Lentiviral Guide

Lentiviral Components

To increase the safety of lentivirus, the components necessary for virus production are split across multiple plasmids (3 for 2nd-generation systems, 4 for 3rd-generation systems). The components of both systems are as follows:

• Lentiviral transfer plasmid encoding your insert of interest. The transgene sequence is flanked by long terminal repeat (LTR) sequences, which facilitate integration of the transfer plasmid sequences into the host genome. Typically it is the sequences between and including the LTRs that is integrated into the host genome upon viral transduction. Many lentiviral transfer plasmids are based on the HIV-1 virus. For safety reasons, transfer plasmids are all replication incompetent and may contain an additional deletion in the 3'LTR, rendering the virus “self-inactivating” (SIN) after integration.
• Packaging plasmid(s)
• Envelope plasmid

Addgene’s packaging and envelope plasmids are generalized and appropriate for varied cell types and systems. When planning your experiment, the important component to consider and optimize is the transfer plasmid. 2nd generation lentiviral plasmids utilize the viral LTR promoter for gene expression, whereas 3rd-generation transfer plasmids utilize a hybrid LTR promoter (more information on this below). Additional or specialized promoters may also be included within a transfer plasmid: for example, the U6 promoter is included in the pSico plasmid to drive shRNA expression. Other features that can be included in transfer plasmids include: Tet- or Cre-based regulation and fluorescent fusions or reporters.
Browse lentivirus plasmids available from Addgene.

2nd Generation

The graphic to the right shows how the lentiviral genome is edited down and distributed across the three plasmids comprising the 2nd-generation lentiviral system. This system contains a single packaging plasmid encoding the Gag, Pol, Rev, and Tat genes. The transfer plasmid contains the viral LTRs and psi packaging signal (not pictured). Unless an internal promoter is provided, gene expression is driven by the 5'LTR, which is a weak promoter and requires the presence of Tat to activate expression. The envelope protein Env (usually VSV‐G due to its wide infectivity) is encoded on a third, separate, envelope plasmid. All 2nd generation lentiviral transfer plasmids must be used with a 2nd generation packaging system because transgene expression from the LTR is Tat-dependent.

Second Generation Lentiviral Plasmids

3rd Generation

The 3rd generation system further improves on the safety of the 2nd generation in a few key ways. First, the packaging system is split into two plasmids: one encoding Rev and one encoding Gag and Pol. Although safer, this system can be more cumbersome to use and result in lower viral titers due to the addition of one additional plasmid.

Second, Tat is eliminated from the 3rd generation system through the addition of a chimeric 5' LTR fused to a heterologous promoter on the transfer plasmid. Expression of the transgene from this promoter is no longer dependent on Tat transactivation. The 3rd generation transfer plasmid can be packaged by either a 2nd generation or 3rd generation packaging system. For a comparison of the key differences between the 2nd and 3rd generation packaging systems, see the table below.

Third Generation Lentiviral Plasmids

2nd vs 3rd Generation Lentiviral Systems

Feature	2nd Generation	3rd Generation
Transfer Plasmid	Can be packaged ONLY by a second generation packaging system that includes TAT	Can be packaged by both 2nd and 3rd generation packaging systems
Packaging Plasmid	All on one plasmid: Gag, Pol, Rev, Tat	Two plasmids: one encoding Gag and Pol and another encoding Rev
Envelope Plasmid	Interchangeable: usually encodes for VSV-G	Interchangeable: usually encodes for VSV-G
Safety	Safe. Replication incompetent: Uses 3 separate plasmids encoding various HIV genes.	Safer. Replication incompetent and always SIN: Uses 4 plasmids instead of 3 and eliminates the requirement for Tat.
LTR Viral Promoter	Wild type	Hybrid: 5'LTR is partially deleted and fused to a heterologous enhancer/promoter such as CMV or RSV

Viral Production

The process of producing infectious transgenic lentivirus is outlined in the simple schematic to the right. 3-4 plasmids are transfected into A293T cells:

• one transfer plasmid
• one or two packaging plasmid(s)
• one envelope plasmid

After media change and a brief incubation period, supernatant containing the virus is removed and stored or centrifuged to concentrate virus. Crude or concentrated virus can then be used to transduce the cells of interest. For determination of viral titer and full details, see the protocol available at the Trono lab website.

Viral Production Schematic

Frequently Asked Questions (FAQ) about Lentiviral Plasmids

What is the difference between 2nd generation and 3rd generation lentiviral systems?

For a full description of 2nd and 3rd generation lentiviruses, please review 2nd vs 3rd generation above. Briefly, 2nd generation lentiviral systems use more HIV proteins (on fewer plasmids) in order to produce functional lentiviral particles than 3rd generation systems.

• 2nd generation packaging systems: express the HIV gag, pol, rev, and tat genes all from a single packaging plasmid such as psPAX2.
• 3rd generation packaging systems: express gag and pol from one packaging plasmid and rev from another, such as pMDLg/pRRE and pRSV-Rev. 3rd generation packaging systems DO NOT express tat. Third generation lentiviral systems are considered safer than second generation systems, but may be more difficult to use because they require transfection with four separate plasmids in order to create functional lentiviral particles.

IMPORTANT : A 3rd generation transfer plasmid can be used with a 2nd generation packaging system, but a 2nd generation transfer plasmid cannot be used with a 3rd generation packaging system.

What is the difference between a lentivirus and a retrovirus?

Lentiviruses are a subtype of retrovirus. From an experimental standpoint the main difference between lentiviruses and standard retroviruses (γ-retroviruses) is that lentiviruses are capable of infecting non-dividing and actively dividing cell types whereas standard retroviruses can only infect mitotically active cell types. This means that lentiviruses can infect a greater variety of cell types than retroviruses.

Both lentiviruses and standard retroviruses use the gag, pol, and env genes for packaging; however, they are different viruses and thus use slightly different isoforms of these packaging components. Therefore, lentivirus may not be efficiently packaged by retroviral packaging systems, and vice versa.

Which bacterial strain should be used for cloning and producing my lentiviral plasmids?

Due to the long terminal repeats found in lentiviral plasmids, we recommend using a strain that reduces the frequency of homologous recombination of unstable regions, such as Invitrogen Stbl3™ or NEB Stable cells. This will ensure that the repeats will be maintained and often results in a greater yield of DNA. However, if the plasmid contains a Gateway cassette containing the ccdB gene, a ccdB survival strain is necessary.

What cell line should be used in order to produce lentivirus?

293T cells are usually used to produce lentivirus.

What dictates lentiviral host cell range (tropism)?

Lentiviral tropism is determined by the ability of the viral envelope protein to interact with receptors at the host cell surface. The VSV-G envelope protein is commonly used in lentiviral particle production because it confers broad tropism over a range of species and cell types. For more information, see the Cronin, et al. article on different envelopes and their tropism.

How can lentivirus be used to make stable cell lines?

Lentiviruses can be used to make stable cell lines in the same manner as standard retroviruses. That is, many lentiviral genomes have selectable markers, such as the puromycin resistance gene, conferring antibiotic resistance to infected host cells. When these antibiotics are added to the growth medium of the host cells, they kill off any cells that have not incorporated the lentiviral genome and those cells that survive can be expanded to create stable cell lines, which have incorporated the lentiviral genome and harbor the genetic information encoded by that genome.

Many lentiviral transfer plasmids do not have selectable markers conferring resistance to an antibiotic, but do encode another marker, such as GFP. A researcher can use FACS to sort cells expressing GFP and later expand these cells into a cell line.

Where does lentivirus integrate?

Genome-wide studies of viral integration have shown that lentiviruses most often integrate into actively transcribed genes, and that this preference is conserved across target species. Although chromatin availability facilitates integration, it does not explain the lentiviral preference for transcribed genes. Studies comparing the lentivirus HIV and the retrovirus MMLV indicate that the viral integrase plays a role in shaping integration site preferences. A major cellular determinant is LEDGF/p75, a lentiviral tethering protein that recruits the pre-integration complex to transcriptional units and facilitates integration. LEDGF/p75 binding sites are enriched in gene bodies and mostly absent in promoters and intergenic regions, mirroring patterns of lentiviral integration.

Can lentiviral plasmids be used in direct transfections as opposed to making virus?

Some (but not all) lentiviral transfer plasmids can be used in transient transfections to achieve expression of the transgene, and those that can are primarily third generation constructs. Lentiviral transfer plasmids are not designed specifically for transient transfections. Therefore, there may be limited transgene expression due to the lentiviral LTRs. While possible, it is not explicitly recommended that you use lentiviral transfer plasmids for simple transfections.

Is it feasible to express cDNA from a lentiviral transfer plasmids normally used for shRNA expression?

Yes, it is feasible, but first the promoter within the transfer plasmid must be modified. Most shRNA‐expressing lentiviral plasmids, such as pLKO.1, use a U6 or H1 promoter in order to drive RNA pol III-directed transcription of shRNAs. cDNA expression requires RNA pol II, and thus requires an RNA pol II promoter, such as CMV or RSV.

What techniques can be used to clone an insert into a lentiviral plasmid containing only one restriction site?

If a lentiviral transfer plasmid contains a single restriction site, one can use standard cloning techniques to ligate the insert into this site. If it is not immediately feasible to digest and clone the insert from a parent vector, some possible approaches to using this site include subcloning or appending compatible restriction sites onto the insert of interest using PCR. The process of subcloning consists of digesting the insert of interest from its parent vector and ligating into a second vector in such as way that the insert may later be digested from this new vector and cloned into the lentiviral vector. This is basically shuffling restriction sites between vectors until the gene of interest is flanked by sites compatible with those in the vector into which one ultimately wants to ligate the insert. Often times it is less time consuming and easier to simply add restriction sites onto the insert of interest using PCR. This is accomplished by PCR amplifying the insert sequence using primers that contain the restriction sites needed. Functional restriction sites must be a certain number of bases from the ends of the primers used. Alternatively, you could ligate a multiple cloning site (MCS) from a separate vector into the single site in the lentiviral vector and generate more useful restriction sites.

For more information about cloning and changing a MCS, visit Addgene's Plasmid Reference and Protocol Guide.

How do I clone my insert into a transfer vector with no viable restriction sites but compatible with the Gateway® cloning system?

Gateway® compatible vectors use recombination in order to generate clones containing the insert of interest. In brief, the insert is first cloned into an entry vector at a region flanked by sequences (called attP1 and attP2) that allow the insert to recombine with the destination vector (in this case the destination vector would be the lentiviral transfer vector). The destination vector contains attB sequences with which the attP sequences recombine. Visit Invitrogen's website for more information on the Gateway® cloning system.

What safety concerns surround the use of lentiviral vectors?

As noted by the NIH, the two main safety concerns surrounding the use of lentiviral are:

1. The potential for generation of replication-competent lentivirus
2. The potential for oncogenesis

The potential for generation of replication-competent lentivirus is addressed by the design of the vectors and by safe laboratory practice. In terms of vector design, 2nd and 3rd generation lentiviral systems provided by Addgene separate transfer, envelope, and packaging components of the virus onto different vectors. The transfer vector encodes the gene of interest and contains the sequences that will incorporate into the host cell genome, but cannot produce functional viral particles without the genes encoded in the envelope and packaging vectors. Unless recombination occurs between the packaging, envelope, and transfer vectors, and the resulting construct is packaged into a viral particle, it is not possible for viruses normally produced from these systems to replicate and produce more virus after the initial infection. In this regard, 3rd generation systems are considered safer than 2nd generation systems because the packaging vector has been divided into two separate plasmids (resulting in a four plasmid system in total). In addition, 3rd generation systems do not use the HIV protein tat in order to produce full length virus from the transfer vector during the viral production stage.

Many of the lentiviral transfer vectors that have been deposited with Addgene are self-inactivating (SIN) vectors. These vectors have a deletion in the 3'LTR of the viral genome that is transferred into the 5'LTR after one round of reverse transcription. This deletion abolishes transcription of the full-length virus after it has incorporated into a host cell.

The potential for oncogenesis is largely based on the specific insert contained within the lentiviral transfer vector (dependent upon whether or not it is an oncogene) and should be considered on a case by case basis.

Biosafety should always be considered with respect to the precise nature of experiments being performed, and your biosafety office can provide more information on your institution's best practices with regard to lentiviral research. The NIH has additional information on lentiviral safety considerations.

Additional Resources

Web References

• Find lentiviral plasmids available from Addgene
• Find ready-to-use lentivirus available through Addgene's viral service
Addgene has put together a webinar ( Lentivirus 101: Plasmids and Viral Production ) with Bitesize Bio focused on understanding the components of lentiviruses and how they are produced in the lab. The webinar covers:
1. Plasmids required to generate lentivirus (both 2nd and 3rd generation systems)
2. Safety Concerns
3. Lentiviral-based applications

• Read our detailed protocol for using the popular cloning vector pLKO.1.
• Trono Lab Resources:
  • Lentivectors Toolbox and Maps
  • Standard Lentiviral Production & Titration Protocols

Publications

• Retroviral integration site selection. Desfarges S, Ciuffi A. Viruses. 2010. Jan;2(1):111-30. PubMed.

• Altering the tropism of lentiviral vectors through pseudotyping. Cronin J, Zhang XY, Reiser J. Curr Gene Ther. 2005. 5(4): 387-398. PubMed.

• HIV-1 Genome Nuclear Import Is Mediated by a Central DNA Flap. Zennou V, Petit C, Guetard D, Nerhbass U, Montagnier L, Charneau P. Cell. 2000. 101(2): 173-185. PubMed.

• Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element Enhances Expression of Transgenes Delivered by Retroviral Vectors. Zufferey R, Donello JE, Trono D, and Hope TJ. J Virol. 1999. 73(4):2886-92. PubMed.

• A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L. J Virol. 1998. 72(11):8463-8471. PubMed.

• Self-Inactivating Lentivirus for Safe and Efficient In Vivo Gene Delivery. Zufferey R, Dull T, Mandel RJ, Bukovsky A, Quiroz D, Naldini L, and Trono D. J Virol. 1998. 72(12): 9873-9880. PubMed.

• Retroviruses. Coffin JM, Hughes SH, Varmus HE. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, 1997. Available online: Genetic Organization and Genetic & Structural Features.

• In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Naldini L, Blömer U, Gallay P, Ory D, Mulligan R, Gage FH, Verma IM, and Trono D. Science. 1996. 272(5259): 263-267. PubMed.

Glossary

Plasmid Type	Element	Delivery relative to transgene	Purpose
Envelope	VSVG	in trans	Vesicular stomatitis virus G glycoprotein; Broad tropism envelope protein used to psuedotype most lentiviral vectors.
Packaging	Gag	in trans	Precursor structural protein of the lentiviral particle containing Matrix, Capsid, and Nucleocapsid components.
	Pol	in trans	Precursor protein containing Reverse Transcriptase and Integrase components.
	Rev	in trans	On separate plasmid from Gag/Pol in third generation system; Binds to the Rev Response Element (RRE) within unspliced and partially spliced transcripts to facilitate nuclear export.
	Tat	in trans	Second generation only; Trans-activator; binds TAR to activate transcription from the LTR promoter.
Transfer	cPPT	in cis	Central polypurine tract; recognition site for proviral DNA synthesis. Increases transduction efficiency and transgene expression.
	Psi (Ψ)	in cis	RNA target site for packaging by Nucleocapsid.
	RRE	in cis	Rev Response Element; sequence to which the Rev protein binds.
	WPRE	in cis	Woodchuck hepatitis virus post‐transcriptional regulatory element; sequence that stimulates the expression of transgenes via increased nuclear export.
	LTR Sub-components: U3 R Sub-element: TAR U5	in cis	LTR; Long terminal repeats; U3-R-U5 regions found on either side of a retroviral provirus (see below). Cloning capacity between the LTRs is ∼8.5kb, but inserts bigger than ∼3kb are packaged less efficiently. Sub-components: U3; Unique 3'; region at the 3' end of viral genomic RNA (but found at both the 5' and 3' ends of the provirus). Contains sequences necessary for activation of viral genomic RNA transcription. R; Repeat region found within both the 5' and 3' LTRs of retro/lentiviral vectors. Tat binds to this region. Sub-element: TAR; 2nd generation only; Trans-activating response element; located in the R region of the LTR and acts as a binding site for Tat. U5; Unique 5'; region at the 5' end of the viral genomic RNA (but found at both the 5' and 3' ends of the provirus).
	5' LTR	in cis	Acts as an RNA pol II promoter. The transcript begins, by definition, at the beginning of R, is capped, and proceeds through U5 and the rest of the provirus. Third generation vectors use a hybrid 5' LTR with a constitutive promoter such as CMV or RSV.
	3' LTR	in cis	Terminates transcription started by 5' LTR by the addition of a poly A tract just after the R sequence.