomy 12p mosaicism due to 2 copies of an apparent i(12p) [46,XX/48,XX, +i(12p),+i(12p)] In every cell that contained the i(12p), 2 copies of the marker were found. The hexasomy was found in amniocytes (16%) and skin fibroblasts (95%) but not in peripheral blood lymphocytes. The chromosomal origin of the marker was confirmed with the use of in situ hybridization of alpha-satellite specific for the centromere of chromosome 12. The present case was diagnosed following chromosome analysis for anomalies on ultrasound. The hexasomy 12p patient showed striking phenotypic similarities with severely affected tetrasomy 12p cases and died shortly after birth. We propose that the more severe presentation of this case is due to the 4 extra copies of 12p. © 1993 Wiley-Liss, Inc.     

Prezygotic origin of the isochromosome 12p in Pallister-Killian syndrome

Pallister-Killian syndrome is a rare disorder comprising multiple congenital anomalies, streaks of hypo(hyper)pigmentation, seizures, profound mental retardation, and the presence of an extra metacentric chromosome i(12)(p10), usually limited to skin fibroblasts. The mechanism and parental origin of the extra chromosome i(12)(p10) are unknown. Here, we present a girl with Pallister-Killian syndrome and the i(12)(p10) in 50% of cultured skin fibroblasts. Using micro-satellite DNA markers of chromosome 12p, we detected 3 alleles—including 2 different alleles of maternal origin—in cultured skin fibroblasts, suggesting that the tetrasomy 12p is the result of a prezygotic event, with a nondisjunction event during maternal meiosis. Am. J. Med. Genet. 69:166–168, 1997. © 1997 Wiley-Liss, Inc.     

Tetrasomy for the short arm of chromosome 12 with accessory isochromosome (+i(12p)) and a marked LDH-B gene dosage effect

A fetus with tetrasomy for the short arm of chromosome 12 due to a de novo accessory isochromosome i(12p) is described. Involvement of the 12p in this chromosome aberration was suggested by banding analysis and substantiated by detection of a marked increase of LDH-B in the fetal fibroblasts. The syndrome shown by this fetus includes many of the minor anomalies described for live-born patients with partial trisomy 12p, and in addition malformations including brachymelia, anal atresia and double kidneys.     

Tissue specificity and stability of mosaicism in Pallister–Killian +i(12p) syndrome: Relevance for prenatal diagnosis

We ascertained +i(12p) mosaicism during third trimester in a case of polyhydramnios and diaphragmatic hernia. Primary cultures of amniocytes had colonies with +i(12p), colonies without +i(12p), and mixed colonies with 46/47, +i(12p). The likely explanation was instability and loss of i(12p) during somatic divisions of amniocytes. Fetal blood in third trimester retained +i(12p) in 13% of cells. A review of mosaicism in published cases indicates that factors influencing the presence of +i(12p) include tissue type and in vitro and in vivo age. In blood, amniocyte, and probably bone marrow cultures, +i(12p) is less stable than in fibroblast-like cultures derived from skin and other tissues. Young cultures at early passage are more likely to have +i(12p) than old cultures. Cultures from young (especially fetal) donors are more likely to retain +i(12p) than cultures from adult donors. These rules will be important in determining appropriate tissues for diagnosis and interpretation of mosaicism in this disorder.     

Interleukin-12p40 mediates transient protection against Mycobacterium avium infection in the absence of interleukin-12

Produced by macrophages and dendritic cells, interleukin (IL)-12 is composed of a p35 and a p40 subunit and promotes protection against intracellular pathogens through the development of interferon-gamma (IFNgamma) -producing T cells. The p40 subunit is also shared by the dimeric cytokines IL-12p40 homodimer and IL-23. In man, genetic defects in IL-12p40-mediated mechanisms are responsible for the familial occurrence of nontuberculous mycobacterial infections, the most common of which is infection with Mycobacterium avium. To experimentally differentiate the contribution of IL-12p40-containing cytokines in the outcome of M. avium infection, we studied wild-type, p35- and p35/p40 doubly deficient mice in an intravenous infection model which reflects many parameters of the disseminated infection in humans. Our study shows that in contrast to p35/p40 doubly deficient mice, p35-deficient mice mount a transient antibacterially protective response against M. avium although such animals were unable to produce detectable levels of IFNgamma or generate efficient granulomas. In conclusion, our results identify an antibacterial effector mechanism preserved in p35-deficient mice that is absent in mice devoid of p35 and p40. This phenotype probably reflects an IL-12p40-dependent effect on macrophage activation at the level of innate immunity.     

In vitro binding of simian immunodeficiency virus matrix protein to the cytoplasmic domain of the envelope glycoprotein

Incorporation of the envelope (Env) glycoprotein into budding virions is a key step in the replication cycle of lentiviruses. Previously, we provided genetic and biochemical evidence indicating that Env packaging into simian immunodeficiency virus (SIV) particles is mediated by the association of the Env cytoplasmic domain (CD) with the matrix (MA) domain of Gag. In this study, we developed an in vitro binding assay that, based on recombinant proteins expressed in bacteria, allowed us to demonstrate the physical interaction between the SIV Env CD and the MA in the absence of other viral or cellular proteins. We show that this association is blocked by mutations in each of the interacting domains that have been reported to interfere in vivo with the incorporation of Env into SIV virions. Moreover, we determined that the binding of SIV MA to the Env CD is saturable with a dissociation constant of 7x10(-7) M. Interestingly, the SIV MA is capable of specifically interacting in vitro with the human immunodeficiency virus type 1 Env CD, but not with that of the distantly related feline immunodeficiency virus. Our results strongly support the notion that the association between the SIV MA and Env CD plays a central role in the process of SIV Env incorporation into Gag-made particles.     

A 2a/1b full-length p7 inter-genotypic chimeric genome of hepatitis C virus is infectious in vitro

The p7 protein of hepatitis C virus (HCV) functions as an ion channel in planar lipid bilayers, and its function is vital for the virus life cycle. In this study, we replaced either the entire or partial p7 of genotype 2a (strain JFH1), an HCV strain that replicates and produces virus progeny in vitro, with the corresponding regions of the p7 protein from genotype 1b (Australian isolate, HCV-A). Compared to wild type, the chimeric viruses reached their peak of infectivity with a delay but they produced a comparable titer to the wild type virus and the progeny viruses were able to infect naive permissive cells. Amantadine treatment of wild type and chimeric viruses reduced the virus titers by about 50% and 45%, respectively. Therefore, in this study, for the first time, we demonstrated that genotype 2a (JFH1 strain) genome encoding a full-length genotype 1b p7 gene produces infectious particles in vitro. These chimeric viruses are valuable instruments for comparative studies of the p7 proteins.