A quantitative method for normalization of transfection efficiency using enhanced green fluorescent protein

Dandekar, Dineshkumar; Kumar, Manish; Ladha, Jayashree; Ganesh, Krishna N.; Mitra, Debashis

doi:10.1016/j.ab.2005.02.006

Cited by 25 publications

(26 citation statements)

References 12 publications

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“…Normalization of transfection efficiency was performed using EGFP reporter (pEGFP-N1) co-transfection and quantitation as described earlier. 44 The culture supernatants were also collected at the time of luciferase assay from HIV-1 NL4-3 and Nef-deleted NL4-3 molecular-clonetransfected cells to determine virus production. 45 …”

Section: Transient Transfection and Luciferase Assaymentioning

confidence: 99%

Reciprocal Regulation of Human Immunodeficiency Virus-1 Gene Expression and Replication by Heat Shock Proteins 40 and 70

Kumar

Rawat

Khan

et al. 2011

Journal of Molecular Biology

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Section: Transient Transfection and Luciferase Assaymentioning

confidence: 99%

Reciprocal Regulation of Human Immunodeficiency Virus-1 Gene Expression and Replication by Heat Shock Proteins 40 and 70

Kumar

Rawat

Khan

et al. 2011

Journal of Molecular Biology

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“…The cells were then lysed in cell lysis reagent (Promega), and luciferase assays were performed using Luclite substrate (PerkinElmer Life Sciences). Normalization of transfection efficiency was done using enhanced green fluorescent protein reporter (pEGFP-N1) co-transfection and quantitation as described earlier (42). For RNAi experiments, Jurkat-1G5 cells were transfected with 100 nM siRNAs along with expression vectors using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.…”

Section: Methodsmentioning

confidence: 99%

Cyclin K Inhibits HIV-1 Gene Expression and Replication by Interfering with Cyclin-dependent Kinase 9 (CDK9)-Cyclin T1 Interaction in Nef-dependent Manner

Khan

Mitra

2011

Journal of Biological Chemistry

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Human immunodeficiency virus type-1 (HIV-1) perpetuates its survival in the host by utilizing multiple strategies such as irreversible integration of its DNA into the host cell genome to establish the provirus (1), diversification of its genome to escape or tolerate adaptive immune responses (2), and use of the accessory genes (nef, vif, vpu, and vpr) to modulate host cell immune responses further (3-5). These viral accessory genes are frequently seen as dispensable in many in vitro cell culture systems but seem to be indispensable, in the context of natural infections in vivo. Among these accessory genes, pleiotropic functions of Nef have been studied extensively. It is a 27-30-kDa, N-terminally myristoylated protein having a well characterized CD4 down-regulatory activity (6). Nef does not possess any enzymatic activity, but it modulates diverse signaling pathways and the gene expression of host cell proteins (7,8). Furthermore, Nef influences the activation state of the host cell by bringing together different host cell proteins, protein kinases, and components of the endocytic machinery and making the cells permissible to the virus (9, 10). All these functions of Nef are manifested by a number of events such as activation of signaling molecules, modulation of apoptosis, activation of transcription factors, mitigation of transcriptional repressors, and an increase in the infectivity of virions (11). Although these functions of Nef have been well studied, controversy exists on its role in viral infectivity and replication as both negative (12, 13) and positive (14, 15) roles have been attributed in the literature. Although a number of reports show that Nef increases viral replication by activating T-cells, the molecular basis of Nef-induced viral gene expression and replication remains to be clearly elucidated. Nef was also shown to physically interact with Tat and augment viral gene expression (16), although availability of Tat is critical for the elongation step of long terminal repeat (LTR) 2 -driven transcription by RNA polymerase II. Several cellular factors have been identified for their roles in this process, including positive transcription elongation factor b (P-TEFb) complex. Different P-TEFb complexes isolated from mammalian cells contain a common catalytic subunit, cyclin-dependent kinase 9 (CDK9), and one of the regulatory cyclins, CycT1, CycT2a, CycT2b,. However, CycT1-containing P-TEFb complex is the key complex responsible for HIV-1 transcription elongation (19). Tat is expressed early in the replicative cycle of HIV and is essential for viral gene expression from the LTR promoter. It recognizes the 5Ј-bulge in the transactivation response stem loop RNA, which is located at the 5Ј-end of all viral transcripts. Tat binds to CycT1, and together they form the combinatorial surface that interacts with the transactivation response RNA with high affinity and specificity. The obligate partner of CycT1, CDK9, then hyperphosphorylates the C-terminal domain (CTD) of RNA polymerase II, resulting in incre...

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“…After 4 h of transfection, the transfected cells were either treated or mock treated with HSP inhibitor KNK437 (Calbiochem) at 100 M final concentration for 32 h. The cells were then lysed in cell lysis reagent (Promega), and luciferase assays were performed using Luclite substrate (PerkinElmer Life Sciences). Normalization of transfection efficiency was done using the enhanced green fluorescent protein reporter (pEGFP-N1) co-transfection and quantitation as described earlier (38). The culture supernatants were also collected at the time of luciferase assay from HIV-1 NL4-3 and the nef-deleted NL4-3 molecular clone-transfected cells to determine virus production.…”

Section: Methodsmentioning

confidence: 99%

Heat Shock Protein 40 Is Necessary for Human Immunodeficiency Virus-1 Nef-mediated Enhancement of Viral Gene Expression and Replication

Kumar¹,

Mitra

2005

Journal of Biological Chemistry

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The human immunodeficiency virus-1 (HIV-1) Nef protein, originally identified as a negative factor, has now emerged as one of the most important viral proteins necessary for viral pathogenesis and disease progression. Nef has been also implicated in viral infectivity and replication, however, the molecular mechanism of Nef-induced viral gene expression and replication is not clearly understood. Although involvement of heat shock proteins in viral pathogenesis has been reported earlier, a clear understanding of their role remains to be elucidated. Here we report for the first time that Nef not only interacts with heat shock protein 40 (Hsp40) but it also induces the expression of Hsp40 in HIV-1-infected cells. The interaction between Nef and Hsp40 is important for increased Hsp40 translocation into the nucleus of infected cells, which seems to facilitate viral gene expression by becoming part of the cyclin-dependent kinase 9-associated transcription complex regulating long terminal repeat-mediated gene expression. The finding is consistent with the failure of the nef-deleted virus to induce Hsp40, resulting in reduced virus production. Our data further shows that, whereas, Hsp40 overexpression induces viral gene expression, silencing of Hsp40 reduces the gene expression in a Nef-dependent manner. Thus our results clearly indicate that Hsp40 is crucial for Nef-mediated enhancement of viral gene expression and replication.Viruses are known to modulate cellular proteins for successful replication within the host cells. The sequence of events in the establishment of a productive infection by human immunodeficiency virus type 1 (HIV-1) 3 not only involves interaction between a number of viral and cellular factors but is also accompanied by complex and dynamic changes in the patterns of cellular gene expression. Nef, a 27-30-kDa myristoylated phosphoprotein, encoded by HIV-1 has been shown to play a crucial role in viral pathogenesis by modulating cellular gene expression and signaling pathways (1, 2). nef-deleted viruses fail to replicate efficiently in vivo, and do not develop symptoms of acquired immunodeficiency syndrome (AIDS) (3, 4). Nef is also thought to contribute to viral pathogenesis by down-regulation of CD4 and major histocompatibility complex class I surface molecules preventing viral superinfection and by helping the virus to evade host immune system. Nef has also been implicated in the activation of T cells, making the cells permissible to the virus (5, 6). All these functions of Nef are manifested by a number of important events such as activation of upstream signaling molecules, inhibition of apoptosis in the infected cell, activation and up-regulation of transcription factors, alleviation of repressors of transcription, as well as increase of the infectivity of newly produced virions (1, 7). Even though these functions of Nef have been well studied, controversy exists on its role in viral infectivity and replication as both negative (8, 9) and positive (10, 11) effects are available in the literature...

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A quantitative method for normalization of transfection efficiency using enhanced green fluorescent protein

Cited by 25 publications

References 12 publications

Reciprocal Regulation of Human Immunodeficiency Virus-1 Gene Expression and Replication by Heat Shock Proteins 40 and 70

Reciprocal Regulation of Human Immunodeficiency Virus-1 Gene Expression and Replication by Heat Shock Proteins 40 and 70

Cyclin K Inhibits HIV-1 Gene Expression and Replication by Interfering with Cyclin-dependent Kinase 9 (CDK9)-Cyclin T1 Interaction in Nef-dependent Manner

Heat Shock Protein 40 Is Necessary for Human Immunodeficiency Virus-1 Nef-mediated Enhancement of Viral Gene Expression and Replication

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