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 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.

scholarly journals Concerted Action of Multiple cis-Acting Sequences Is Required for Rev Dependence of Late Human Immunodeficiency Virus Type 1 Gene Expression

2000 ◽  
Vol 74 (22) ◽  
pp. 10822-10826 ◽  
Author(s):  
Marcus Graf ◽  
Alexandra Bojak ◽  
Ludwig Deml ◽  
Kurt Bieler ◽  
Hans Wolf ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Export ◽  
Regulatory Elements ◽  
Cis Acting ◽  
Immunodeficiency Virus ◽  
Responsive Element ◽  
Hiv 1

ABSTRACT Based on the human immunodeficiency virus type 1 (HIV-1)gag gene, subgenomic reporter constructs have been established allowing the contributions of differentcis-acting elements to the Rev dependency of late HIV-1 gene products to be determined. Modification of intragenic regulatory elements achieved by adapting the codon usage of the complete gene to highly expressed mammalian genes resulted in constitutive nuclear export allowing high levels of Gag expression independent from the Rev/Rev-responsive element system and irrespective of the absence or presence of the isolated major splice donor. Leptomycin B inhibitor studies revealed that the RNAs derived from the codon-optimizedgag gene lacking AU-rich inhibitory elements are directed to a distinct, CRM1-independent, nuclear export pathway.

scholarly journals Inhibition of Human Immunodeficiency Virus Type 1 (HIV-1) Replication by HIV-1-Based Lentivirus Vectors Expressing Transdominant Rev

2001 ◽  
Vol 75 (8) ◽  
pp. 3590-3599 ◽  
Author(s):  
Mario R. Mautino ◽  
Nicholas Keiser ◽  
Richard A. Morgan
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Export ◽  
Wild Type Virus ◽  
Wild Type ◽  
Helper Plasmid ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Retrovirus vectors expressing transdominant-negative mutants of Rev (TdRev) inhibit human immunodeficiency virus type 1 (HIV-1) replication by preventing the nuclear export of unspliced viral transcripts, thus inhibiting the synthesis of Gag-Pol, Env, and genomic RNA. The use of HIV-1–based vectors to express TdRev would have the advantage of allowing access to nondividing hematopoietic cells. It would also provide additional levels of protection by sequestering the viral regulatory proteins Tat and Rev, competing for encapsidation into wild-type virions, and inhibiting reverse transcription. Here we describe HIV-1-based vectors that express TdRev. These vectors contain mutations in the splicing signals or replacement of the Rev-responsive element by the simian retrovirus type 1 constitutive transport element, making them less sensitive to the inhibitory effects of TdRev. In addition, overexpression of Rev and the use of an HIV-1 helper plasmid that drives high levels of Gag-Pol synthesis were used to transiently overcome the inhibition by TdRev of the synthesis of Gag-Pol during vector production. SupT1 cells transduced with these vectors were more resistant to HIV-1 replication than cells transduced with Moloney murine leukemia virus-based vectors expressing TdRev. Furthermore, we show that these vectors can be mobilized by the wild-type virus, reducing the infectivity of virions escaping inhibition and conferring protection against HIV-1 replication to previously untransduced cells.

scholarly journals Nuclear Export of Vpr Is Required for Efficient Replication of Human Immunodeficiency Virus Type 1 in Tissue Macrophages

2003 ◽  
Vol 77 (13) ◽  
pp. 7582-7589 ◽  
Author(s):  
Michael P. Sherman ◽  
Carlos M. C. de Noronha ◽  
Lauren A. Eckstein ◽  
Jason Hataye ◽  
Pamela Mundt ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Nucleocytoplasmic Shuttling ◽  
Viral Spread ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Retroviruses must gain access to the host cell nucleus for subsequent replication and viral propagation. Human immunodeficiency virus type 1 (HIV-1) and other primate lentiviruses are distinguished from the gammaretroviruses by their ability to infect nondividing cells such as macrophages, an important viral reservoir in vivo. Rather than requiring nuclear membrane breakdown during cell division, the HIV-1 preintegration complex (PIC) enters the nucleus by traversing the central aqueous channel of the limiting nuclear pore complex. The HIV-1 PIC contains three nucleophilic proteins, matrix, integrase, and Vpr, all of which have been implicated in nuclear targeting. The mechanism by which Vpr can display such nucleophilic properties and yet also be available for incorporation into virions assembling at the plasma membrane is unresolved. We recently characterized Vpr as a nucleocytoplasmic shuttling protein that contains two novel nuclear import signals and an exportin-1-dependent nuclear export signal (NES). We now demonstrate that mutation of this NES impairs the incorporation of Vpr into newly formed virions. Furthermore, we find that the Vpr NES is required for efficient HIV replication in tissue macrophages present in human spleens and tonsils. These findings underscore how the nucleocytoplasmic shuttling of Vpr not only contributes to nuclear import of the HIV-1 PIC but also enables Vpr to be present in the cytoplasm for incorporation into virions, leading to enhancement of viral spread within nondividing tissue macrophages.

scholarly journals A Nuclear Kinesin-Like Protein Interacts with and Stimulates the Activity of the Leucine-Rich Nuclear Export Signal of the Human Immunodeficiency Virus Type 1 Rev Protein

2003 ◽  
Vol 77 (13) ◽  
pp. 7236-7243 ◽  
Author(s):  
L. K. Venkatesh ◽  
T. Gettemeier ◽  
G. Chinnadurai
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Export ◽  
Cell Leukemia Virus Type ◽  
Rev Response Element ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Rev Protein

ABSTRACT The Rev protein of human immunodeficiency virus type 1 (HIV-1) is essential for the nucleocytoplasmic transport of unspliced and partially spliced HIV mRNAs containing the Rev response element (RRE). In a yeast two-hybrid screen of a HeLa cell-derived cDNA expression library for human factors interacting with the Rev leucine-rich nuclear export sequence (NES), we identified a kinesin-like protein, REBP (Rev/Rex effector binding protein), highly homologous to Kid, the carboxy-terminal 75-residue region of which interacts specifically with the NESs of HIV-1 Rev, human T-cell leukemia virus type 1 Rex, and equine infectious anemia virus Rev but not with functionally inactive mutants thereof. REBP is a nuclear protein that colocalizes with Rev in the nucleoplasm and nuclear periphery of transfected cells. Specific, albeit weak, interaction between REBP and Rev could be demonstrated in coimmunoprecipitation assays in BSC-40 cells. REBP can modestly enhance Rev-dependent RRE-linked reporter gene expression both independently and in cooperation with the nucleoporin cofactor Rab/hRIP. Thus, REBP displays the characteristics expected of an authentic mediator of Rev NES function and may play a role in RRE RNA transport during HIV infection.

scholarly journals Matrix Mediates the Functional Link between Human Immunodeficiency Virus Type 1 RNA Nuclear Export Elements and the Assembly Competency of Gag in Murine Cells

2009 ◽  
Vol 83 (17) ◽  
pp. 8525-8535 ◽  
Author(s):  
Nathan M. Sherer ◽  
Chad M. Swanson ◽  
Stelios Papaioannou ◽  
Michael H. Malim
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Export ◽  
Particle Production ◽  
Functional Link ◽  
Rev Response Element ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) assembles poorly in murine cells, reflecting inefficient targeting of the Gag structural polyprotein to the plasma membrane. Virus particle production can be restored by replacing the cis-acting Rev response element (RRE) in Gag-Pol mRNAs with multiple copies of the CTE (4×CTE), suggesting a mechanistic link between HIV-1 RNA trafficking and productive Gag assembly. In this report, we demonstrate that Gag molecules generated from RRE-dependent transcripts are intrinsically defective for assembly in murine 3T3 cells. When controlled for the intracellular Gag level, modulations of the Gag matrix (MA) domain that enhance Gag membrane association (e.g., deletion of the MA globular head) substantially improve assembly for Gag derived from RRE- but not 4×CTE-dependent transcripts. Gag mutants carrying a leucine zipper replacement of the nucleocapsid (NC) domain remain largely assembly defective when derived from RRE-dependent transcripts, indicating that the defect does not reflect aberrant NC/RNA-driven Gag multimerization. We further demonstrate that single changes in uncharged amino acids implicated in Gag/MA myristoyl switch regulation, most notably replacing the leucine at position 21 with serine, improve assembly for Gag derived from RRE-dependent transcripts. In sum, we provide genetic evidence to suggest that HIV-1 RNA metabolism specifically modulates the activation of MA-dependent membrane targeting.

scholarly journals PSF Acts through the Human Immunodeficiency Virus Type 1 mRNA Instability Elements To Regulate Virus Expression

2003 ◽  
Vol 23 (18) ◽  
pp. 6618-6630 ◽  
Author(s):  
Andrei S. Zolotukhin ◽  
Daniel Michalowski ◽  
Jenifer Bear ◽  
Sergey V. Smulevitch ◽  
Abdulmaged M. Traish ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Export ◽  
Regulatory Elements ◽  
Nucleocytoplasmic Transport ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV) gag/pol and env mRNAs contain cis-acting regulatory elements (INS) that impair stability, nucleocytoplasmic transport, and translation by unknown mechanisms. This downregulation can be counteracted by the viral Rev protein, resulting in efficient export and expression of these mRNAs. Here, we show that the INS region in HIV-1 gag mRNA is a high-affinity ligand of p54nrb/PSF, a heterodimeric transcription/splicing factor. Both subunits bound INS RNA in vitro with similar affinity and specificity. Using an INS-containing subgenomic gag mRNA, we show that it specifically associated with p54nrb in vivo and that PSF inhibited its expression, acting via INS. Studying the authentic HIV-1 mRNAs produced from an infectious molecular clone, we found that PSF affected specifically the INS-containing, Rev-dependent transcripts encoding Gag-Pol and Env. Both subunits contained nuclear export and nuclear retention signals, whereas p54nrb was continuously exported from the nucleus and associated with INS-containing mRNA in the cytoplasm, suggesting its additional role at late steps of mRNA metabolism. Thus, p54nrb and PSF have properties of key factors mediating INS function and likely define a novel mRNA regulatory pathway that is hijacked by HIV-1.

scholarly journals Nucleocytoplasmic Shuttling by Human Immunodeficiency Virus Type 1 Vpr

2001 ◽  
Vol 75 (3) ◽  
pp. 1522-1532 ◽  
Author(s):  
Michael P. Sherman ◽  
Carlos M. C. de Noronha ◽  
Marina I. Heusch ◽  
Spencer Greene ◽  
Warner C. Greene
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Fusion Proteins ◽  
Nucleocytoplasmic Shuttling ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) is capable of infecting nondividing cells such as macrophages because the viral preintegration complex is able to actively traverse the limiting nuclear pore due to the redundant and possibly overlapping nuclear import signals present in Vpr, matrix, and integrase. We have previously recognized the presence of at least two distinct and novel nuclear import signals residing within Vpr that, unlike matrix and integrase, bypass the classical importin α/β-dependent signals and do not require energy or a RanGTP gradient. We now report that the carboxy-terminal region of Vpr (amino acids 73 to 96) contains a bipartite nuclear localization signal (NLS) composed of multiple arginine residues. Surprisingly, when the leucine-rich Vpr(1–71) fragment, previously shown to harbor an NLS, or full-length Vpr is fused to the C terminus of a green fluorescent protein-pyruvate kinase (GFP-PK) chimera, the resultant protein is almost exclusively detected in the cytoplasm. However, the addition of leptomycin B (LMB), a potent inhibitor of CRM1-dependent nuclear export, produces a shift from a cytoplasmic localization to a nuclear pattern, suggesting that these Vpr fusion proteins shuttle into and out of the nucleus. Studies of nuclear import with GFP-PK–Vpr fusion proteins in the presence of LMB reveals that both of the leucine-rich α-helices are required for effective nuclear uptake and thus define a unique NLS. Using a modified heterokaryon analysis, we have localized the Vpr nuclear export signal to the second leucine-rich helix, overlapping a portion of the amino-terminal nuclear import signal. These studies thus define HIV-1 Vpr as a nucleocytoplasmic shuttling protein.

scholarly journals The Carboxy-Terminal Region of the Human Immunodeficiency Virus Type 1 Protein Rev Has Multiple Roles in Mediating CRM1-Related Rev Functions

2002 ◽  
Vol 76 (16) ◽  
pp. 8079-8089 ◽  
Author(s):  
Yoshiyuki Hakata ◽  
Masami Yamada ◽  
Naoto Mabuchi ◽  
Hisatoshi Shida
Keyword(s):  
Human Immunodeficiency Virus ◽  
Complex Formation ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Export ◽  
Terminal Region ◽  
Immunodeficiency Virus ◽  
Carboxy Terminal ◽  
Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) regulatory protein, Rev, mediates the nuclear export of unspliced and singly spliced viral mRNAs by bridging viral RNA and export receptor human CRM1 (hCRM1). Ribonucleoprotein complex formation, including the oligomerization of Rev proteins on viral RNA, must occur to allow export. We show here that Rev-Rev interactions, which are a basis of complex formation, can be initiated without cellular factors and are subsequently enhanced by hCRM1-Ran-GTP. Furthermore, we reveal functions for the Rev carboxy-terminal (C-terminal) region, which is well conserved among many HIV-1 strains, and for which no function has been reported. This region is required for the efficient binding of Rev to hCRM1 and consequently for nuclear export, Rev-Rev dimerization, and full Rev transactivator activity. Consistent with these results, a HIV-1 proviral plasmid that expresses a C-terminally truncated Rev mutant protein produces smaller amounts of the p24 antigen than does a plasmid that possesses an intact rev gene. These results indicate the functional importance of the C-terminal region for full Rev activity, which leads to efficient HIV-1 replication.

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