The biology of lentiviral infection:

1 - Molecular aspects

s-acting elements : sequences present in the genome required for reverse transcription,
viral
packaging and integration. Most of them are essential in the design of
lentiviral vectors and are usually included in the transfer vector (part of
the lenti/retroviral vector which will integrate in the host cell genome
and encodes the GOI.
Molecular Function Role in vector
determinant
LTR Contain sequences for viral gene expression; reverse trascrption; Essential
integration
-U5 A unique, non-coding region of variable lenght which is the first part of
the genome to be reverse transcribed, forming the 3' end of the
provirus genome.

-R A short (18-250nt) sequence which forms a direct repeat at the both

ends of the genome, which is therefore 'terminally redundant'. I nt is
+1 transcription start site.

-U3 A unique non-coding region of 200-1,200nt which forms the 5' end of
the provirus after reverse transcription; contains the promoter
Other Cis-acting alements

vpu
PBS

cPPT/CTS

Molecular Function Role in vector

determinant
PBS (Primer Sequence complementary to the 3' end of the specific tRNA primer Essential
Binding Site) (tRNALys) used by the virus to begin reverse transcription of the
minus-strand RNA. Upstream U5

PSI () Packaging signal required for incapsidation of the genomic transfer Essential
RNA
RRE (rev 234 bp sequence in the env gene that is constitute by several stem- Beneficial
responsive loop structure. It binds Rev for the the efficient transport of single packaging than
element ) splice and unspliced RNAs from nucleus to the cytoplasm. MLV

cPPT & CTS Required for priming of the plus-strand synthesis (so for the second- Essential
strand. It allows the nuclear import of the new synthetized dsDNA cPPT: no entry
CTS marks the termination of strand displacement synthesis 99bp in the nucleus
from cPPT. and low
integration
ans-acting elements:
Encode three different groups of proteins: structural, regulatory and
accessory. Some of them are essentially required for particle formation
and can be delivered from the outside.

Molecular Function Role in vector

determinant
Gag Translated from the unspliced mRNA as a precursor protein, it encodes Essential
structural proteins such as:
- matrix (MA) essential for virion assembly,
- capsid (CA) which forms the hydrophobic core of the virion and it is
essential for assembly and maturation
-nucleocapsid (NC) which coats viral RNA
- p1,p2 and p6 --> unknown functions

Pol Encodes 3 enzymes required for viral replication: Essential

- protease (PRO) which cleaves Gag-Pol polyproteins.
- reverse transcriptase (RT) --> it contains three enzymatic activities
1. RNA-dependent DNA polymerase
2. Rnase H
3. DNA dependent DNA polymerase
- integrase (IN)

Env Is essential for viral binding and entry into the host cells. It encodes Essential
envelope glycoprotein: gp160 (surface protein), gp120 (SU) and gp41
(transmembrane protein). The surface glycoprotein is required for
binding to cellular receptors whereas the transmebrane one is
responsible for the fusion with the cellular membrane
Other Trans-acting elements

Thats why these

genes are called
accessory! They are
nonessential for
virus replication in
cell coltures
The biology of lentiviral infection:

2 Life cycle
Attachment and entry:The virus enters the target cells by fusion between the cellular and
viral membrane and delivers the nucleoprotein core containing the
genomic RNA ino the cytoplasm.

Receptor

Furthermore, the virus entry is pH dependent

Uncoating and Reverse Trascription

After virion-bound matrix and capsid proteins

disassemble and the nucleoprotein complex is
delivered into the cells, reverse transcription
begins in the citoplasm thanks to the RTC

PIC
(PreIntegration Complex

Cellular Viral

Importin

Nup98

The PIC reach the

PIC can cross the intact nuclear nucleus by active
envelop through the NPC in an energy transport along
dependent active process. microtubules

Integration (BAF/Emerin implicated in

anchoring DNA PIC to chromatin)
The biology of lentiviral infection:

3 Lentiviral vector
 TRANSIENT TRANSFECTION> Packaging
stable production cell

VSV-
G
- CEM CD4+ cell line
Viral vectors must be Replication deficient otherwise
- p24 evaluation
RCL particle
- safe
- all ORFs
mantained
except env
- RCR

- dispensable
elements
mantained

- RCR

- Codon
optimized (CO)
- Fusogenic
- Stamble at -80C
- Concentrated higher
title
 8 Kb

Promoter: RS < CNV

V
 LTR vs SIN

> Insertional oncogenesis >

 The circular form doesnt allow the integration in
cell host.
The RT is mantained
Used for a temporary delivery
Titration

Nonfunctional methods

1) ELISA p24

2) QRT-PCR

Functional methods

flow cytometry
1) Transduction Unit (TU/mL) gene reporter
analysis of
transduced cells