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Method Article

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

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DOI:

10.3791/53682

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March 2nd, 2016

 , 
1Department of Medicine, Division of Immunobiology, University of Vermont, 2Department of Microbiology & Molecular Genetics, University of Vermont

In This Article

Summary

The first step in the arenavirus life cycle is attachment of viral particles to host cells. We report a quantitative (q)RT-PCR-based assay for ultrasensitive detection and quantitation of arenavirus attachment events.

Abstract

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.

Introduction

Arenaviruses are a family of enveloped, single-stranded RNA viruses that are maintained primarily in rodents in nature1-3. While these viruses typically establish an asymptomatic, persistent infection in rodent reservoirs1,2, they can cause severe disease in humans3. Important pathogenic species include the New World Junin virus (JUNV)4 and the Old World Lassa virus5, which are the etiologic agents of Argentine hemorrhagic fever in South America and Lassa fever in Africa, respectively. Lymphocytic choriomeningitis virus (LCMV), the prototypic virus of the family6, has a worldwide distribution and is responsible for a variety of disease states including aseptic meningitis in immunocompetent individuals6, severe birth defects in the developing fetus7, or high lethality in immunosuppressed individuals following solid-organ transplantation8,9. The lack of United States Food and Drug Administration (FDA)-approved vaccines or effective antivirals to combat these viruses highlights the critical need to develop novel strategies to therapeutically target these emerging/re-emerging pathogens.

The arenavirus genome consists of two single-stranded RNA segments, the large (L) (~7.2 kb) and small (S) (~3.6 kb) segments10. The S segment encodes the viral nucleoprotein (NP) and envelope glycoprotein (GP) while the L segment encodes the viral polymerase (L) and matrix protein (Z). The L and S genomic RNA segments (vRNAs) are packaged into virions10-12. The initial step of arenavirus infection is attachment of viral particles to host cells through an interaction between the viral GP and its corresponding host cell receptors which, for JUNV, includes the human transferrin receptor 1 (hTfR1)13,14. Following attachment, JUNV particles are taken into the cell via clathrin-mediated endocytosis15. Particles then traffic to the late endosome where the low pH of this compartment triggers the activation of GP16,17. Once activated, the GP-encoded fusion peptide inserts into the endosomal membrane, initiating fusion between the viral and endosomal membranes. Successful fusion results in the release of the virion-packaged vRNAs into the cytoplasm, where they serve as templates for genomic replication and transcription. When sufficient quantities of viral structural proteins and vRNAs are generated, new particles assemble and bud from the infected cell to complete the life cycle18.

To facilitate the discovery of new strategies to therapeutically target the pathogenic arenaviruses, our group has focused on the identification of host factors that are critical for virus propagation, but dispensable for the host. In this context, defining the precise stage(s) of the viral life cycle controlled by such host factors is necessary to guide the development of antiviral strategies. Herein, we describe a quantitative (q)RT-PCR-based assay that can be used to measure arenavirus-cell attachment for the purpose of identifying whether selected host proteins and/or antiviral molecules influence this initial stage of viral entry19. Alternative approaches to measure arenavirus-cell attachment have featured virions labeled with biotin20, fluorescent dyes21, or radioactive isotopes22. Drawbacks to these methods can include the requirement for large quantities of input virus (e.g. high multiplicities of infection (MOI)), the use of radioactivity, and/or additional handling steps to prepare input virus (e.g. concentration and/or labeling of virus). In particular, the use of high MOI makes it difficult to distinguish productive versus nonproductive attachment events15,23. The qRT-PCR-based assay described here can detect attachment events using as few as 20 plaque forming units (PFUs) of virus, which translates into an MOI of 0.0004 for a 48-well plate. Therefore, the strong sensitivity of the assay overcomes the requirement for high MOI as well as any virus labeling or concentration steps. Further, the assay can be i) adapted to measure virion endocytosis as well as release of virus genome into the cytoplasm following fusion and ii) tailored for use with other viruses through the development of virus-specific qRT-PCR reagents (for examples, see 24-27).

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Protocol

Note: It is critical that the described protocol be carried out using strict pre-PCR technique (see 28 for an excellent overview on how to set up a pre-PCR laboratory and how to conduct experiments using good pre-PCR technique). It is imperative that all reagents and equipment are free of amplified DNA containing the qPCR target sequence and that appropriate controls are in place to detect such contamination. An overview of the protocol is shown in Figure 1.

1. Prepare Media and Seed Cells in 48-well Plates

  1. Prepare 500 ml of complete Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid) buffer solution by adding 50 ml of heat inactivated fetal bovine serum and 5 ml each of a 100x Penicillin-Streptomycin and 100x HEPES buffer solution to a 500 ml bottle of DMEM. This reagent can be stored at 4 °C for months.
  2. Seed 2.5 x 104 Vero E6 cells per well in a 48-well tissue culture plate in a final volume of 500 µl of complete DMEM. Incubate the plate at 37 °C in a humidified incubator containing 5% CO2 for 10 to 18 hr.
    1. Seed the plate at the end of the workday. Test each virus sample in either duplicate or triplicate wells.
      Note: While a 48-well-based platform for this assay is described, it should be possible to scale the assay down for use in 96- or 384-well plates.

2. Carry Out Virus-cell Attachment

Note: The virus used in this protocol, JUNV strain Candid #1 (C#1), has been successfully used as a live attenuated vaccine for the prevention of JUNV disease29. JUNV C#1 was derived from the virulent JUNV strain XJ through serial passage both in vivo and in vitro and differs from the parental XJ strain by 12 amino acids30. Because JUNV C#1 is a biosafety level (BSL)-2-rated pathogen, the work described for this section must be carried out using appropriate BSL-2 practices31. This includes the use of personal protective equipment (e.g. eye protection, laboratory coat, double gloves), a class II biosafety cabinet, and ensuring that all personnel have received the institutional training and approvals required to safely handle this organism.

  1. Dilute the JUNV C#1 sample(s) to be tested to the desired PFUs or focus forming units in ice-cold complete DMEM and store on ice until ready to add to each well.
    Note: As an alternative to conducting the binding studies with virus diluted in complete DMEM, use a minimal medium containing PBS with a reduced serum concentration (2%) and an appropriate buffer to establish whether serum may cause interference with virus attachment. Similar attachment protocols for alternative viruses have reported the use of RPMI 1640 medium without bicarbonate containing 0.2% bovine serum albumin, 10 mM (2-(morpholino)ethanesulfonic acid), and 10 mM HEPES, pH 6.832. This binding media may be superior to prevent pH changes that could occur when media is removed from the CO2-environment of the incubator.
    1. Inoculate wells with PFUs ranging from 200 to 2,000 to limit nonproductive attachment events. As shown in Figure 2, this assay is capable of detecting attachment using as few as 20 PFU (or as many as 2 x 106 PFU).
    2. Create a master mix of virus for inoculation onto cells. Each virus sample will be inoculated onto duplicate wells in a volume of 50 µl per well. Therefore, if the addition of 2,000 PFUs of virus per well is desired, dilute 4,800 PFUs into a total volume of 120 µl of ice cold complete DMEM.
      1. To control for the specificity of the assay, use a matched pair of Chinese hamster ovary cell lines that either do not express TfR1 (TRVb) or express hTfR1 (TRVb-1) as JUNV attachment to these cells should either be reduced or not, respectively13,33. Alternatively, treat cells with a protease such as proteinase K to prevent arenavirus attachment34 or entry35. Soluble hTfR1 or the monocolonal antibody ch128.1, which is specific for hTfR1, could also be considered as they are known to block entry of JUNV into cells13,36. Additionally, pretreatment of cells with free mannose14 or incubation of particles with JUNV-specific neutralizing antibodies37 can inhibit virus entry.
      2. Please note, however, that these latter reagents and approaches, despite blocking JUNV entry, have not been confirmed to block attachment, although this is a likely explanation. The protocol described here could be used to formally determine whether these various approaches impact virion attachment.
  2. Remove plates from the 37 °C incubator and place either in a 4 °C refrigerator or on ice for 15 min to inhibit the ability of the plated cells to carry out endocytosis.
  3. Next, aspirate the complete DMEM from each well and rapidly wash each well twice using 100 µl of ice cold PBS (phosphate buffered saline) pH 7.4. Then add 50 µl of the pre-diluted virus samples (prepared in step 2.1) to the appropriate wells.
  4. Wrap the plate in plastic wrap and immediately place either back on ice or in a 4 °C refrigerator for 1 to 1.5 hr to allow virus attachment to occur. Keep the plate, wash buffer, and virus samples cold at 4 °C throughout this step.
  5. After the completion of the 4 °C incubation, aspirate the virus inoculum and wash each well 3 times with 200 µl of ice-cold PBS.

3. Viral RNA Extraction

  1. Purify viral RNA from JUNV C#1 particles attached to the monolayers by using the commercial kit (e.g., RNeasy Mini kit) according to manufacturer's protocol. Specifically, follow the "purification of total RNA from animal cells using spin technology protocol" on pages 23-28 in the RNeasy Mini Handbook (4th Edition, April 2006) with the modifications listed below.
    1. Lyse the cells as directed in step 1B by adding 350 µl of lysis buffer RLT directly to each well. Mix the buffer several times to ensure cell lysis. Note that cells easily lyse in this buffer and complete lysis can be verified by viewing wells under an inverted microscope.
    2. Transfer the resulting cell lysate to the kit column (step 3a) and follow the rest of the protocol as described. At this point, the virus has been neutralized by the lysis buffer so the remaining steps of the protocol can be done outside of the class II biosafety cabinet provided the kit tubes were not contaminated with infectious virus.
    3. Include the optional step 9 from the manufacturer's protocol, which is an additional centrifugation of the spin column to eliminate any possible carryover of Buffer RPE.
    4. In the final step the manufacturer's protocol (step 10), elute the purified RNA from the spin column using 30 µl of nuclease-free ddH2O. Store RNA at -80 °C at this point or use directly in the qRT-PCR assay. To prevent degradation of purified viral RNA samples by nucleases, use nuclease-free reagents and equipment and keep thawed RNA samples on ice.
      Note: Buffers RLT and RW1 contain guanidine salt and should not be mixed with bleach. Buffer RW1 contains ethanol.

4. qRT-PCR Measurement of Viral Genome

  1. To convert JUNV C#1 S segment RNA into cDNA for subsequent qPCR, subject the viral RNA (generated in step 3.1.4) to RT in a total volume of 50 µl.
    1. To set up the RT reaction, first create a master mix containing each of the following reagents per reaction: 6.75 µl of nuclease-free ddH2O, 5 µl of a 2 µM stock of the RT primer S 2098- to 2075- (5'-AAGGGTTTAAAAATGGTAGCAGAC-3'), which is complementary to the NP region of the S segment genomic RNA, 5 µl of 10X PCR buffer II, 11 µl of a 25 mM MgCl2 solution, 1.25 µl (62.5 Units) of RT enzyme, 1 µl (0.4 Units) of RNase inhibitor, and 10 µl of a 500 µM stock of dNTPs.
      Note: During the course of arenavirus replication, 4 viral S segment RNA species are generated: a full length genomic RNA (vRNA), a full length antigenomic RNA (vcRNA) (that is the reverse complement of the vRNA), and two subgenomic mRNAs encoding the NP and GP genes, respectively. The RT primer listed here is specific for only the S segment vRNA, but not the vcRNA, NP mRNA, or GP mRNA.
    2. Gently mix the RT master mix and then aliquot 40 µl into individual 0.2 ml PCR tubes. Add 10 µl of viral RNA to each tube. Include i) a no template control tube (e.g. add 10 µl of nuclease-free ddH2O to 40 µl of the master mix) and ii) a no RT enzyme control (e.g. add 10 µl of a known-positive viral RNA sample to 40 µl of master mix that did not receive any RT enzyme) to screen for contamination of the RT reagents with amplified DNA containing the viral target sequence. Include RNA extracted from uninfected cells with the full RT master mix to control for assay specificity in the presence of cellular RNA.
    3. Additionally, include a known-positive viral RNA sample to the full master mix to verify the quality of the RT reagents. Note that the volume of the RT reaction can be reduced to 25 µl if necessary.
  2. Subject the RT tubes to the following cycling conditions: 25 °C for 10 min, 48 °C for 30 min, and 95 °C for 5 min. Note that samples can be stored at -20 °C at this point or left overnight in the thermocycler by adding a 4 °C step.
  3. Perform qPCR in a total volume of 25 µl.
    1. Make a qPCR master mix by combining the following: 2.5 µl (of a 9 µM stock) of each the forward PCR primer S1937+ to 1958+ (5'-CATGGAGGTCAAACAACTTCCT-3') and reverse PCR primer S 2001- to 1982- (5'-GCCTCCAGACATGGTTGTGA-3'), 2.5 µl (of a 2 µM stock) of the qPCR probe S 1960+ to 1975+ (5'-6FAM-ATGTCATCGGATCCTT-MGBNFQ-3'), and 12.5 µl of the 2X universal qPCR master mix. Note that the forward primer reported here, which is specific for JUNV C#1, differs by 1 nt from the originally reported primer sequence38, which targets the virulent JUNV strain Romero.
    2. Gently mix the solution and then add 20 µl to each qPCR well. Add 5 µl of each RT reaction to the appropriate qPCR wells. Conduct qPCR in triplicate for each sample tested. Note that the PCR reaction volume can be reduced to as little as 12.5 µl if needed.
      Note: The software associated with the qPCR machine will generate absolute copy numbers of JUNV C#1 S segment genomic RNA by comparing the value of each unknown sample to a series of standard dilutions of plasmid encoding the JUNV C#1 NP gene, which is the target of the qPCR primer and probe set. The qPCR assay can only provide quantitation for values that fall within the range of the standard curve. For this reason, include the following quantities of plasmid (in triplicate wells): 5, 50, 5 x 103, 5 x 105, and 5 x 107 copies per well.
  4. Load the qPCR plate into the thermocycler and run the following reaction conditions: 95 °C for 10 min and 40 cycles of 95 °C for 15 sec and 60 °C for 1 min.
    1. Collect and analyze the data to determine the quantity of viral RNA present in each sample according to manufacturer's protocol.
  5. To establish a standard curve using a newly purified sample of plasmid containing the qPCR target sequence, first determine the plasmid copy number in this solution.
    1. Calculate the molecular weight of the plasmid. If the sequence of the entire plasmid is known, calculate this using the following formula: MW = ([A * 312.2] + [G * 328.2] + [C * 288.2] + [T * 303.2] - 61.13). Remember to include the total number of a particular nucleotide found on each strand of the double stranded plasmid.
    2. If the size of the plasmid is known but its exact sequence is not, calculate the MW under the assumption that each dsDNA base pair has a MW of 600.
      Note: The pCAGGS-JUNV C#1 NP plasmid used here has a MW of 4.2 x 106.
    3. Once the MW of the plasmid has been determined, calculate how many copies per µl are present in the stock plasmid solution.
    4. First calculate the total µg of plasmid in the solution, then convert this to moles of plasmid (e.g. if 69 µg of plasmid are contained in 300 µl of water, then 6.9 x 10-5 g of plasmid * 1 mole/4.2 x 106 g of plasmid = 1.6 x 10-11 moles of plasmid), which can be converted to copy number (e.g. 1.6 x 10-11 moles of plasmid * 6.022 x 1023 molecules/mole = 9.8 x 1012 molecules (or copies)).
    5. Divide the total copies of plasmid in the plasmid solution by the volume of that solution to get copies per µl (e.g. 9.8 x 1012 copies/300 µl = 3.28 x 1010 copies).
    6. Dilute this solution appropriately so that 5 µl volumes can be loaded onto the PCR plate to deliver the range of copy numbers needed to establish the standard curve.

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Results

To demonstrate the sensitivity and dynamic range of the qPCR assay, known quantities of a plasmid encoding the NP gene of JUNV C#1 (range 5-5 x 107 copies) were subjected to qPCR as described in Section 4 of the protocol. The assay is sensitive and can reliably detect as few as 5 copies of the target nucleic acid (Figure 2). Further, the dynamic range of the assay is large and, as shown in Figure 2, can cover at least 7 logs of template. While ...

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Discussion

The protocol we describe is straightforward and robust provided the following critical considerations are observed. First, strict pre-PCR technique must be employed (see 28 for an excellent review). It is imperative that all reagents and equipment are free of amplified DNA containing the qPCR target sequence and that appropriate controls are in place to detect such contamination. Second, for the virus-cell attachment portion of the protocol, the virus, cells, and media must be kept at 4 °C to prevent...

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Disclosures

The authors declare that they have no competing financial interests.

Acknowledgements

We thank Markus Thali, Nathan Roy, Christopher Ziegler, Emily Bruce, and Benjamin King for helpful discussions and the National Institutes of Health for support of these studies through grants T32 AI055402 (JK), R21 AI088059 (JB), and P20RR021905 (JB).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
DMEMLife Technologies11965-118
Penicillin-StreptomycinLife Technologies15140-163100x
HEPES Buffer SolutionLife Technologies15630-130100x
PBS pH 7.4Life Technologies10010-0231x
Vero E6 cellsAmerican Type Culture CollectionCRL-1586
Costar 48-well plateCorning3548
RNeasy mini kitQiagen74106do not mix buffers RLT or  RW1 with bleach
QIAshredder homogenizerQiagen79654
Taqman Universal PCR Master MixLife Technologies4326614
Multiscribe Reverse Transcriptase (50U/µl)Life Technologies4311235
dNTP Mixture (10 mM; 2.5 mM each dNTP)Life TechnologiesN808-0260
GeneAmp 10X PCR Buffer II and 25 mM MgCl2 solutionLife TechnologiesN808-0010
RNase Inhibitor (5000U/100 µl)Life TechnologiesN808-0119
RT primer 5’-AAGGGTTTAAAAAT
GGTAGCAGAC-3’		Integrated DNA Technologies250 nmol DNA oligostandard desalting
Forward qPCR primer  5’-CATGGAGGTCAAACAACTTCCT-3’Life Technologies4304971standard desalting
Reverse qPCR primer 5’-GCCTCCAGACATGGTTGTGA-3’Life Technologies4304971standard desalting
TaqMan Probe 5’-6FAM-ATGTCATCGGATCCTT-MGBNFQ-3’Life Technologies4316033HPLC purified
MicroAmp Fast Optical 96-well reaction plate (0.1 mL)Life Technologies4346906
StepOnePlus Real-Time PCR SystemLife Technologies4376598or other qPCR instrument

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