An In Vitro Assay to Study Arenavirus Attachment to the Host Cell

Resumo

Source: Klaus, J. P., et al Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment. J. Vis. Exp. (2016).

This video demonstrates how to measure arenavirus attachment to monkey kidney cells. The assay uses low temperatures to block endocytosis and isolate the virus binding step. The approach enables precise quantification of surface-bound virus particles under conditions that restrict internalization.

Protocolo

Note: It is critical that the described protocol be carried out using strict pre-PCR technique 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 disease. 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 acids. 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 practices. 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 plaque-forming units (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 the 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 roswell Park Memorial Institute (RPMI) 1640 medium without bicarbonate containing 0.2% bovine serum albumin, 10 mM (2-(morpholino)ethanesulfonic acid), and 10 mM HEPES, pH 6.8. This binding media may be superior to prevent pH changes that could occur when the 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.
   2. Create a master mix of the 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, respectively. Alternatively, treat cells with a protease such as proteinase K to prevent arenavirus attachment or entry. 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 cells. Additionally, pretreatment of cells with free mannose or incubation of particles with JUNV-specific neutralizing antibodies 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.

Resultados

Figure 1: Depiction of protocol workflow. The primary steps of the virus-cell attachment assay are 1) incubation of virus particles with cells at 4 °C to allow virus-cell attachment to occur followed by washes to remove unbound virus, ...

Materiais

Lista de materiais utilizados neste artigo

Nome	Empresa	Número de catálogo	Comentários
DMEM	Life Technologies	11965-118
Penicillin-Streptomycin	Life Technologies	15140-163	100x
HEPES Buffer Solution	Life Technologies	15630-130	100x
PBS pH 7.4	Life Technologies	10010-023	1x
Vero E6 cells	American Type Culture Collection	CRL-1586
Costar 48-well plate	Corning	3548