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Delaying Photobleaching of a Light-Switch Complex for Real-Time Imaging of Single Viral Particle Uncoating Yingxin Ma, Zongqiang Cui, Zhike He, Wei Li, Zhi-Ping Zhang, Xiaowei Zhang, Xian-En Zhang, and Tianwei Tan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03127 • Publication Date (Web): 04 Oct 2016 Downloaded from http://pubs.acs.org on October 9, 2016

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Analytical Chemistry

Delaying Photobleaching of a Light-Switch Complex for Real-Time Imaging of Single Viral Particle Uncoating Yingxin Ma1, 2†, Zongqiang Cui2†, Zhike He4, Wei Li2, Zhiping Zhang2, Xiaowei Zhang2, Xian-En Zhang3,*, Tianwei Tan1,* 1

Beijing Key Lab of Bioprocess, College of Life Science and Technology, Beijing University of

Chemical Technology, Beijing, P.R.China; 2

State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences,

Wuhan, P.R.China; 3

National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules,

Institute of Biophysics, Chinese Academy of Sciences, Beijing, P.R.China; 4

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College

of Chemistry and Sciences, Wuhan University, Wuhan, Hubei, P.R.China

†These authors contributed equally to this work Email: [email protected], [email protected]

ABSTRACT: Photobleaching is a major obstacle in the real-time imaging of biological events, particularly at the single-molecule/particle level. Here, we report a strategy to delay photobleaching of a light-switch complex, [Ru(phen)2dppx]2+, by insertion of a six-cysteine peptide into virus particles. The six-cysteine peptide was inserted into viral protein R of HIV-1, and assembled into infectious HIV-1 viral particles, where it effectively delayed the photobleaching of the [Ru(phen)2dppx]2+ complex used to label viral genomic RNAs. This delay in photobleaching allowed for a mono-fluorescent assay to be constructed for the real-time monitoring of viral uncoating, a poorly understood process. This novel strategy to delay photobleaching in infectious viral particles provides a powerful method to analyze viral uncoating at the single-particle level in real time.

INTRODUCTION Photobleaching is a major obstacle in the real-time imaging of biological events, particularly at the

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single-molecule/particle level. Strategies to delay the photobleaching of fluorescent dyes have been investigated but little progress has been made. Some reagents, such as β-mercaptoethanol and 11-mercaptoundecanoic acids, have been reported to delay luminescence photobleaching.1-3 However, these reagents are not appropriate for real-time imaging of living cells. Recently, molecular light-switch complexes, such as [Ru(phen)2dppx]2+, have been employed for labeling DNA or RNA for fluorescence microscopy imaging owing to their unique photo-physical properties.4-9 As a kind of light-switch, these metal complexes display no luminescence in aqueous solution, but show remarkable enhancement of fluorescence intensity upon association with DNA or RNA.10,11 These light-switch complexes have been shown to be effective for DNA and RNA labeling and their application has been extended to live virus genome labeling and virus tracking.12 In our recent report, the Ru(II) complex was used to label HIV-1 genomic RNAs to allow for imaging of the viral uncoating process.13 Compared with the quantum dot or other fluorescent dyes, the Ru(II) complex is more suitable for labeling of viral genomic contents, since it can be incorporated in the viral natural DNAs or RNAs without genetic or chemical modification, present specific fluorescent signals, and do not obviously affect viral infectivity. However, luminescent photobleaching of Ru(II) complexes, owing to the oxidation of [Ru(phen)2dppx]2+ to non-luminescent [Ru(phen)2dppx]3+,3 remains an obstacle in the real-time imaging of single virus particles. Here, we report a strategy to delay the photobleaching of Ru(II) complexes by the introduction of a six-cysteine peptide into virus particles. The six-cysteine peptide with reducing ability was inserted into viral protein R (Vpr) of HIV-1, and bound to the HIV genomic RNAs to delay the photobleaching of the Ru(II) complexes labeled on the RNAs. Based on this delay in photobleaching during virus infection, we developed a novel uncoating assay for HIV virus using Ru(II) complex-labeled mono-fluorescent viral particles without compromising virus infectivity. EXPERIMENTAL SECTION Plasmids. The plasmid pAD8WT was used to produce reporter viruses. To construct pAD8TC, the tetracysteine

motif

was

inserted

into

the

cyclophilin-binding

loop

of

the

capsid

(H87C/A88C/I91C/A92C) by segment overlap PCR using the BssHII-SpeI restriction enzyme sites. To construct pcDNA3.1-Vpr-nC, the cysteine motif was introduced into the C-terminal domain of Vpr by


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PCR, and the complete coding sequence was inserted into the plasmid pcDNA3.1 using the HindIII-BamHI restriction enzyme sites. Virus preparation. HIV-1 viral particles were produced in 293T cells by transfection with the plasmid pAD8WT using lipofectamine 2000 (Invitrogen). The plasmid transfections and virus experiments were performed in BSL-2 or BSL-3 labs. Briefly, 30 µg of pAD8WT was transfected into 5 × 106 293T cells cultured in 10-cm plates to generate HIVWT or HIVRu virus. Then, 27 µg of pAD8WT and 3 µg of pcDNA3.1-Vpr-nC were transfected to generate HIVnC-Ru; 20 µg of pAD8WT and 10 µg of pAD8TC were transfected to generate HIVTC virus; and 18 µg of pAD8WT, 9 µg of pAD8TC, and 3 µg of pcDNA3.1-Vpr-6C were transfected to generate HIV6C-Ru-TC virus. The viruses were harvested 16–18 h after transfection, and then passed through a 0.45-µm-pore-size filter (Millipore). The virus concentration was determined by p24CA ELISA (PerkinElmer). Fluorescence labeling and purification of viruses. For labeling of viral genomic RNAs, 1 mL of 10-4 M [Ru(phen)2(dppx)]2+ labeling solution was added into the culture medium of 293T cells 6 h after transfection. Then, 1 µL of FlAsH (Invitrogen) labeling solution (2 mM) was added to 2 mL of virus and incubated for 2 h at 37°C for TC-tag labeling. Unbound fluorescent dyes were removed by centrifugation of the virus in PEG 20000 solution, and the virions were resuspended in 1 mL of opti-MEM. Virus infection and fluorescence imaging. TZM-bl cells were incubated with the fluorescent viral particles at 4°C for 30 min, and unbound particles were removed and replaced with fresh media prior to incubation in a heated chamber at 37°C with 5% CO2. Cells and viruses incubated in a confocal dish sealed by parafilm were visualized under a spinning disk confocal microscope. RESULTS AND DISCUSSION To delay the photobleaching of Ru(II) complexes labeled on the viral genomic RNA of HIV particles, we inserted different numbers of cysteine residues (n=0, 3, 6, 12) into HIV-1 Vpr, which binds to p6 in the Gag protein and is incorporated into the viral core (Scheme 1).14,15 Different types of HIV-1 particles containing various Vpr-cysteine peptide fusion proteins (HIVRu, HIV3C-Ru, HIV6C-Ru, and HIV12C-Ru) were acquired, and their genomic RNAs were labeled with [Ru(phen)2dppx]2+ according to our previously established method (Fig. S-1).13 We also tested the infectivity of these HIV particles and



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our results showed that these particles possessed similar infectivity to wild-type HIV particles (HIVWT) (Fig. S-2).16 Then, these viral particles were analyzed for photobleaching under a microscope. As shown in Figure 1, the luminescent intensity of the Ru(II) complex decreased by 40% within 240 s for virions with no cysteines inserted into Vpr (HIVRu). However, when different numbers of cysteine residues were inserted into Vpr, the luminescent intensity of the Ru(II) complex decreased by 30%, 10%, and 20% for HIV3C-Ru, HIV6C-Ru, and HIV12C-Ru, respectively. These results showed that the insertion of cysteines into Vpr delayed photobleaching of the Ru(II) complex labeled on viral genomic RNAs. This effect was most significant when the six-cysteine peptide was inserted into Vpr. It has been suggested that photobleaching of the Ru(II) complex is mainly owing to the oxidation of [Ru(phen)2dppx]2+ to [Ru(phen)2dppx]3+. Here, we tested the oxidation of the [Ru(phen)2dppx]2+ during illumination according to the method built by Jacqueline et al.17 As shown in Fig. S-3A, the molar extinction coefficient (ε) of the complex solution decreased obviously upon the illumination, suggesting that a decrease of the [Ru(phen)2dppx]2+ concentration and an increase of the [Ru(phen)2dppx]3+

concentration,

due

to

[Ru(phen)2dppx]2+

has

a

higher

ε

value

than

[Ru(phen)2dppx]3+. When there was cysteine, the ε value had a slower and less decrease upon the illumination. Further, the molar extinction coefficient (ε) could be recovered by addition of cysteine after removing the illumination (Fig. S-3B). These results showed that cysteine could cause the reduction of [Ru(phen)2dppx]3+ to [Ru(phen)2dppx]2+, and this reduction effect should contribute to the photobleaching delay of the Ru(II) complex. Next, we applied this photobleaching-delay method to monitor HIV-1 uncoating. HIV-1 uncoating, an obligatory step of viral early infection, is defined as the loss of viral capsid that occurs within the cytoplasm of infected cells before the viral genome enters into cell nucleus. First, we constructed a dual-fluorescent viral particle, HIV6C-Ru-TC, resulting in labeling of both genomic RNAs and capsid. A tetracysteine (TC) motif was inserted into the capsid, and the FlAsH fluorescent dye was used to label the capsid through its interaction with the TC-tag.18,19 A dual-fluorescent viral particle, HIVRu-TC, without insertion of cysteines into Vpr, was produced as a control. Co-localization of the Ru(II) complex and FlAsH in HIVRu-TC and HIV6C-Ru-TC virions confirmed that the genomic RNAs and the capsid were successfully labeled (Figure 2A). The infectivity assays were also carried out for these


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viral particles, and the results showed that the labeling of [Ru(phen)2dppx]2+ did not affect viral infectivity; the insertion of cysteine had a minor effect on viral infectivity; whereas the introduction of a TC tag in the capsid significantly impaired viral infectivity (Figure 2B and Fig. S-2). However, HIVRu-TC and HIV6C-Ru-TC virions retained approximately 50% of the infectivity of HIVWT, which could be used to monitor further uncoating. Then, we infected TZM-bl cells with HIV6C-Ru-TC and analyzed viral uncoating in real time. Particles displaying fluorescent signals from both the Ru(II) complex and FlAsH were counted as HIV6C-Ru-TC virions and tracked as merged yellow fluorescent dots. As shown in Figure 3A and 3B and movie S-1, during viral uncoating, the red and green fluorescent signals were separate. The luminescent intensity of the red Ru(II) signal remained unchanged before separation, but dramatically reduced during the viral uncoating process (Figure 3C). The change in luminescence intensity coincided with the initiation of viral uncoating. We also used HIVRu-TC particles to infect TZM-bl cells, and the red Ru(II) signal was also separated from the green FlAsH signal in the cytoplasm (Figure 3D and 3E and movie S-2). The luminescent intensity of the Ru(II) complex was significantly reduced both before and after the separation (Figure 3F). These results showed that, during real-time imaging of the uncoating process, the six-cysteine motif fused to Vpr could delay photobleaching of the Ru(II) complex in HIV6C-Ru-TC before RNA and capsid separation. In this study, 30 separations from 27,000 individual HIV6C-Ru-TC virions, and 30 separations from 29,000 individual HIVRu-TC virions, were observed at 60–120 min post-infection in the cytoplasm. These two types of dual-fluorescent viral particles exhibited similar time and place of uncoating process, while the luminescent intensity of [Ru(phen)2dppx]2+ in HIV6C-Ru-TC and HIVRu-TC virions differed during viral uncoating. These results suggested that the insertion of cysteine into Vpr can delay photobleaching of the Ru(II) complex before viral uncoating, and does not affect the dynamics of the uncoating process. Based on the photobleaching-delay effect, the change in luminescence intensity is consistent with initiation of the uncoating process and can therefore serve as an indicator of viral uncoating. Then, we used the change in luminescent intensity to study HIV-1 uncoating with single-color fluorescently labeled HIV6C-Ru particles. This method did not require the capsid to be labeled to impair



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viral infectivity, which meant that the monitoring assay could be performed under more natural conditions. The single-color HIV6C-Ru particles were used to infect TZM-bl cells and the fluorescence intensity of [Ru(phen)2dppx]2+ was tracked in real time. As shown in Figure 4A–4C and movie S-3, the red fluorescence signal of [Ru(phen)2dppx]2+ translocated to the cytoplasm, and its luminescence intensity was analyzed accordingly. The change in luminescent intensity was recorded to be at 60–120 min post-infection, indicating that HIV-1 uncoating occurred during this time period. In this experiment, 30 uncoating events among 10,000 HIV6C-Ru virions were observed at 60–120 min post-infection. These results demonstrated that the uncoating process has been successfully revealed by tracking mono-fluorescent virions. This method provided more natural uncoating information than that obtained using dual-color-labeled virus particles that exhibit capsid modification and diminished infectivity. CONCLUSION Photobleaching of luminescent dyes is the most commonly encountered difficulty during luminescence analysis. Although some reagents have been tested for their ability to delay luminescence photobleaching, to date no method has been reported that can be applied to the real-time imaging in living cells. Here, we have developed a strategy to delay the photobleaching of [Ru(phen)2dppx]2+ in infectious HIV-1 virus particles. The six cysteine peptide, which was inserted into Vpr and encapsulated in viral particles, can effectively delay the photobleaching of [Ru(phen)2dppx]2+ labeled on the HIV-1 genomic RNA. Vpr with the cysteine residues inserted may aid the reduction of [Ru(phen)2dppx]3+ to [Ru(phen)2dppx]2+ and the recovery of fluorescence. Based on the delaying photobleaching and change in fluorescence intensity associated with [Ru(phen)2dppx]2+ during viral uncoating, a powerful mono-fluorescent assay was built for the real-time analysis of viral uncoating at the single-particle level.


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FIGURES

Vpr cysteine Ru

HIV nC-Ru Assembly

Nucleus

Scheme 1. Construction of mono-fluorescent HIVnC-Ru viral particles. Ru = [Ru(phen)2(dppx)]2+



B 120

3C

110 FL Intensity (%)

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6C

12C

WT

100 90 80 70 60 50 40

0

50

100

150

200 250 Time (S)

300

350

400

Figure 1. Photobleaching of [Ru(phen)2(dppx)]2+ in the HIVRu, HIV3C-Ru, HIV6C-Ru, and HIV12C-Ru viral particles.


FlAsH

Merge

B 120 100

FlAsH

Merge

80 56.23

60

50.12

40 20

TC

TC u-

6C

R

W

T

0 u-

Ru

100

-R

Ru

HIV 6C-Ru-TC

A

HIV Ru-TC

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Luciferase activity (% of HIVWT)

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Figure 2. (A) Colocalization of Ru and FlAsH signals in dual-labeled HIV6C-Ru-TC and HIVRu-TC particles (scale bar: 2 µm). (B) The capacity of HIVWT, HIVRu-TC, and HIV6C-Ru-TC to infect target cells was assessed by measuring luciferase activity in the indicator TZM-bl cells.



A

0s

10 s

60 s

70 s

80 s

100 s

160 s

170 s

B

C 130 FL Intensity (%)

120

6C-Ru-TC

110 100 90 80 70 60

D

E

0

20

40

60

80 100 Time (S)

120

140

160

0s

20 s

40 s

70 s

100 s

120 s

140 s

170 s

F

110 100

FL Intensity (%)

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Ru-TC

90 80 70 60 50 40 30

0

20

40

60

80 100 Time (S)

120

140

160

Figure 3. (A) Snapshots of the real-time separation of Ru and FlAsH signals in HIV6C-Ru-TC particles. Scale bar: 1 µm. (B) DIC image of the infected cell. The separation site is marked, and the boundary of the cell is highlighted by a white line. (C) Luminescence intensity of the Ru(II) complex, detected in (A). The moment of viral uncoating is marked by the dashed line. (D) Snapshots of the real-time separation of Ru and FlAsH signals in HIVRu-TC particles. Scale bar: 1 µm. (E) DIC image of the infected cell. The separation site is marked, and the boundary of the cell is highlighted by a white line. (F) Luminescence intensity of the Ru(II) complex, detected in (D). The moment of viral uncoating is marked by the dashed line.


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A

B

0s

54 s

114 s

156 s

198 s

258 s

324 s

396 s

C

130 6C-Ru

120

FL Intensity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


110 100 90 80 70 60

0

50

100

150

200 250 Time (S)

300

350

400

Figure 4. (A) Snapshots of the real-time imaging of Ru signals in HIV6C-Ru particles. Scale bar: 1 µm. (B) DIC image of the infected cell. The virus location is marked, and the boundary of the cell is highlighted by a white line. (C) Luminescence intensity of the Ru(II) complex, detected in (A). The moment of viral uncoating is marked by the dashed line.



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AUTHOR INFORMATION Corresponding Author *Correspondence: [email protected] (Xian-En Zhang), [email protected] (Tian-Wei Tan). Notes The authors declare no competing financial interest ACKNOWLEDGMENT ZQ Cui is supported by the National Natural Science Foundation of China (NSFC) (no. 31470269) and the Youth Innovation Promotion Association CAS. XE Zhang is grateful for support from the Chinese Academy of Sciences (KJZD-EW-TZ-L04). We thank the Core Facility and Technical Support, Wuhan Institute of Virology for excellent technical support. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge via the Internet at http://pubs.acs.org. Figures S-1 to S-3 and Movies S-1 to S-3. REFERENCES 1

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Table of Contents

Vpr cysteine Ru

HIV nC-Ru Assembly

Nucleus


Delaying Photobleaching of a Light-Switch ... - ACS Publications

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