scholarly journals Localization of HIV-1 RNA in mammalian nuclei.

1996 ◽  
Vol 135 (1) ◽  
pp. 9-18 ◽  
Author(s):  
G Zhang ◽  
M L Zapp ◽  
G Yan ◽  
M R Green
Keyword(s):  
Nuclear Export ◽  
Splice Junction ◽  
Stable Population ◽  
Beta Globin ◽  
Nuclear Retention ◽  
Viral Rnas ◽  
Immunodeficiency Virus ◽  
Nascent Transcripts ◽  
Hiv 1

The Rev protein of human immunodeficiency virus type 1 (HIV-1) facilitates the nuclear export of unspliced and partially spliced viral RNAs. In the absence of Rev, these intron-containing HIV-1 RNAs are retained in the nucleus. The basis for nuclear retention is unclear and is an important aspect of Rev regulation. Here we use in situ hybridization and digital imaging microscopy to examine the intranuclear distributions of intron-containing HIV RNAs and to determine their spatial relationships to intranuclear structures. HeLa cells were transfected with an HIV-1 expression vector, and viral transcripts were localized using oligonucleotide probes specific for the unspliced or spliced forms of a particular viral RNA. In the absence of Rev, the unspliced viral RNAs were predominantly nuclear and had two distinct distributions. First, a population of viral transcripts was distributed as approximately 10-20 intranuclear punctate signals. Actinomycin D chase experiments indicate that these signals represent nascent transcripts. A second, stable population of viral transcripts was dispersed throughout the nucleoplasm excluding nucleoli. Rev promoted the export of this stable population of viral RNAs to the cytoplasm in a time-dependent fashion. Significantly, the distributions of neither the nascent nor the stable populations of viral RNAs coincided with intranuclear speckles in which splicing factors are enriched. Using splice-junction-specific probes, splicing of human beta-globin pre-mRNA occurred cotranscriptionally, whereas splicing of HIV-1 pre-mRNA did not. Taken together, our results indicate that the nucleolus and intranuclear speckles are not involved in Rev regulation, and provide further evidence that efficient splicing signals are critical for cotranscriptional splicing.

Rev and the fate of pre-mRNA in the nucleus: implications for the regulation of RNA processing in eukaryotes

1993 ◽  
Vol 13 (10) ◽  
pp. 6180-6189 ◽  
Author(s):  
M H Malim ◽  
B R Cullen
Keyword(s):  
T Cells ◽  
Regulation Of Gene Expression ◽  
Nucleocytoplasmic Transport ◽  
Viral Mrnas ◽  
Nuclear Retention ◽  
Human T Cells ◽  
Cellular Factors ◽  
Immunodeficiency Virus ◽  
Nuclear Degradation ◽  
Hiv 1

Although a great deal is known about the regulation of gene expression in terms of transcription, relatively little is known about the modulation of pre-mRNA processing. In this study, we exploited a genetically regulated system, human immunodeficiency virus type 1 (HIV-1) and its trans-activator Rev, to examine events that occur between the synthesis of pre-mRNA in the nucleus and the translation of mRNA in the cytoplasm. Unlike the majority of eukaryotic pre-mRNAs whose introns are efficiently recognized and spliced prior to nucleocytoplasmic transport, HIV-1 mRNAs containing functional introns must be exported to the cytoplasm for the expression of many viral proteins. Using human T cells containing stably integrated proviruses, we demonstrate that such incompletely spliced viral mRNAs are exported to the cytoplasm only in the presence of the Rev trans-activator. In the absence of Rev, these intron-containing RNAs are sequestered in the T-cell nucleus and either spliced or, more commonly, degraded. Because Rev does not inhibit the expression of fully spliced viral mRNA species in T cells, we propose that Rev, rather than inhibiting viral pre-mRNA splicing, is acting here both to prevent the nuclear degradation of HIV-1 pre-mRNAs and to induce their translocation to the cytoplasm. Taken together, these findings indicate that the cellular factors responsible for the nuclear retention of unspliced pre-mRNAs, although most probably splicing factors, do not invariably commit these RNAs to productive splicing and can, instead, program such transcripts for degradation.

scholarly journals Reassessment of the Roles of Integrase and the Central DNA Flap in Human Immunodeficiency Virus Type 1 Nuclear Import

2002 ◽  
Vol 76 (23) ◽  
pp. 12087-12096 ◽  
Author(s):  
Jeffrey D. Dvorin ◽  
Peter Bell ◽  
Gerd G. Maul ◽  
Masahiro Yamashita ◽  
Michael Emerman ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
In Situ Hybridization ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Import ◽  
Immunodeficiency Virus ◽  
Central Polypurine Tract ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) can infect nondividing cells productively because the nuclear import of viral nucleic acids occurs in the absence of cell division. A number of viral factors that are present in HIV-1 preintegration complexes (PICs) have been assigned functions in nuclear import, including an essential valine at position 165 in integrase (IN-V165) and the central polypurine tract (cPPT). In this article, we report a comparison of the replication and infection characteristics of viruses with disruptions in the cPPT and IN-V165. We found that viruses with cPPT mutations still replicated productively in both dividing and nondividing cells, while viruses with a mutation at IN-V165 did not. Direct observation of the subcellular localization of HIV-1 cDNAs by fluorescence in situ hybridization revealed that cDNAs synthesized by both mutant viruses were readily detected in the nucleus. Thus, neither the cPPT nor the valine residue at position 165 of integrase is essential for the nuclear import of HIV-1 PICs.

scholarly journals PCR-derived ssDNA Probes for Fluorescent In Situ Hybridization to HIV-1 RNA

2000 ◽  
Vol 48 (2) ◽  
pp. 285-293 ◽  
Author(s):  
Marlyse C. Knuchel ◽  
Brigit Graf ◽  
Erika Schlaepfer ◽  
Herbert Kuster ◽  
Marek Fischer ◽  
...  
Keyword(s):  
T Cells ◽  
In Situ Hybridization ◽  
Fluorescent In Situ Hybridization ◽  
Uracil Dna Glycosylase ◽  
Rna Probes ◽  
Immunodeficiency Virus ◽  
Polymerase Chain ◽  
Rna Probe ◽  
Hiv 1

We developed a simple and rapid technique to synthesize single-stranded DNA (ssDNA) probes for fluorescent in situ hybridization (ISH) to human immunodeficiency virus 1 (HIV-1) RNA. The target HIV-1 regions were amplified by the polymerase chain reaction (PCR) and were simultaneously labeled with dUTP. This product served as template for an optimized asymmetric PCR (one-primer PCR) that incorporated digoxigenin (dig)-labeled dUTP. The input DNA was subsequently digested by uracil DNA glycosylase, leaving intact, single-stranded, digoxigenin-labeled DNA probe. A cocktail of ssDNA probes representing 55% of the HIV-1 genome was hybridized to HIV-1-infected 8E5 T-cells and uninfected H9 T-cells. For comparison, parallel hybridizations were done with a plasmid-derived RNA probe mix covering 85% of the genome and a PCR-derived RNA probe mix covering 63% of the genome. All three probe types produced bright signals, but the best signal-to-noise ratios and the highest sensitivities were obtained with the ssDNA probe. In addition, the ssDNA probe syntheses generated large amounts of probe (0.5 to 1 μg ssDNA probe per synthesis) and were easier to perform than the RNA probe syntheses. These results suggest that ssDNA probes may be preferable to RNA probes for fluorescent ISH.

scholarly journals C19ORF66 is an Interferon-Stimulated Gene (ISG) which Inhibits Human Immunodeficiency Virus-1

2016 ◽  
Author(s):  
Weidong Xiong ◽  
Deisy Contreras ◽  
Joseph Ignatius Irudayam ◽  
Ayub Ali ◽  
Otto O. Yang ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Nuclear Export ◽  
Splice Variants ◽  
Nuclear Export Signal ◽  
Full Length ◽  
Type I ◽  
Immune Factor ◽  
Immunodeficiency Virus ◽  
P24 Protein ◽  
Hiv 1

ABSTRACTInnate immunity is the first line of defense against invading microbes1. The type I interferon (IFN) pathway plays a key role in controlling Human Immunodeficiency Virus type 1 (HIV-1) replication2,3. We identified an IFN-α stimulated gene C19ORF66 that we term Suppressor of Viral Activity (SVA). Full length SVA-1 protein inhibits HIV-1 by blocking virion production. SVA splice variants truncated at the C-terminus and/or disrupted at the nuclear export signal (NES) lose antiviral activity and localize to nucleus, while full length SVA-1 co-localizes with HIV-1 p24 protein in the cytoplasmic compartment of infected cells. SVA-1 is structurally and functionally conserved across species, including mouse and chimpanzee. We provide the first description of the effector function of the gene SVA/C190RF66 as an innate immune factor with anti-HIV-1 activity.

scholarly journals SRp40 and SRp55 Promote the Translation of Unspliced Human Immunodeficiency Virus Type 1 RNA

2010 ◽  
Vol 84 (13) ◽  
pp. 6748-6759 ◽  
Author(s):  
Chad M. Swanson ◽  
Nathan M. Sherer ◽  
Michael H. Malim
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Export ◽  
Sr Proteins ◽  
Rna Recognition Motif ◽  
Genomic Rnas ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Nuclear RNA processing events, such as 5′ cap formation, 3′ polyadenylation, and pre-mRNA splicing, mark mRNA for efficient translation. Splicing enhances translation via the deposition of the exon-junction complex and other multifunctional splicing factors, including SR proteins. All retroviruses synthesize their structural and enzymatic proteins from unspliced genomic RNAs (gRNAs) and must therefore exploit unconventional strategies to ensure their effective expression. Here, we report that specific SR proteins, particularly SRp40 and SRp55, promote human immunodeficiency virus type 1 (HIV-1) Gag translation from unspliced (intron-containing) viral RNA. This activity does not correlate with nucleocytoplasmic shuttling capacity and, in the case of SRp40, is dependent on the second RNA recognition motif and the arginine-serine (RS) domain. While SR proteins enhance Gag expression independent of RNA nuclear export pathway choice, altering the nucleotide sequence of the gag-pol coding region by codon optimization abolishes this effect. We therefore propose that SR proteins couple HIV-1 gRNA biogenesis to translational utilization.

scholarly journals Feline Immunodeficiency Virus Gag Is a Nuclear Shuttling Protein

2012 ◽  
Vol 86 (16) ◽  
pp. 8402-8411 ◽  
Author(s):  
Iris Kemler ◽  
Dyana Saenz ◽  
Eric Poeschla
Keyword(s):  
Feline Immunodeficiency Virus ◽  
Nuclear Export ◽  
Leptomycin B ◽  
Genomic Rnas ◽  
Intracellular Location ◽  
Export Pathway ◽  
Immunodeficiency Virus ◽  
Nuclear Shuttling ◽  
Rna Interaction ◽  
Hiv 1

Lentiviral genomic RNAs are encapsidated by the viral Gag protein during virion assembly. The intracellular location of the initial Gag-RNA interaction is unknown. We previously observed feline immunodeficiency virus (FIV) Gag accumulating at the nuclear envelope during live-cell imaging, which suggested that trafficking of human immunodeficiency virus type 1 (HIV-1) and FIV Gag may differ. Here we analyzed the nucleocytoplasmic transport properties of both Gag proteins. We discovered that inhibition of the CRM1 nuclear export pathway with leptomycin B causes FIV Gag but not HIV-1 Gag to accumulate in the nucleus. Virtually all FIV Gag rapidly became intranuclear when the CRM1 export pathway was blocked, implying that most if not all FIV Gag normally undergoes nuclear cycling. In FIV-infected feline cells, some intranuclear Gag was detected in the steady state without leptomycin B treatment. When expressed individually, the FIV matrix (MA), capsid (CA), and nucleocapsid-p2 (NC-p2) domains were not capable of mediating leptomycin B-sensitive nuclear export of a fluorescent protein. In contrast, CA-NC-p2 did mediate nuclear export, with MA being dispensable. We conclude that HIV-1 and FIV Gag differ strikingly in a key intracellular trafficking property. FIV Gag is a nuclear shuttling protein that utilizes the CRM1 nuclear export pathway, while HIV-1 Gag is excluded from the nucleus. These findings expand the spectrum of lentiviral Gag behaviors and raise the possibility that FIV genome encapsidation may initiate in the nucleus.

scholarly journals Restriction of Human Immunodeficiency Virus Type 1 Rev Function in Murine A9 Cells Involves the Rev C-Terminal Domain

2003 ◽  
Vol 77 (5) ◽  
pp. 3084-3090 ◽  
Author(s):  
Sandra M. P. Marques ◽  
Jean-Luc Veyrune ◽  
Ram R. Shukla ◽  
Ajit Kumar
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Export ◽  
Productive Infection ◽  
Cell Leukemia Virus Type ◽  
Terminal Domain ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) Rev and human T-cell leukemia virus type 1 (HTLV-1) Rex proteins are essential for the expression of viral structural proteins and productive infection. Both contain a nuclear export signal (NES) in their C-terminal domain and a nuclear localization signal (NLS) in their N-terminal domain. The NES and NLS are necessary for shuttling between nucleus and cytoplasm and are therefore indispensable for the transport of unspliced and singly spliced viral transcripts. HIV-1 Rev function is restricted in A9 cells, a murine fibroblast cell line, whereas HTLV-1 Rex is functional in these cells. Immunofluorescence studies with RevGFP fusion protein demonstrate normal import and export of Rev in A9 cells. To ascertain which domains of Rev are necessary for the restriction of Rev function in A9 cells, we studied a chimeric construct in which the NES domain of Rev was exchanged with Rex C-terminal amino acids 79 to 95, the Rev1-79/Rex79-95 chimera, which restored Rev function in A9 cells. In addition, overexpression of a truncated Rev containing the Rev C-terminal domain in the presence of wild-type Rev, led to restoration of Rev function in A9 cells. These results suggest that the C-terminal domain of HIV-1 Rev plays an important role in restricting Rev function in murine cells.

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