The distribution of Sindbis virus proteins in mosquito cells as determined by immunofluorescence and immunoelectron microscopy

Miller, Mary Lou; Brown, Dennis T.

doi:10.1099/0022-1317-74-2-293

Cited by 14 publications

(17 citation statements)

References 19 publications

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“…Cells infected with either poliovirus or Sindbis contain de novo synthesized vesicular structures containing polypeptides which are components of the viral RNA biosynthetic machinery (Bienz et al, 1992;Miller and Brown, 1993). For poliovirus it is clear that these structures are sites of RNA replication (Takeda et al, 1986;Bienz et al, 1992).…”

Section: Discussionmentioning

confidence: 99%

Localization of mouse hepatitis virus open reading frame 1A derived proteins

Bi¹,

Piñón²,

Hughes³

et al. 1998

J Neurovirol

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We have investigated the intracellular localization of proteolytic cleavage products encoded in the 5' portion of mouse hepatitis virus (MHV) gene 1. Immuno¯uorescent labeling of cells with an antiserum which recognizes p28, the ORF1a N-terminal cleavage product, resulted in widespread somewhat granular cytoplasmic staining, indicating that this protein is widely distributed in the cytoplasm of MHV-infected, but not control uninfected cells. Immunouorescent staining of infected cells with antisera which recognize the downstream polypeptides, p65, p240 and p290 labeled discrete vesicular perinuclear structures. Double immuno¯uorescent labeling of BHK cells expressing the MHV receptor (BHK MHVR1 ) and infected with MHV-A59 with a Golgi-speci®c anti-mannosidase II monoclonal antibody and with antiserum recognizing each of these anti-MHV ORF1a polypeptides, showed that the p240 and p290 polypeptides were localized in discrete vesicular structures that overlapped the Golgi complex. Labeling with antibodies speci®c for p65 colocalized with the Golgi region, and showed staining of the perinuclear cytoplasm as well. Plasmids containing sequences contained in the ®rst 6.75 kb of ORF1a have been expressed using the coupled vaccinia virus-T7 polymerase system. Immuno¯uorescent labeling of transfectants with the anti-ORF1a antisera showed patterns of antigen distribution similar to those observed in cells infected with MHV-A59. A deletion analysis with constructs containing only portions of the ORF1a sequence indicated that 303 amino acids containing the ®rst papain-like protease domain (PLP-1) was suf®cient to associate this protein with the Golgi.

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Section: Discussionmentioning

confidence: 99%

Localization of mouse hepatitis virus open reading frame 1A derived proteins

Bi¹,

Piñón²,

Hughes³

et al. 1998

J Neurovirol

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“…Briefly, plasmid DNA was linearized by digestion with NotI, and equal amounts of DNA were used as a template to synthesize RNA transcripts, using a T7 mMessage mMachine in vitro transcription kit (Ambion) according to the manufacturer's directions. A portion of each transcription reaction was radiolabeled using 35 S-UTP (GE Healthcare). To measure 35 S-UTP incorporation, labeled transcripts were bound to glass filters, washed, and quantified using a scintillation counter.…”

Section: Methodsmentioning

confidence: 99%

“…In spite of this, approximately one-half of nsP2 is found in the nucleus of infected mammalian cells, and nsP2 is the only alphavirus nonstructural protein present in the nucleus (1,20,35,42,50,51). Studies performed with SFV, an Old World alphavirus, identified PRRRV as the nuclear localization signal (NLS) in nsP2 and defined the central arginine residue in the sequence as essential for nsP2 nuclear localization.…”

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confidence: 99%

Nuclear Import and Export of Venezuelan Equine Encephalitis Virus Nonstructural Protein 2

Montgomery

Johnston

2007

J Virol

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Many RNA viruses, which replicate predominantly in the cytoplasm, have nuclear components that contribute to their life cycle or pathogenesis. We investigated the intracellular localization of the multifunctional nonstructural protein 2 (nsP2) in mammalian cells infected with Venezuelan equine encephalitis virus (VEE), an important, naturally emerging zoonotic alphavirus. VEE nsP2 localizes to both the cytoplasm and the nucleus of mammalian cells in the context of infection and also when expressed alone. Through the analysis of a series of enhanced green fluorescent protein fusions, a segment of nsP2 that completely localizes to the nucleus of mammalian cells was identified. Within this region, mutation of the putative nuclear localization signal (NLS) PGKMV diminished, but did not obliterate, the ability of the protein to localize to the nucleus, suggesting that this sequence contributes to the nuclear localization of VEE nsP2. Furthermore, VEE nsP2 specifically interacted with the nuclear import protein karyopherin-␣1 but not with karyopherin-␣2, -3, or -4, suggesting that karyopherin-␣1 transports nsP2 to the nucleus during infection. Additionally, a novel nuclear export signal (NES) was identified, which included residues L526 and L528 of VEE nsP2. Leptomycin B treatment resulted in nuclear accumulation of nsP2, demonstrating that nuclear export of nsP2 is mediated via the CRM1 nuclear export pathway. Disruption of either the NLS or the NES in nsP2 compromised essential viral functions. Taken together, these results establish the bidirectional transport of nsP2 across the nuclear membrane, suggesting that a critical function of nsP2 during infection involves its shuttling between the cytoplasm and the nucleus.Venezuelan equine encephalitis virus (VEE), a New World member of the genus Alphavirus in the Togaviridae family, is a pathogen that is responsible for significant disease in equines and can cause a wide range of human symptoms, from inapparent infection to acute encephalitis. In nature, VEE is transmitted between susceptible hosts through a mosquito vector and is responsible for periodic outbreaks of widespread human and equine disease throughout the Americas.The alphaviruses are enveloped viruses with an 11.5-kb genome of single-stranded message-sense RNA. The viral genome, which resembles cellular messages because it contains 5Ј-and 3Ј-untranslated regions, a 5Ј-terminal methylguanylate cap, and a 3Ј-terminal polyadenylate tail, encodes four nonstructural proteins (nsP1 through 4) and three mature structural proteins (capsid, E2, and E1). Upon infection, this RNA genome is directly translated in the cell to produce the nonstructural polyproteins P1234 and P123, depending on whether there is readthrough at the opal stop codon present between nsP3 and nsP4 in most alphaviruses (10, 32). The polyprotein is processed by nsP2, which contains a protease domain in its carboxy terminus, to form the four individual nonstructural proteins, nsP1 to -4 (22). Early during infection, negative-sense viral RNA is sy...

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“…nsP2 also possesses poorly understood auxiliary functions that may affect the outcome of infection. Although the replication cycle occurs in the cytoplasm, nsP2 is the only alphavirus nonstructural protein that is present in both the cytoplasm and the nucleus of infected mammalian cells (3,34,45; S. A. Montgomery and R. E. Johnston, unpublished data). Although the role of nuclear nsP2 remains elusive, disruption of nsP2 nuclear localization compromises the ability of the virus to spread in the brain (11).…”

mentioning

confidence: 99%

“…For electroporation, BHK-21 cells were cultured overnight in medium containing 10% fetal bovine serum (FBS), harvested when subconfluent, and prepared for electroporation as previously described (8,29). HEK-293 cells (ATCC, passage [30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45] were incubated at 37°C under 5% CO 2 and maintained in Dulbecco modified Eagle medium (Gibco) supplemented with 10% FBS, 100 U of penicillin per ml, and 0.5 mg of streptomycin per ml.…”

mentioning

confidence: 99%

Ribosomal Protein S6 Associates with Alphavirus Nonstructural Protein 2 and Mediates Expression from Alphavirus Messages

Montgomery

Berglund

Beard

et al. 2006

J Virol

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Although alphaviruses dramatically alter cellular function within hours of infection, interactions between alphaviruses and specific host cellular proteins are poorly understood. Although the alphavirus nonstructural protein 2 (nsP2) is an essential component of the viral replication complex, it also has critical auxiliary functions that determine the outcome of infection in the host. To gain a better understanding of nsP2 function, we sought to identify cellular proteins with which Venezuelan equine encephalitis virus nsP2 interacted. We demonstrate here that nsP2 associates with ribosomal protein S6 (RpS6) and that nsP2 is present in the ribosome-containing fractions of a polysome gradient, suggesting that nsP2 associates with RpS6 in the context of the whole ribosome. This result was noteworthy, since viral replicase proteins have seldom been described in direct association with components of the ribosome. The association of RpS6 with nsP2 was detected throughout the course of infection, and neither the synthesis of the viral structural proteins nor the presence of the other nonstructural proteins was required for RpS6 interaction with nsP2. nsP1 also was associated with RpS6, but other nonstructural proteins were not. RpS6 phosphorylation was dramatically diminished within hours after infection with alphaviruses. Furthermore, a reduction in the level of RpS6 protein expression led to diminished expression from alphavirus subgenomic messages, whereas no dramatic diminution in cellular translation was observed. Taken together, these data suggest that alphaviruses alter the ribosome during infection and that this alteration may contribute to differential translation of host and viral messages.The Alphavirus genus contains over 25 recognized viruses with a wide geographic distribution. Venezuelan equine encephalitis virus (VEE) is a New World alphavirus that is maintained in nature by cycling between a mosquito vector and susceptible vertebrate hosts. VEE is responsible for periodic outbreaks of disease in humans and equines and is classified as a select agent, making it a prominent pathogen among the alphaviruses.Alphaviruses possess a genome of single-stranded messagesense RNA that is approximately 11.5 kb in length. The alphavirus genome is organized such that the 5Ј two-thirds of the genome encodes four nonstructural proteins (nsP1 through nsP4), whereas the 3Ј one-third encodes three mature structural proteins (capsid, E2, and E1) that are expressed at high levels after transcription from an internal 26S subgenomic mRNA promoter. The viral genome appears to be similar to cellular mRNAs, since it contains 5Ј and 3Ј untranslated regions, with a 5Ј-terminal methylguanylate cap and a 3Ј-terminal polyadenylate tail. Upon release into a host cell, the viral genomic RNA is directly translated by the cellular translation machinery. The nonstructural proteins are synthesized as two polyproteins, termed P123 and P1234. P123 results when translation terminates at an opal termination codon between nsP3 and nsP4. When t...

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The distribution of Sindbis virus proteins in mosquito cells as determined by immunofluorescence and immunoelectron microscopy

Cited by 14 publications

References 19 publications

Localization of mouse hepatitis virus open reading frame 1A derived proteins

Localization of mouse hepatitis virus open reading frame 1A derived proteins

Nuclear Import and Export of Venezuelan Equine Encephalitis Virus Nonstructural Protein 2

Ribosomal Protein S6 Associates with Alphavirus Nonstructural Protein 2 and Mediates Expression from Alphavirus Messages

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