Cytoplasmic Utilization of Human Immunodeficiency Virus Type 1 Genomic RNA Is Not Dependent on a Nuclear Interaction with Gag

Grewe, Bastian; Hoffmann, Birgit; Ohs, Inga; Blissenbach, Maik; Brandt, Sabine; Tippler, Bettina; Grünwald, Thomas; Überla, Klaus

doi:10.1128/jvi.06874-11

Cited by 33 publications

(45 citation statements)

References 69 publications

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“…Two studies in the past year confirmed that HIV-1 Gag lacks a CRM1-dependent nuclear export signal (2,18). In addition, HIV-1 Gag lacking the reported nuclear import and export signals can mediate efficient packaging (18). Our present results with HIV-1 Gag support the current consensus that a nuclear function of this protein in assembly can be excluded unless this occurs at a level that is below the detection limits of present methodologies.…”

Section: Discussionsupporting

confidence: 85%

“…3A to F, 4B, and 6C to F). The results suggest that (10,8,18,21,25,11,18, and 26 cells for the columns going from left to right, respectively), and the ratio of the MFI in the nucleus to the MFI in the total cell was determined and expressed as a percentage (mean Ϯ standard deviation). (K to Q) Subcellular localization of FIV MA-CFP, CA-CFP, and NCp2-CFP.…”

Section: Discussionmentioning

confidence: 95%

“…The HIV-1 MA domain was reported previously to have two NLSs (5,21) and one NES (13), but multiple subsequent reports have shown that HIV-1 MA-GFP is excluded from the nucleus (2,10) and that it does not contain a conventional NLS (15,22). Two studies in the past year confirmed that HIV-1 Gag lacks a CRM1-dependent nuclear export signal (2,18). In addition, HIV-1 Gag lacking the reported nuclear import and export signals can mediate efficient packaging (18).…”

Section: Discussionmentioning

confidence: 97%

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Feline Immunodeficiency Virus Gag Is a Nuclear Shuttling Protein

Kemler

Saenz

Poeschla

2012

J Virol

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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.G ag is both necessary and sufficient for retrovirus particle formation and budding. The protein is translated on free polysomes and targeted to the plasma membrane, where virus particles are assembled (16). A central question is whether Gag and the viral genomic RNA (gRNA) associate at the plasma membrane or earlier during assembly and, if so, whether this occurs in the cytosol; in association with organelles, e.g., cotranslationally; or even in the nucleus, as was described previously for an alpharetrovirus (17). Human immunodeficiency virus type 1 (HIV-1) gRNAs become anchored at the plasma membrane before particle assembly is detectable, but it is not clear whether the Gag-gRNA complexes of this and other lentiviruses first form in the cytoplasm and then transit to the plasma membrane or the gRNA traffics there independently (26,27). Biochemical experiments supported the former scenario, with a monomer or low-order multimers forming on HIV-1 gRNAs and higher-order multimer formation depending on subsequent plasma membrane interactions (33).Gag is encoded by the full-length unspliced viral RNA. To circumvent the cellular checkpoint that prevents the export of intron-containing mRNA, lentiviruses express Rev, which functions as an adaptor between the cellular karyopherin CRM1/exportin-1 and a Rev response element (RRE) located in the 3= region of unspliced viral RNAs (9). However, the main cellular function of the CRM1 nuclear export pathway is the export of cellular proteins containing a...

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Section: Discussionsupporting

confidence: 85%

Section: Discussionmentioning

confidence: 95%

Section: Discussionmentioning

confidence: 97%

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Feline Immunodeficiency Virus Gag Is a Nuclear Shuttling Protein

Kemler

Saenz

Poeschla

2012

J Virol

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“…An hRSV plasmid expressing the codon-optimized hRSV-F ectodomain fused to a His tag was transfected in 293T cells using polyethylenimine as described previously (4,37). Supernatants were harvested and placed on a column packed with 5 ml of cobalt resin (Thermo Scientific, Germany) as recommended by the manufacturer.…”

Section: Methodsmentioning

confidence: 99%

Novel Vaccine Regimen Elicits Strong Airway Immune Responses and Control of Respiratory Syncytial Virus in Nonhuman Primates

Grünwald

Tenbusch

Schulte

et al. 2014

J Virol

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Induction of long-lasting immunity against viral respiratory tract infections؉ and CD8 ؉ T cell response in the lower respiratory tract and protection from intranasal hRSV challenge. Thus, parenteral DNA priming followed by booster immunization targeted to a mucosal inductive site constitutes an effective vaccine regimen for eliciting protective immune responses at mucosal effector sites. IMPORTANCEThe human respiratory syncytial virus (hRSV) is the most common cause of severe respiratory tract disease in infancy and leads to substantial morbidity and morality in the elderly. In this study, we compared the immunogenicity and efficacy of several genebased immunization protocols in rhesus macaques. Thereby, we found that the combination of an initially parenterally delivered DNA vaccine with a subsequent atraumatic tonsillar adenoviral vector immunization results in a strong systemic immune response accompanied by an exceptional high T-cell response in the mucosa. Strikingly, these animals were protected against a RSV challenge infection controlling the viral replication indicated by a 1,000-fold-lower viral load in the lower respiratory tract. Since mucosal cellular responses of this strength had not been described in earlier RSV vaccine studies, this heterologous DNA prime-tonsillar boost vaccine strategy is very promising and should be pursued for further preclinical and clinical testing.

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“…The initial recognition of the HIV-1 gRNA by Pr55 Gag , during which discrimination against cellular and spliced vRNAs takes place, occurs in the cytoplasm and involves a very limited number of Pr55 Gag molecules [42][43][44][45] . Once these complexes become anchored in the plasma membrane, Pr55 Gag molecules are rapidly recruited as viral particle assembly proceeds 43,46 .…”

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confidence: 99%