Nuclear Polyhedrosis Virus: A Possible Example of De Novo Intranuclear Membrane Morphogenesis (original) (raw)
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Journal of Virology, 2001
Partial deletions within Autographa californica open reading frame 61 (FP25K) alter the expression and accumulation profile of several viral proteins and the transport of occlusion-derived virus (ODV)-E66 to intranuclear membranes during infection (S. C. Braunagel et al., J. Virol. 73:8559-8570, 1999). Here we show the effects of a full deletion and overexpression of FP25K on the transport and expression of two ODV envelope proteins, ODV-E66 (E66) and ODV-E25 (E25). Deletion and overexpression of FP25K substantially altered the levels of expression of E66 during infection. Compared with cells infected with wild-type (wt) virus, the levels of E66 were reduced fivefold in cells infected with a viral mutant lacking FP25K (⌬FP25K) and were slightly increased in cells infected with a viral mutant overexpressing FP25K (FP25K polh). In contrast, no significant changes were observed in the levels of E25 among wt-, ⌬FP25K-, and FP25K polh-infected cells. The changes observed in the levels of E66 among the different viral mutants were not accompanied by changes in either the time of synthesis, membrane association, protein turnover, or steady-state transcript abundance. Deletion of FP25K also substantially altered the transport and localization of E66 during infection. In cells infected with the ⌬FP25K mutant virus, E66 accumulated in localized regions at the nuclear periphery and the outer nuclear membrane and did not traffic to intranuclear membranes. In contrast, in cells infected with the FP25K polh mutant virus E66 trafficked to intranuclear membranes. For comparison, E25 was normally transported to intranuclear membranes in both ⌬FP25K-and FP25K polh-infected cells. Altogether these studies suggest that FP25K affects the synthesis of E66 at a posttranscriptional level, probably by altering the translation of E66; additionally, the block in transport of E66 at the nuclear envelope in ⌬FP25K-infected cells suggests that the pathway of E66 trafficking to the inner nuclear membrane and intranuclear microvesicles is specifically regulated and must be influenced by factors that do not control the traffic of E25.
Inhibition of Nuclear Import and Alteration of Nuclear Pore Complex Composition by Rhinovirus
Journal of Virology, 2002
Nucleocytoplasmic trafficking pathways and the status of nuclear pore complex (NPC) components were examined in cells infected with rhinovirus type 14. A variety of shuttling and nonshuttling nuclear proteins, using multiple nuclear import pathways, accumulated in the cytoplasm of cells infected with rhinovirus. An in vitro nuclear import assay with semipermeabilized infected cells confirmed that nuclear import was inhibited and that docking of nuclear import receptor-cargo complexes at the cytoplasmic face of the NPC was prevented in rhinovirus-infected cells. The relocation of cellular proteins and inhibition of nuclear import correlated with the degradation of two NPC components, Nup153 and p62. The degradation of Nup153 and p62 was not due to induction of apoptosis, because p62 was not proteolyzed in apoptotic HeLa cells, and Nup153 was cleaved to produce a 130-kDa cleavage product that was not observed in cells infected with poliovirus or rhinovirus. The finding that both poliovirus and rhinovirus cause inhibition of nuclear import and degradation of NPC components suggests that this may be a common feature of the replicative cycle of picornaviruses. Inhibition of nuclear import is predicted to result in the cytoplasmic accumulation of a large number of nuclear proteins that could have functions in viral translation, RNA synthesis, packaging, or assembly. Additionally, inhibition of nuclear import also presents a novel strategy whereby cytoplasmic RNA viruses can evade host immune defenses by preventing signal transduction into the nucleus.
Journal of Virology, 1977
We report here an in vitro system designed to study the interactions of vesicular stomatitis virus (VSV) proteins with cellular membranes. We have synthesized the VSV nucleocapsid (N) protein, nonstructural (NS) protein, glycoprotein (G protein), and membrane (M) protein in a wheat germ, cell-free, protein-synthesizing system directed by VSV 12 to 18S RNA. When incubated at low salt concentrations with purified cytoplasmic membranes derived from Chinese hamster ovary cells, the VSV M and G proteins bind to membranes, whereas the VSV N and NS proteins do not. The VSV M protein binds to membranes in low or high divalent cation concentrations, whereas binding of significant amounts of G protein requires at least 5 mM magnesium acetate concentrations. Vesicular stomatitis virus (VSV) is a simple, lar membranes, whereas the VSV N and NS enveloped virus that contains two membrane proteins do not. proteins: the glycoprotein (G protein), which forms the spikes of the virion (4, 23), and the MATERIALS AND METHODS membrane (M) protein, which lines the inner Cells and viruses. Membranes were prepared surface of the viral membrane (3). There are from CHO cells. Stocks of VSV (pure B particles of three other known viral proteins, the VSV nuthe Indiana serotype) were grown in CHO cells and cleocapsid (N) protein, the nonstructural (NS) purified as described previously (20). protein, and the viral transcriptase (L) protein. Preparation of VSV 12-186 polyribosomal RNA. These three proteins are associated with the The procedure described by Palmiter (15) was used core ofthe virus particle (16, 23, 24). Each of the with several modifications for the preparation of five viral proteins is synthesized from a mono-VSV 12-18S polyribosomal RNA. CHO cells growing cistronic mRNA (9, 13, 14). at 37°C were infected with VSV at a multiplicity of 3 risngtic m arNAl(9 13, 14). PFU/cell as described previously (20), except that 5 During the early stages in the maturation Of~.tg of actinomycin D per ml was added at the beginthis virus, host cell membranes are modified ning of infection. [3H]uridine (70 Ci/mmol, 25 ,uCi/ with the VSV G and M proteins. Cell fractionaml; New England Nuclear Corp.) was added 2 h tion studies of VSV-infected cells have shown postinfection. Infected cells were harvested at 4.5 h that the VSV M and G proteins rapidly become postinfection, suspended in sucrose-TKM buffer associated with the membrane fraction of the (0.05 M Tris [pH 7.5], 0.025 M KCl, 0.005 M magnecells after their synthesis (5, 11, 12, 24). Nucleo-sium acetate, 0.25 M sucrose), and disrupted with 10 capsid structures containing the genome RNA strokes of a tight-fitting Dounce homogenizer. Nuas well as the viral N, NS, and L proteins are clei were removed by centrifugation (1,000 x g for 2 asswemlld as the vira plaNsm. andsLptroteisures min). The resulting cytoplasmic extract was centriassembled in the cytoplasm. These structures fuged at 20,000 x g for 20 min, and the pellet (mem
Proceedings of the National Academy of Sciences, 2007
Although the nuclear envelope is a dynamic structure that disassembles and reforms during mitosis, the formation of membranous vesicles derived from the nuclear envelope has not yet been described in noninfected cells. However, during herpesvirus maturation, intranuclear capsids initiate transit to the cytosol for final maturation by budding at the inner nuclear membrane. Two conserved herpesvirus proteins are required for this primary envelopment, designated in the alphaherpesviruses as pUL31 and pUL34. Here, we show that simultaneous expression of pUL31 and pUL34 of the alphaherpesvirus pseudorabies virus in stably transfected rabbit kidney cells resulted in the formation of vesicles in the perinuclear space that resemble primary envelopes without a nucleocapsid. They contain pUL31 and pUL34 as shown by immunolabeling and are derived from the nuclear envelope. Thus, coexpression of only two conserved herpesvirus proteins without any other viral factor is sufficient to induce the formation of vesicles from the nuclear membrane. This argues for the contribution of cellular factors in this process either recruited from their natural cytoplasmic location or not yet identified as components of the nuclear compartment.
Okajimas folia anatomica Japonica, 1994
Translocation of baculovirus nucleocapsids (45 nm in diameter, approximate length of 280-300 nm) from nucleoplasm to cytoplasm was studied morphologically using cryofixation and gold labeled wheat germ agglutinin (WGA-gold) during recombinant Autographa californica nuclear polyhedrosis virus infection in Sf9 cells. Nucleocapsids formed in the nucleoplasm migrated into protrusions of the nuclear envelope, but not into nuclear pore complexes. We found cross-like membranous structures. Small pores seemed to be in the protruding nuclear double membranes. The middle piece of a nucleocapsid was located within the small pore whereas the upper part was in the cytoplasm. Other nucleocapsids were situated within pores without colocalization of WGA-gold in the nuclear envelope. These results suggest that baculovirus nucleocapsids use small pores in the nuclear-derived membranes or incomplete nuclear pores in the nuclear envelope to migrate from the nucleoplasm to the cytoplasm, but not complet...