Recombinant HIV-1 nucleocapsid protein p15 produced as a fusion protein with glutathione S-transferase in Escherichia coli mediates dimerization and enhances reverse transcription of retroviral RNA

Weiß, Sabine; König, Bernhard; Morikawa, Yuko; Jones, Ian M.

doi:10.1016/0378-1119(92)90123-7

Cited by 46 publications

(37 citation statements)

References 32 publications

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“…It is uncertain whether MA is a component of the RSV PIC, although genetic evidence supports this idea (12). Most certainly, NC is present within the PIC because it is bound to the RNA genome in the virion and is involved in reverse transcription and integration (2,6,16,34,48). An NLS in RSV integrase was previously identified (20), and thus there are three candidate karyophilic proteins that might promote nuclear transport of the viral genome early in infection.…”

Section: Discussionmentioning

confidence: 99%

Importin-β Family Members Mediate Alpharetrovirus Gag Nuclear Entry via Interactions with Matrix and Nucleocapsid

Butterfield-Gerson¹,

Scheifele²,

Ryan³

et al. 2006

J Virol

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The retroviral Gag polyprotein orchestrates the assembly and release of virus particles from infected cells. We previously reported that nuclear transport of the Rous sarcoma virus (RSV) Gag protein is intrinsic to the virus assembly pathway. To identify cis-and trans-acting factors governing nucleocytoplasmic trafficking, we developed novel vectors to express regions of Gag in Saccharomyces cerevisiae. The localization of Gag proteins was examined in the wild type and in mutant strains deficient in members of the importin-␤ family. We confirmed the Crm1p dependence of the previously identified Gag p10 nuclear export signal. The known nuclear localization signal (NLS) in MA (matrix) was also functional in S. cerevisiae, and additionally we discovered a novel NLS within the NC (nucleocapsid) domain of Gag. MA utilizes Kap120p and Mtr10p import receptors while nuclear entry of NC involves the classical importin-␣/␤ (Kap60p/95p) pathway. NC also possesses nuclear targeting activity in avian cells and contains the primary signal for the import of the Gag polyprotein. Thus, the nucleocytoplasmic dynamics of RSV Gag depend upon the counterbalance of Crm1p-mediated export with two independent NLSs, each interacting with distinct nuclear import factors.

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Section: Discussionmentioning

confidence: 99%

Importin-β Family Members Mediate Alpharetrovirus Gag Nuclear Entry via Interactions with Matrix and Nucleocapsid

Butterfield-Gerson¹,

Scheifele²,

Ryan³

et al. 2006

J Virol

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“…However, a GST-Fdg1-PRD fusion protein shows a potential to activate cortactin in an efficiency similar to dynamin 2 (Kim et al, 2004). As GST is known to form a stable dimer (Weiss et al, 1992), the apparent effect of GST-Fdg1-PRD was thought to be the result of a possible dimerization of cortactin induced by the GST fusion. We have frequently observed that cortactin mutant with deletion of the SH3 domain showed a slight increase (B30%) as compared to wild type cortactin (Figure 4a, data not shown), suggesting that the SH3 domain may impose a weak self-inhibition to the function of cortactin.…”

Section: Discussionmentioning

confidence: 99%

Differential regulation of cortactin and N-WASP-mediated actin polymerization by missing in metastasis (MIM) protein

et al. 2005

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“…The RNase H activity of RT is essential for minus-strand DNA transfer (5, 29, 34). Through various in vitro assays, NC was also clearly demonstrated to facilitate the efficiency of minus-strand DNA transfer (2,8,12,16,20,25,33,35,38,39,43,46).…”

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confidence: 99%

Utilization of Nonviral Sequences for Minus-Strand DNA Transfer and Gene Reconstitution during Retroviral Replication

Cheslock

Anderson

Hwang

et al. 2000

J Virol

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Minus-strand DNA transfer, an essential step in retroviral reverse transcription, is mediated by the two repeat (R) regions in the viral genome. It is unclear whether R simply serves as a homologous sequence to mediate the strand transfer or contains specific sequences to promote strand transfer. To test the hypothesis that the molecular mechanism by which R mediates strand transfer is based on homology rather than specific sequences, we examined whether nonviral sequences can be used to facilitate minus-strand DNA transfer. The green fluorescent protein (GFP) gene was divided into GF and FP fragments, containing the 5 and 3 portions of GFP, respectively, with an overlapping F fragment (85 bp). FP and GF were inserted into the 5 and 3 long terminal repeats, respectively, of a murine leukemia virus-based vector. Utilization of the F fragment to mediate minus-strand DNA transfer should reconstitute GFP during reverse transcription. Flow cytometry analyses demonstrated that GFP was expressed in 73 to 92% of the infected cells, depending on the structure of the viral construct. This indicated that GFP was reconstituted at a high frequency; molecular characterization further confirmed the accurate reconstitution of GFP. These data indicated that nonviral sequences could be used to efficiently mediate minus-strand DNA transfer. Therefore, placement and homology, not specific sequence context, are the important elements in R for minus-strand DNA transfer. In addition, these experiments demonstrate that minus-strand DNA transfer can be used to efficiently reconstitute genes for gene therapy applications.All retroviruses replicate their genome using an RNA form to generate a DNA form in a process called reverse transcription (42). The viral RNA is characterized by short repeat (R) regions at the 5Ј and 3Ј ends (7,14). The R region at the 5Ј end is immediately followed by a unique 5Ј sequence named U5. Because the viral RNA is the mRNA or the plus strand, the first strand of DNA synthesized is complementary to the viral RNA and is referred to as the minus-strand DNA. Viral DNA synthesis initiates near the 5Ј end of the viral RNA, using a tRNA primer that binds to the primer-binding site (PBS) in the viral RNA (7). Reverse transcriptase (RT) copies R and U5 and quickly reaches the 5Ј end of the RNA template. This short stretch of DNA that contains R and U5 is referred to as minus-strand strong-stop DNA. It is thought that the RNase H activity of RT degrades the RNA template in the RNA-DNA hybrid and exposes the strong-stop DNA. The newly synthesized R in the viral DNA is complementary to the R near the 3Ј end of the viral RNA. Presumably, the complementarity facilitates alignment and hybridization of the two nucleic acids and allows RT to continue DNA synthesis, using sequences near the 3Ј end of the viral RNA as a template. This switching of the RT complex from the 5Ј end to near the 3Ј end of the viral RNA, known as minus-strand DNA transfer, is an essential step in reverse transcription (7,14). Minus-strand DNA transfer...

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Recombinant HIV-1 nucleocapsid protein p15 produced as a fusion protein with glutathione S-transferase in Escherichia coli mediates dimerization and enhances reverse transcription of retroviral RNA

Cited by 46 publications

References 32 publications

Importin-β Family Members Mediate Alpharetrovirus Gag Nuclear Entry via Interactions with Matrix and Nucleocapsid

Importin-β Family Members Mediate Alpharetrovirus Gag Nuclear Entry via Interactions with Matrix and Nucleocapsid

Differential regulation of cortactin and N-WASP-mediated actin polymerization by missing in metastasis (MIM) protein

Utilization of Nonviral Sequences for Minus-Strand DNA Transfer and Gene Reconstitution during Retroviral Replication

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