Distinct Intracellular Trafficking of Equine Infectious Anemia Virus and Human Immunodeficiency Virus Type 1 Gag during Viral Assembly and Budding Revealed by Bimolecular Fluorescence Complementation Assays

Jin, Jing; Sturgeon, Timothy J.; Chen, Chaoping; Watkins, Simon C.; Weisz, Ora A.; Montelaro, Ronald C.

doi:10.1128/jvi.00431-07

Cited by 41 publications

(60 citation statements)

References 56 publications

(87 reference statements)

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“…Since cytoplasmic levels of Gag were similar between the two constructs, these findings suggest that how an mRNA is delivered to the cytoplasm can significantly impact the protein interactions that underlie virus formation. Subsequent studies by Jin et al 12 confirmed these observations and implicated that the defect in murine cells was due to a block in Gag multimerization. More recently, Ward et al 13 have observed that the pathway of RNA export has a significant effect on antisense RNA function, Using human derived cell lines, it was found that exportin-1 exported antisense RNA was able to suppress expression of its target gene, while the same sequence delivered via Nxf-1 was not.…”

supporting

confidence: 61%

How does the journey affect the message(RNA)?

Cochrane¹

2009

RNA Biology

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supporting

confidence: 61%

How does the journey affect the message(RNA)?

Cochrane¹

2009

RNA Biology

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“…Replacement of the RRE by the hepatitis B virus posttranscriptional regulatory element (PRE) results in normal Gag expression but reduces the budding efficiency by 10-fold (24). Cell-and species-specific effects have also been observed.…”

mentioning

confidence: 99%

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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“…Separately, the encoded fragments are unable to fluoresce; however, co-expression of interacting proteins individually fused to these fragments generates detectable fluorescence signal when the two fluorescent protein fragments are placed in close proximity (less than 15 nm). The BiFC assay has been used previously to demonstrate co-assembly of HIV-1 and HIV-2 Gag 319 (27). We prepared a panel of constructs expressing fusion proteins with the optimized N-(VN) and C-(VC) terminal fragments of the Venus fluorescence protein for BiFC assay.…”

Section: Resultsmentioning

confidence: 99%

Correlation of the Tight Junction-like Distribution of Claudin-1 to the Cellular Tropism of Hepatitis C Virus

Yang

Qiu

Biswas

et al. 2008

Journal of Biological Chemistry

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Claudin-1 (CLDN1), a tight junction (TJ) protein, has recently been identified as an entry co-receptor for hepatitis C virus (HCV). Ectopic expression of CLDN1 rendered several non-hepatic cell lines permissive to HCV infection. However, little is known about the mechanism by which CLDN1 mediates HCV entry. It is believed that an additional entry receptor(s) is required because ectopic expression of CLDN1 in both HeLa and NIH3T3 cells failed to confer susceptibility to viral infection. Here we found that CLDN1 was co-immunoprecipitated with both HCV envelope proteins when expressed in 293T cells. Results from biomolecular fluorescence complementation assay showed that overexpressed CLDN1 also formed complexes with CD81 and low density lipoprotein receptor. Subsequent imaging analysis revealed that CLDN1 was highly enriched at sites of cell-cell contact in permissive cell lines, co-localizing with the TJ marker, ZO-1. However, in both HeLa and NIH3T3 cells the ectopically expressed CLDN1 appeared to reside predominantly in intracellular vesicles. The CLDN1-CD81 complex formed in HeLa cells was also exclusively distributed intracellularly, co-localizing with EEA1, an early endosomal marker. Correspondingly, transepithelial electric resistance, obtained from the naturally susceptible human liver cell line, Huh7, was much higher than that of the HeLa-CLDN1 cell line, suggesting that Huh7 is likely to form functional tight junctions. Finally, the disruption of TJ-enriched CLDN1 by tumor necrosis factor-␣ treatment markedly reduced the susceptibility of Huh7.5.1 cells to HCV infection. Our results suggest that the specific localization pattern of CLDN1 may be crucial in the regulation of HCV cellular tropism.Hepatitis C virus (HCV), 3 a major human pathogen, specifically infects hepatocytes. In nature, the virus may exist in several forms: enveloped lipoprotein-free virus, enveloped lipoprotein-associated virus, non-enveloped lipoprotein-free virus, and non-enveloped lipoprotein-associated virus (1). While the nature of these different forms remains elusive, they may infect cells via different means (1). Importantly, pseudoviral particles that consist of an HIV core and HCV E1 and E2 (i.e. HCVpp) have been found to strictly infect human hepatocytes and a few hepatoma-derived cell lines (2-4). HCVpp most likely resembles enveloped lipoprotein-free viruses whose entry is dependent upon two of the HCV envelope proteins, E1 and E2 (5-9). A plethora of evidence that has been accumulated from studies employing HCVpp has allowed for a proposed model of HCV entry: HCV attaches to hepatocytes via specific receptor(s) followed by clathrin-dependent internalization (endocytosis) (10, 11). Internalized virions then traffic to early endosomes where the virion envelope proteins undergo conformational changes and then fuse with the cellular membrane for viral entry (4, 12). In numerous attempts to identify an HCV entry receptor(s), both human tetraspanin CD81 and the human scavenger receptor, SR-BI, were isolated in screens based on...

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Distinct Intracellular Trafficking of Equine Infectious Anemia Virus and Human Immunodeficiency Virus Type 1 Gag during Viral Assembly and Budding Revealed by Bimolecular Fluorescence Complementation Assays

Cited by 41 publications

References 56 publications

How does the journey affect the message(RNA)?

How does the journey affect the message(RNA)?

Feline Immunodeficiency Virus Gag Is a Nuclear Shuttling Protein

Correlation of the Tight Junction-like Distribution of Claudin-1 to the Cellular Tropism of Hepatitis C Virus

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