This document discusses the structure and classification of viruses. It notes that viruses can have DNA or RNA genomes and be enveloped or non-enveloped. Important human viruses discussed include HIV, influenza, hepatitis B, herpes viruses, and papillomaviruses. The document also examines the life cycle of viruses, including entry into host cells, genome replication, protein synthesis, and virus assembly and release. Finally, it reviews potential targets for antiviral drugs, such as inhibitors of viral attachment, fusion, reverse transcription, protease, polymerase and neuraminidase.

1. Antiviral Drugs

2. Structure of viruses
•DNA-viruses: poxviruses, herpes, adenoviruses,
papillomaviruses.
Contain mostly double-stranded DNA, a small number
single-stranded DNA.
DNA viruses enter the cell nucleus and direct the
generation of new viruses.
•RNA-viruses: influenza, measles, mumps, cold,
meningitis, poliomyelitis, retroviruses (AIDS, T-cell
leukemia), arenaviruses.
Contain largely single-stranded RNA (ssRNA).
RNA viruses do not enter the cell nucleus (except the
influenza virus).
RNA retroviruses uses the viral reverse transcriptase to
make a DNA copy of the viral RNA, which is then
integrated into the host genome.
structure of the influenza virus

3. Types of viruses

4. Types of important viruses
Table 3 Classification of selected animal viruses with RNA genomes
HIV, human immunodeficiency virus; ds, double-stranded; ss, single-stranded; neg, negative; pos, positive.
Virus family Capsid shape Envelope Virion size (nm) RNA RNA length (kb) Example
Picornaviruses Icosahedral No 27 ss; pos 7 Poliovirus
Togaviruses Icosahedral Yes 70 ss; pos 12 Equine encephalitis
virus
Flaviviruses Icosahedral Yes 45 ss; pos 11 Yellow fever virus
Bunyaviruses Helical Yes 100 ss; neg 14 Hantavirus
Orthomyxoviruses Helical Yes 100 ss; neg 14 Influenza virus
Paramyxoviruses Helical Yes 120–250 ss; neg 15 Measles virus
Rhabdoviruses Helical Yes 80 × 180 ss; neg 11 Rabies virus
Filoviruses Helical Yes Long filaments ss; neg 13 Ebola virus
Retroviruses Icosahedral Yes 90 ss; pos 9 HIV
Reoviruses Icosahedral Yes 80 ds 23 kbp Human rotavirus
Table 2 Classification of animal viruses with DNA genomes
ds, double stranded; ss, single-stranded.
Virus family Capsid shape Envelope Virion size (nm) DNA DNA length (kbp) Example
Andenoviruses Icosahedral No 80 ds 36 Human adenovirus
Herpesviruses Icosahedral Yes 200 ds 130–230 Herpes simplex virus
Poxviruses Complex Yes 300 ds 130–280 Smallpox virus
Papovaviruses Icosahedral No 45–60 ds 5–8 Human papillomavirus
Parvoviruses Icosahedral No 22 ss 5 kb Canine parvovirus
Hepadnaviruses Icosahedral Yes 42 ds 3 Hepatitis B virus

5. Virus Structure
Poxvirus
Rabiesvirus
Mumpsvirus
Poxvirus
T-virus Coliphage
Lambda
Herpes Simplex
Adenovirus HIV Influenzavirus
Tobacco Mosaic Simian Rhino
upper respiratory infections
(Tollwut)

6. The structure of viruses
tobacco virus
Herpes simplex
capsid

7. Some disturbing figures about AIDS...(2013)
AIDS infected in 2000
Lifetime risk of dying of AIDS for 15-year-old boys
In 2015, it was estimated that
36.7 million people lived with the
disease worldwide, and that AIDS
killed an estimated 1.1 million
people in 2015, including 210,000
children

8. The ranges around the estimates in this table define the boundaries within which the actual numbers lie, based on the best available information.
Regional HIV and AIDS statistics and features 2012
TOTAL 35.3 million
[32.2 million – 38.8 million]
2.3 million
[1.9 million – 2.7 million]
Adults and children
newly infected with HIV
Adults and children
living with HIV
Sub-Saharan Africa
Middle East and North Africa
South and South-East Asia
East Asia
Latin America
Caribbean
Eastern Europe and Central Asia
Western and Central Europe
North America
Oceania
25.0 million
[23.5 million – 26.6 million]
3.9 million
[2.9 million – 5.2 million]
1.5 million
[1.2 million – 1.9 million]
1.3 million
[1.0 million – 1.7 million]
1.3 million
[980 000 – 1.9 million]
1.6 million
[1.4 million – 1.8 million]
270 000
[160 000 – 440 000]
86 000
[57 000 – 150 000]
130 000
[89 000 – 190 000]
48 000
[15 000 – 100 000]
260 000
[200 000 – 380 000]
880 000
[650 000 – 1.2 million]
250 000
[220 000 – 280 000]
860 000
[800 000 – 930 000]
51 000
[43 000 – 59 000]
32 000
[22 000 – 47 000]
81 000
[34 000 – 160 000]
12 000
[9400 – 14 000]
29 000
[25 000 – 35 000]
2100
[1500 – 2700]
1.6 million
[1.4 million – 1.9 million]
Adult & child
deaths due to AIDS
1.2 million
[1.1 million – 1.3 million]
220 000
[150 000 – 310 000]
52 000
[35 000 – 75 000]
91 000
[66 000 – 120 000]
20 000
[16 000 – 27 000]
17 000
[12 000 – 26 000]
41 000
[25 000 – 64 000]
11 000
[9400 – 14 000]
7600
[6900 – 8300]
1200
[<1000 – 1800]
0.8%
[0.7% - 0.9%]
Adult prevalence
(15 49) [%]
4.7%
[4.4% – 5.0%]
0.3%
[0.2% – 0.4%]
0.4%
[0.3% – 0.5%]
0.7%
[0.6% – 1.0%]
0.5%
[0.4% – 0.8%]
0.1%
[0.1% – 0.2%]
<0.1%
[<0.1% – 0.1%]
1.0%
[0.9% – 1.1%]
0.2%
[0.2% – 0.2%]
0.2%
[0.2% – 0.3%]

9. Total: 35.3 million [32.2 million – 38.8 million]
Western &
Central Europe
860 000
[800 000 – 930 000]
Middle East & North Africa
260 000
[200 000 – 380 000]
Sub-Saharan Africa
25.0 million
[23.5 million – 26.6 million]
Eastern Europe
& Central Asia
1.3 million
[1.0 million – 1.7 million]
South & South-East Asia
3.9 million
[2.9 million – 5.2 million]
Oceania
51 000
[43 000 – 59 000]
North America
1.3 million
[980 000 – 1.9 million]
Latin America
1.5 million
[1.2 million – 1.9 million]
East Asia
880 000
[650 000 – 1.2 million]
Caribbean
250 000
[220 000 – 280 000]
Adults and children estimated to be living with HIV 2012

10. Estimated adult and child deaths from AIDS 2012
Western &
Central Europe
7600
[6900 – 8300]
Middle East & North Africa
17 000
[12 000 – 26 000]
Sub-Saharan Africa
1.2 million
[1.1 million – 1.3 million]
Eastern Europe
& Central Asia
91 000
[66 000 – 120 000]
South & South-East Asia
220 000
[150 000 – 310 000]
Oceania
1200
[<1000 – 1800]
North America
20 000
[16 000 – 27 000]
Latin America
52 000
[35 000 – 75 000]
East Asia
41 000
[25 000 – 64 000]
Caribbean
11 000
[9400 – 14 000]
Total: 1.6 million [1.4 million – 1.9 million]

11. About 6,300 new HIV infections a day in 2012
About 95% are in low- and middle-income countries
About 700 are in children under 15 years of age
About 5,500 are in adults aged 15 years and older, of whom:
almost 47% are among women
about 39% are among young people (15-24)

12. Time course of the HIV infection
•Once HIV has killed so many CD4+ T cells that there are fewer than 200 of these cells per microliter of blood,
cellular immunity is lost. Acute HIV infection progresses over time to clinical latent HIV infection.
•In the absence of antiretroviral therapy, the median time of progression from HIV infection to AIDS is nine to
ten years, and the median survival time after developing AIDS is only 9.2 months

13. Scanning electron micrograph of HIV-1
budding from a cultured lymphocyte

14. The HIV virus structure and the HIV life cycle

15. Structural Biology of HIV-1
MA matrix protein
TM (ectodomain)
membrane anchor
of gp160
RT reverse
transcriptase
PR protease
IN integrase
CA capsid protein
NC nuclear capsid
SU gp120
Summers, J. Mol.
Biol. 285 (1999),1-32

16. The various stages during viral infection
•Attachment of the virus to the host cell membrane via binding
of molecules of the outer surface of the virion to a receptor
molecule on the host cell (protein or carbohydrate)
•Penetration and uncoating of the virus and subsequent
release of the virions into the host cell. Some viruses inject their
material, others enter the cells intact (via endosomal entry) and
are uncoated inside.
•Retroviruses (e.g. HIV):reverse transcription of viral RNA into
DNA
•Integration of the viral DNA into the host cell genome
•Some viruses (e.g. HIV) use the cellular system for gene
duplication and translation, while others (herpes) uses their own
system to produce mRNA coding for viral proteins (early genes)
• Synthesis and assembly of nucleocapsids (late genes):
Synthesis of capsid proteins that self-assemble to form the
capsid.
• Virion release: Release of the naked virions by cell lysis, where
the cell is destroyed. Alternatively, the viruses with envelopes
can be released by a process known as budding, in which the
nucleocapsid is wrapped by the membrane and pinched off.
Capsidcore
CD4 receptor and
CXCR4 or CCR5
co-receptor proteins Uncoating
HIV gp120
Reverse
transcriptase
Virus adsorption
Reverse
transcription
Integration
(Strand transfer)
Translation
Transcription
Proteolytic processing
by viral protease
Viral proteins and
RNA assemble at
the cell membrane
Budding
RNA
Integr
ase
DNA
Polypeptide
Virus–c
Fusion
inhibitor
s
Co-receptor
antag
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Protease
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s
Integr
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Reverse
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NtRTIs,
NNRTIs)
HIV
CD4+
T cell

17. Viral Entry
enveloped viruses non-enveloped viruses

18. Virus receptors
Host cell receptor Virus
CD4 glycoprotein HIV
CCR5, MCP-1, RANTES HIV
chemokine receptor CXCR4 for
cytokine SDF-1
HIV
acetylcholine receptor rabies virus
complement C3d of B
lymphocytes
glandular fever virus
IL-2 receptor on T-lymphocytes T cell leukemia viruses
beta-adrenoreceptor infantile diarrhea viruses
MHC molecules adenoviruses; T-cell leukemia v.

19. Cell-Exit of Enveloped Viruses

20. Strategies for Antiviral Drug Development
• inhibition of virus adsorption
• inhibition of virus-cell fusion
• inhibition of the HIV integrase (HIV)
• inhibition of viral DNA or RNA synthesis
– inhibitors of viral DNA polymerase
– inhibitors of the reverse transcriptase
– acyclic nucleoside phosphonates
• viral protease inhibition
• viral neuraminidase inhibition (influenza)
• inhibition of IMP dehydrogenase
• inhibition of S-adenosylhomocysteine hydrolase

21. Targets for Antiviral Drugs
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phosphonates papilloma-, herpes-, adeno- HIV: tenofovir
and poxviruses), HIV, HBV
Inhibitors of processes associated HIV, HCV
with viral RNA synthesis
Viral protease inhibitors HIV, herpesviruses, HIV: saquinavir, ritonavir, indinavir, HIV: atazanavir, mozenavir, tipranavir
rhinoviruses, HCV nelfinavir, amprenavir, lopinavir Human rhinovirus: AG7088
Viral neuraminidase inhibitors Influenza A and B virus Zanamivir, oseltamivir§
RWJ270201
IMP dehydrogenase inhibitors HCV, RSV Ribavirin||
Mycophenolic acid, EICAR, VX497
S-adenosylhomocysteine (–)RNA haemorrhagic fever
hydrolase inhibitors viruses (for example, Ebola)
* Brivudin is approved in some countries; for example, Germany.
‡
Lamivudine is also approved for the treatment of HBV.
§
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process, not the viral neuraminidase.
||
Ribavirin is used in combination with interferon-α for HCV.
CMV, cytomegalovirus; EBV, Epstein–Barr virus; EICAR, 5-ethynyl-1-β-D-ribofuranosylimidazole-4-carboxamide; HBV, hepatitis B virus; HCV, hepatitis C virus; HHV, human
herpesvirus; HIV, human immunodeficiency virus; HSV, herpes simplex virus; IMP, inosine 5ʹ-monophosphate; NNRTI, non-nucleoside reverse transcriptase inhibitor; NRTI,
nucleoside reverse transcriptase inhibitor; RSV, respiratory syncytial virus; VZV, varicella-zoster virus.

22. Togavirus (for example, Western RNA polymerase Several*
Equine encephalitis virus)
Rhabdovirus (Rabies virus) RNA polymerase Several*
Filovirus (for example, Ebola virus) RNA polymerase Several*
Hepacivirus (HCV) RNA polymerase, –
RNA helicase,
Viral protease
Orthomyxovirus (Influenza) Matrix (M2) protein –
Neuraminidase
Paramyxovirus (RSV) Fusion polypeptide Several*
Coronavirus (SARS-CoV) Several‡
–
Reovirus (Rotavirus) – Several*
Retrovirus (HIV) Several§||
Several¶
*Inosine 5’-monophosphate (IMP) dehydrogenase, S-adenosylhomocysteine (SAH) hydrolase
decarboxylase and cytosine 5’-triphosphate (CTP) synthetase. ‡
Spike (S) protein, RNA polym
protease. §
Fusion polypeptide (viral glycoprotein gp41), reverse transcriptase and viral proteas
transcription transactivator (TAT). ¶
Integration- and transcription-associated factors.
| Viral and c ellular targets for antiviral agents
Virus Viral target Cellular target Comments
Parvovirus DNA polymerase – Remains to be explored
Polyomavirus DNA polymerase – Remains to be explored
Papillomavirus DNA polymerase, E6, E7 – Remains to be explored
Adenovirus DNA polymerase Cell-associated factors Remains to be explored
α-herpesvirus Thymidine kinase – Target for activation
(HSV-1, HSV-2, VZV) DNA polymerase Target for inhibition
Helicase–primase Target for inhibition
β-herpesvirus Protein kinase – Target for activation
(HCMV, HHV-6, HHV-7) DNA polymerase Target for inhibition
Terminase Target for inhibition
γ-herpesvirus (EBV, HHV-8) DNA polymerase – Target for inhibition
Poxvirus (for example, variola and DNA and RNA polymerases Several* Remains to be explored
vaccinia viruses)
Hepadnavirus (HBV) DNA polymerase – Target for inhibition
(Reverse transcriptase)
Picornavirus Viral capsid – Target for inhibition
(Enteroviruses and Rhinoviruses) RNA polymerase Remains to be explored
Flavivirus (for example, yellow fever RNA polymerase – Remains to be explored
and dengue viruses)
Arenavirus (for example, Lassa) RNA polymerase Several* Remains to be explored
Bunyavirus (for example, RNA polymerase Several* Remains to be explored
Crimean–Congo)
Togavirus (for example, Western RNA polymerase Several* Remains to be explored
Equine encephalitis virus)
Rhabdovirus (Rabies virus) RNA polymerase Several* Remains to be explored
Filovirus (for example, Ebola virus) RNA polymerase Several* Remains to be explored
Hepacivirus (HCV) RNA polymerase, – Being investigated
RNA helicase,
Viral protease
Orthomyxovirus (Influenza) Matrix (M2) protein – Target for inhibition
Neuraminidase Target for inhibition
Paramyxovirus (RSV) Fusion polypeptide Several* Being explored
Coronavirus (SARS-CoV) Several‡
– Being explored
Reovirus (Rotavirus) – Several* Remains to be explored
Retrovirus (HIV) Several§||
Several¶
Established or being
explored

23. Drugs targeting
HIV viruses
Viral
RNA
Chromosoma
l
DNA
Transcription
Assembly
Budding
Viral
proteins
RNA
RNA
RNA
DNA
DNA
CD4 protein
HIV
Viral envelope
proteins (gp160)
HelperT-lymphocyte
HIV protease
Translation
Regula
ted by
Reverse
transcriptase
RNaseH
Integrase
(latency)
Rev
Vif
Tat Nef
CCR5
CXCR4
Virus
adsorption
Virus–cell fusion
Uncoating
Reverse
transcription
Integration

24. •viral proteins
•cellular cofactors
•host cell inhibitors
•approved drugs

25. Retroviral genes of the HIV virus
• gag (group-specific antigen): makes the cone-shaped viral shaped viral
capsid.
• pol(polymerase): codes for viral enzymes, (polymerase): codes for viral
enzymes, reverse transcriptase, integrase, and viral protease.
• env(envelope): makes surface protein gp120 and transmembrane gp41,
enabling HIV to fuse to CD4 cells.
• tat: eliminates the hairpin structure in RNA by phosphorylating CdK9/
CycT. This increases transcription and creates a positive feedback loop
• vpr: is involved in getting the viral RNA into the nucleus of the T cell. It
causes the cell to stop growing, stimulating an immune dysfunction.
• rev: creates a rev Response Unit that exports the HIVrev RNA into the
cytoplasm before it is spliced. It is also positive feedback loop.
• nef: down regulates CD4 on the T cell, inhibiting response to infectious
agents.
• vif: helps HIV infect other cells, though it is still unclear how. It is thought
that it interferes with the T cells proteins that modify RNA.
• vpu: enhances HIV release from the cell.

26. HIV-1 proteins
Swanson et al., Cell 133

27. Antiviral Drugs on the Market in the US (2007)
Name Trade name Company Launched
Nucleoside or nucleotide reverse-transcriptase inhibitors
Zidovudine Retrovir GlaxoSmithKline 1987
Didanosine Videx Bristol–Myers Squibb 1991
Zalcitabine HIVID Roche 1992
Stavudine Zerit Bristol–Myers Squibb 1995
Lamivudine Epivir GlaxoSmithKline,
Shire Pharmaceuticals
1998
Abacavir Ziagen GlaxoSmithKline 1999
Tenofovir disoproxil fumarate Viread Gilead 2001
Emtricitabine Emtriva Gilead 2003
Non-nucleoside reverse-transcriptase inhibitors
Nevirapine Viramune Boehringer Ingelheim 1996
Efavirenz Sustiva, Stocrin Bristol–Myers Squibb, Merck 1998
Delavirdine Rescriptor Pharmacia & Upjohn,
Agouron, Pfizer
1999
Protease inhibitors
Saquinavir Invirase Hoffmann-LaRoche 1995
Indinavir Crixivan Merck 1996
Ritonavir Norvir Abbott, GlaxoSmithKline 1996
Nelfinavir Viracept Agouron, Pfizer 1997
Amprenavir Agenerase, Prozei Vertex 1999
Lopinavir + ritonavir Kaletra, Aluvia Abbott 2000
Atazanavir Reyataz, Zrivada Bristol–Myers Squibb, Novartis 2003
Fosamprenavir Lexiva, Telzir Vertex, GlaxoSmithKline 2003
Tipranavir Aptivus Boehringer Ingelheim 2005
Darunavir Prezista Tibotec 2006
Entry inhibitors
Enfuvirtide Fuzeon Trimeris, Roche 2003
Maraviroc Celsentri,Selzentry Pfizer 2007

28. Virus Adsorption Inhibitors
• polyanionic compounds such as polysulphates, polysulphonates,
polycarboxylates have been shown to bind to the V3 loop of the viral
envelope protein gp120, which is rich in Arg+ and Lys+ residues.
• Thereby, they hamper binding to heparan sulfate, widely expressed
on animal cell surfaces.
• Some polypeptides such as cyanovirin bind to the carbohydrate
portions of gp120, thereby inhibiting their interaction with the human
cell surface receptors CD4.
• Polyanions may become ineffective by mutations in the V3 loop
(K269E, Q278H, N293D), that make these strains resistant to such
treatments.
OSO
n n
3 SO3
Viral adsorption inhibitors
PVAS PVS

29. Virus-Cell Fusion Inhibitors
• in case of HIV gp120 interacts first with a co-receptor; for some
HIV strains its is the chemokine (C-X-C) motif receptor 4 (CXCR4),
for some the CCR5. These receptors usually act as receptors for
the chemokines SDF1 , RANTES or MIP5.
• Initial association of the HIV virions with the cells might be blocked
by antagonists of these receptors.
Virus–cell fusion inhibitors
H
N
N
C
CH3
O
O
TAK779
NH
NH
HN
N
NH
N
HN
HN
AMD3100
3
H
L33 W76
Y108
T82
1
2
3 4
5
6
7
Y37
Interaction of CCR5 with TAK779. A structural
model of CCR5 complexed with TAK779 (FIG.2) , viewed from
within the plane of the membrane12
. The indicated cluster of
amino acids in the TAK779 binding site includes several
aromatic residues (Y37, W86, Y108) that might form
favourable interactions with the aromatic rings of TAK779.

30. Mechanism of fusion of the human and viral membranes
•the fusion peptide
at the N terminus of
gp41 insert into the
cellular membrane.
This event triggers a
structural change,
that brings the two
membranes closer
together.
• several drugs
interfere with the
gp41-mediated fusion
process such as T20
(fuzeon, a 36 aa
peptide that mimics
the ectodomain of
gp41) or the so-called
5-helix, that binds to
the carboxy terminal
region of gp41.

31. Klein, Science, 341 (2013),1199
Antibodies against the HIV-1 envelope spike
Antibody target sites on the HIV-1
envelope spike. Schematic
representation of the HIV-1 envelope
spike. Each of the monomer units of the
trimer is composed of a gp120 and
gp41 trans-membrane protein. The four
best-characterized broadly neutralizing
target sites are highlighted and include
the CD4-binding site (orange), the
glycan-associated epitopes on the
base of the V3 loop (purple), the V1/V2
loop (green), and the membrane-
proximal external region MPER on gp41
(gray). Electron micrograph–derived
illustration of the envelope spike
targeted by representative broadly
neutralizing F(ab)s shown approximately
to scale (bottom: NIH45-46, orange;
PG16, green; PGT128, purple; 2F5, gray).
Examples of first- and second-
generation bNAbs that target these
and other sites are color-coded and
indicated at the bottom.

32. Structure of Fuzeon
•Enfuvirtide therapy costs an estimated US$25,000 per year in the United States
• Dose of Fuzeon for treating adults with HIV or AIDS is Fuzeon 90 mg (1 mL) injected
twice daily just under the skin

33. Inhibitors of viral DNA or RNA synthesis
• RNA viruses: First step is transcription of viral RNA into DNA by the viral reverse transcriptase. All
three phosphorylation steps must be carried out by cellular kinases.

34. Drugs as inhibitors of the reverse transcriptase

35. Non-nucleoside reverse transcriptase inhibitors
•must be hydrophobic
•bind to an allosteric binding site that is adjacent to the substrate (nucleoside)
binding site.
•binding locks the adjacent substrate binding site into an inactive conformation.
•improved specificity for reverse transcriptase over DNA polymerases resulting in
less toxicity
•unfortunately, rapid resistance is developed
N
N
O NH
Br
NH2
N N
N
N
HN NH
N
N
N
HN NH
N
N
Etravirine Rilpivirine Dapivirine

36. Mechanism of action of the HIV integrase
att 3ʹ-processing
Strand transfer
5ʹ
5ʹ
5ʹ
5ʹ 5ʹ 5ʹ
•Integrase is essential for viral replication. No integrases are found in the host
cells.
•It is encoded in the POL gene of HIV, and generated by the HIV protease.
•It is a 32KDa protein with 3 domains: the amino-terminal domain (NTD), the
catalytic core domain (CCD) and the carboxy-terminal domain (CTD). The CCD
forms a dimer.
•It catalyses both 3’ processing of viral DNA in the cytoplasm, and insertion of
the viral DNA into chromosomal DNA in the nucleus
•viral DNA is processed in the cytosol by 3’
endonucleophilic cleavage creating the
reactive intermediate required for the strand
transfer
•the complex formed of processed viral
DNA and integrase diffuses into the nucleus.
In the nucleus the reactive ends attack
chromosomal DNA across the major groove.
Therein, the 3’ OH groups of the viral DNA
are ligated to the 5’DNA phosphate.

37. The HIV Integrase
CCD
CTD

38. HIV Integrase Inhibitors in an advanced Phase of
Development:
Diketoaryl (DKA) and DKA-strand-transfer selective inhibitors
HN
Cl
O OH
N N
NH
N
N
NH
N
O OH
O
F
O
COOH
OH
O
O
COOH
O OH
N3
COOH
O OH
N
F
N
H
O
N
N
R
OH
F
N O
H3C
N
O
CH3
CH3
S
N
O
O
R =
5CITEP
S-1360
L-708,906
MA-DKA
L-731,988
Naphthyridine carboxamides (Merck)
Diketo acids
Diketo aryls (Shionogi)
L-870,810 L-870,812
O
H
N
N
H
N
N N
O
N
O
OH
O
F
Raltegravir (MK-0518)
N
O
OH
F
Cl
O O
OH
H
Elvitegravir (GS-913
7, JTK-303)

39. Mode of action of DKA integrase inhibitors
Viral DNA
(Donor)
Donor site Acceptor site
Host DNA
(Acceptor)
OH
Adenine
5ʹ
O
O
Integrase acidic
residues
(D64, D116, E152)
DKA
Viral DNA (Donor)
e
3ʹ
3ʹ-processing Strand transfer
a
d
b c
DKA
–
O
O
–
O
O
–
O
O
O–
N3
O
O
O
Me2+
Me2+
•The donor site of the integrase
binds viral DNA and catalyses 3’
processing (a).
•The integrase undergoes a
structural change that allows the
binding of the chromosomal DNA
•The DKA’s bind selectively to the
acceptor site
•DKA chelate the metal in the
catalytic site of the integrase in
the CCD domain formed by the
catalytic residues D64, D116 and
E152. Usually the metals
coordinate to these residues and
the phosphodiester backbone of
the DNA substrate.

40. Inhibitors of viral DNA or RNA synthesis
• DNA viruses: First step is
replication of viral DNA. Inhibitors
of the latter are nucleoside
analogues. These are sufficiently
similar to the “normal”
nucleosides, so that they can
serve as substrates for the viral
DNA polymerases. Since the
sugar lacks the 3’ hydroxyl group
they act as chain terminators:

41. Chain terminators for viral DNA polymerases
Acyclovir Cidofovir
(does not require the presence of a
viral thymidine kinase)

42. • The drugs are prodrugs, that can only be converted into the
phosphorylated forms by the viral thymidine kinase and hence do
not interfere with DNA synthesis in non-infected cells:

43. Prodrugs with improved bioavailability

44. Inhibitors of the helicase-primase complex
(HERPES)
Lagging strand
Helicase–primase
complex (UL5, -8, -52)
Leading strand
+ TZP
UL42
ICP8
DNA
polymerase
5 52
8
•The helicase-primase complex unwinds viral DNA at the replication fork
(helicase), and initiates DNA synthesis at both the leading and the
lagging strand (primase).
•TZP enhances binding of the UL5 and UL52 subunits to the strands, resulting
in inhibition of the helicase activity by interrupting the catalytic cycle.
N
N
S
N
H
N
O
O
N
H2
Me
N
N
S
N
H
N
O
O
N
H2
N
S
N
H
NH
O
N
H2
S
B
3
0
1
S
L
I
B
S
B
5
1
1
C
I
B
BILS 179 BS
TZP derivatives
(thiazolyl-phenyl)

45. Protease inhibitors

47. HIV- protease structure with bound darunavir

48. Most drugs against HIV protease are
transition state inhibitors
N
H
N
O
O
N
H
OH
N
O
Substrate
peptidic bond
Inhibitor
hydroxyethylene bond
Saquina
vir Ritonavir Indinavir Nelfinavir Amprenavir
Atazana
vir Fosamprenavir Daruna
vir
Lopina
vir
Q (L) N (F)Y P (I) V (S)V

49. HIV protease Inhibitors
N
OH
N
N
O
N
O NH2
O
O NH
CH3
CH3
H3C
H
H
Saquinavir
hard gel capsules, Invirase ®
soft gelatin capsules, Fortovase ®
H
H
H
S
N
N
S
CH3
N N
O
N
OH
N O
O
CH3
H3C
O
H3C
CH3
Ritonavir
Norvir ®
N
N
N
OH
N
O NH
O
CH3
CH3
H3C
H
HO
Indinavir
Crixivan ®
N
OH
N
CH3 O
S
HO
O NH
CH3
CH3
H3C
H
Nelfinavir
Viracept ®
O
O
O N
H
N
OH CH3
CH3
S
O O
NH2
Amprenavir
Agenerase ®
, Prozei ®
H
NH
O
O
CH3
N
N
H
H3C CH3
N
CH3
O
O
OH
Lopinavir
combined with ritonavir at 4/1 ratio
Kaletra ®
H
H
H
H
N
O N
O
N N
N
N
O OH O
O
O
Atazanavir
Reyataz ®
O
O
O N
H
N
O CH3
CH3
S
O O
P
O
OH
HO
NH2
Fosamprenavir
Lexiva ®
, Telzir ®
O
OH
O
N
S
N
F
F
F
O O
H
Tipranavir (U-140690)
Aptivus ®
S
N
NH2
N
H
O
O
O
O
O
O
HO
Darunavir (TMC-114)
Prezista ®

50. The concept of a transition state
inhibitor
• Transition-state inhibitors are designed to mimic the transition state of an
enzymatic reaction
• They are likely to bind stronger to the active site than the natural enzyme
substrates
• The successful transition-state inhibitor will be a transition-state isostere, that
mimics the tetrahedral centre but cannot be hydrolyzed.

51. The Development of Saquinavir
• Starting from a viral polypeptide sequence that serves as a substrate to the
viral peptidase (Leu-Asn-Phe-Pro-Ile) and retaining the Phe-Pro segment
• Converting it to a
transition-state isostere:
• Adding fragments to fill the S2 and S2’ pockets
and optimizing P1 and P2
• Add fragments to fill S3 subsite
• Increase size of fragment that binds
to the S1’ subsite

52. Commercial PI Drugs
Saquinavir (Roche)
Ritonavir (Abbott)
Indinavir (Merck)
•first PI on the market
•rapid development of resistance
•low oral bioavailability
•strong binding to plasma proteins
•although larger than Saquinavir displays better bio-
availability
•potent inhibitor of cytochrom CYP3A4 and hence shuts
down its own metabolism
•good in combination with other PIs
•strong binding to plasma proteins
•was designed based on Saquinavir
•better oral bioavailability
•less binding to plasma proteins

53. Indinavir (Merck)
HIV combination
therapy (HAART)
Highly Active AntiRetroviral Therapy

54. The principle of vaccination
mimic infection with a pathogen that is attenuated and safe
thereby establishing immunological memory that could respond
in case of an actual infection
• live attenuated virus vaccines (mumps, measles)
• use of killed viruses (e.g. the original Salk killed poliovirus vaccine)
• exposure of the immune system to recombinant viral proteins alone
(hepatitis B)
• the main tool in vaccination is the use of neutralizing antibodies. These
antibodies can bind the free virus, prevent viral entry into the host cell
(“neutralization”). Antibodies can also bind to infected cells triggering
their elimination via the host effector system.
• the second defense line is the cellular immune response, particular
cytotoxic T lymphocytes (CTLs). Immunization with the corresponding T-
cell-inducing vaccine generates a population of memory T cells, that will
rapidly expand upon infection. The CTLs kill infected cells by recognizing
foreign viral proteins.

55. Vaccination
Milestones in the history of virology
Year Event
1796 Vaccination for smallpox (Jenner)
1892 First virus identified ( Tobacco mosaic virus ) (Ivanovsky)
1898 First animal virus identified, foot-and-mouth disease virus (Loeffler and Frosch)
1901 First human virus identified, Yellow fever virus (Reed)
1911 Viral aetiology for malignant sarcoma in chickens (Rous)
1915; 1917 Discovery of viruses that infect bacteria (Twort; d’Herelle)
1918 Worldwide influenza epidemic
1935 Crystallization of Tobacco mosaic virus (Stanley)
1939 First visualization of viruses in the electron microscope
1952 Introduction of cell cultures for growth of animal viruses (Enders; Robbins)
1954; 1956 Salk (inactivated) and Sabin (live attenuated) vaccines for poliomyelitis
1962–1970 Mumps, measles and rubella vaccines licensed
1969 Identification of retrovirus reverse transcriptase (Temin; Baltimore)
1977 World declared free of smallpox
1980–1990 Nucleic acid sequences of many viruses determined
1983 Identification of human immunodeficiency virus, the aetiological agent of
acquired immune deficiency syndrome (AIDS) (Montagnier; Gallo)

56. Development of Vaccines
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57. Development of influenza vaccines
• Vaccines are attenuated (or killed) viruses or viral particles
• Vaccines are usually produced by culturing the virus in chicken
eggs.
• The vaccine is made from the whole killed virus by treating the
virus with ether or detergent to produce a subunit vaccine
• Unfortunately, influenza viruses that are pathogenic in humans
are hard to grow in chicken.
• A way to increase virus yields from eggs is reassortment by mixing
the new virus with a known virus that does grow well in chicken.
The hope is that the reassorted virus contains genes from the
rapidly growing strain, and the hemaglutinin and neuraminidase
surface antigens from the new virus.
• from identifying the swine influenza virus in April 2009 to approval
of an vaccine took only 6 months
• Faster methods for producing vaccines are using live-attenuated
vaccines or vaccines produced in cell cultures

58. Producing a vaccine using reversed genetics

59. Problems for producing HIV vaccines
• the enormous sequence diversity due to a highly error-prone
reverse transcriptase makes it difficult to raise vaccines against
particular epitopes
• within days of expose to HIV viruses massive infection and loss of
memory CD4+ T cells occurs. The latter are critical for
coordinating effective immune responses
• HIV viruses have developed strategies for avoiding immune
elimination. E.g. the accessory protein NEF down-regulates
molecules of the major histocompatibility complex (MHC), that
are important for T cell recognition of infected cells. Moreover,
the surface envelope proteins incorporate multiple features to
avoid antibody recognition. The spikes on the protein surface,
composed of the gp41 TM protein and the glycosylated gp120,
exhibit enormous sequence variability. The recognition surface is
buried under the sugar chains

60. IMP dehydrogenase inhibitors
O
OH OH
HO
N
N
N
H2N
O
O
OH OH
HO
N
N
N
H2N
NH
Ribavirin Viramidine
O
OH OH
O
N
N
N
H2N
O
Ribavirin-MP
ATP
ADP
Nucleoside
kinase
P
PRA
IMP
XMP
GMP
GDP
GTP
RNA synthesis
Succinyl
AMP
AMP
ADP
ATP
IMP dehydrogenase
O
OH
HO
O
O
O
Mycophenolic acid
•IMP (inosine-5-
monophosphate)
dehydrogenase is an
essential enzyme in the
de-novo biosynthesis of
purine mononucleotides.
•Although it is a cellular
target cells infected with
viruses display an
increased need for DNA/
RNA synthesis.
•Used to treat hepatitis
infections in combination
with interferon-α.

61. S-adenosylhomocysteine (SAH) hydrolase
inhibitors
• SAH is a key enzyme for methylation reactions
that depend on S-adenosylmethionine (SAM) as
the methyl donor. Methylations are required for
the maturation of viral mRNA.
• SAH prevents accumulation of itself by auto-
hydrolysis.
• Inhibitors of SAH lead to accumulation of SAH
resulting in inhibition of the methylation reaction.
• Cellular target, but high activity in infected cells.
• Used to treat poxviruses, ebola fever
N
X
N
N
NH2
HO
HO OH
SAH hydrolase inhibitor
)
H
C
r
o
N
=
X
(

62. • interferons belong to the class of cytokines involved in cell-cell
signaling, particularly relating to the early detection of microbial
invasion
• IFNα and IFNβ are produced by most human cells in response to a
viral infection
• production of interferons is mediated through activation of toll-like
receptors
• its production is transient and requires stimulation by viruses, microbial
products and other inducers
• their action is mediated through the JAK/STAT and other signaling
pathways
• they induce expression of 2,5-oligoadenylate synthase, which in turn
produces oligoadenylate that activates ribonuclease L, resulting in
the degradation of single-stranded RNA, and triggers cellular
apoptosis
• IFNs are used to treat hepatitis B and C viral infections
Interferons

63. The influenza lifecycle and drugs

64. Inhibition of Virus Uncoating (Flu)
• Adamantane derivatives block
the migration of protons into the
interior of the virions within
endosomes, thereby preventing
the pH shift required for uncoating.
They act by blocking the M2
(matrix 2) channel.
Unfortunately, rapid resistance
against them occurs.
H
H
NH HCl
2•
H
NH HCl
2•
H
H
H
CH3
Amantadine Rimantadine
a
Ala-30
Gly-34
His-37
Trp-41

65. Viral Neuraminidase Inhibitors (Flu)
• The influenza virus binds to the target cell via interaction of its surface
glycoprotein, haemagglutinin, with the host-cell surface receptor, that
contains sialic acid. Another viral glycoprotein, neuraminidase, cleaves
off the terminal sialic acid, allowing the virus to leave the cell once the
virus has replicated:
• Inhibiting the viral Neuraminidase prevents the virus from leaving the cell
and infecting others.
Nucleus
Budding virus
Neuraminidase cleaves receptor
Neuraminidase inhibitor
Haemagglutinin
Neuraminidase
Receptor
containing
sialic acid
Release of
new virions
Host cell
Nucleus
Virion

66. Commercial Neuraminidase Inhibitors
O O
OH
HO
OH
HO
HO
H
H
N
H3C
O
O O
OH
HO
OH
HO
HO
H
H
N
F3C
O
O
OH
O
HN
O
HN NH2
H C
3
NH
OH
HO
HO
H
H C
3
O
O
CH2
CH3
H2
N
HN
O
O
H3C
H3C
OH
O
HN
H2N NH
OH
HN
O
CH3 CH3
CH3
H
OH
O
HN
H2N NH
R
Rʹ
NH
CH3
O
OH
O
HN
H2N NH
R
NH
H
CH3
O
O
OH
HN
H2N NH
N
O
O
Rʹ
R NH
CH3
H
N
H2N OH
O
H
N
F3C
O
N
O
N
H
CH3
OH
O
H
N
H3C
H3C
H
O
O
CH3
H
DANA
Zanamvir
Peramivir
RWJ-270201
Cyclopentane amide
derivative
Pyrrolidine derivative
A-192558
A-315675
Cyclopentane
derivative
Cyclopentane
derivative
FANA
Oseltamivir
(Relenza) (Tamiflu)

67. The structure of the surface protein Hemaglutinin
haemagglutinin trimer complexed
with N-acetylneuramic acid (in CPK form)
monomer unit

68. Complexation mode of α-Neu5Ac

69. Development of Neuramidase inhibitors
From the proposed catalytic mechanism (based on a X-ray
structure of the enzyme in complex to sialic acid) a transition-
state inhibitor was developed:

70. (Relenza)
Tamiflu has better bioavailability (is the ethylester prodrug, cleaved by
esterases). Can be administered orally.
neuramic acid

71. Tamiflu is a good mimic of α-Neu5Ac2en
O
HO
O
OH
NH
O
OH
HO
OH
HO
O
NH2
NH
O
O
Neu5Ac2en
Oseltamivir (free acid)
Arg224
Glu27
6

72. Influenza pandemics

73. Flu Epidemics
H1N1 H2N2 H3N?
1918
H1N1
Spanish
1957
H2N2
Asian
PB1
HA
NA
PB1
HA
1968
H3N2
Hong Kong
H3N2
1950s
H1N1
1977
H1N1
Russian
H1N1
•50’000 deaths in US
•33’000 deaths in US
•H5N1 (bird flu): originated in Hong Kong 1977, and re-appeared 2003. Highly pathogenic. Severe
respiratory infections with high mortality. (261 deaths so far)
•H1N1 (swine flu): originated from Mexico
•50’000 000 worldwide

74. Components of the H1N1 virus
•belongs to the class of influenza A viruses
•genome is composed of eight segments of single-stranded,
negative-sense RNA, that each encode 1 or 2 proteins (NP=
nucleoprotein; PB components of the polymerase complex)

75. Pathogenicity
•Human influenza viruses preferentially bind to sialic acid that is linked to galactose
by an α2,6 linkage (SAα2,6Gal)
•this is matched by corresponding carbohydrates on epithelial cells in the human
trachea
•avian viruses preferentially recognize SAα2,3Gal in the intestinal tract of waterfowl
(birds)
•differences in receptor-binding specificities are determined by the amino acids in
the HA receptor binding pocket
•HA cleavage efficiency is essential for viral activity. Low pathogenic avian viruses
and non-avian influenza viruses posses a single Arg at the cleavage site. Highly
pathogenic H5 and H7 viruses posses several basic amino acids at the HA cleavage
site.
Table 2 | Determinants of viral pathogenicity
Protein Position Pathogenicity Swine-origin H1N1 viruses Function
Low High
PB2 627 Glu Lys Glu Replicative ability in some mammals, including
humans
701 Asp Asn Asp Nuclear import kinetics affecting replicative
ability in mice
PB1-F2 66 Asn Ser Truncated (11 aa) Induction of apoptosis
HA Cleavage site Single basic aa Multiple basic aa Single basic aa HA cleavage
NS1 92 Asp Glu Asp Unknown (interferon response?)
C terminus Arg-Ser-Glu-Val,
deletion
Glu-Ser-Glu-Val 11 C-terminal aa truncated Unknown
aa, amino acid(s).

76. History of the swine flu
(2009)
Table 1 |
Date Event
Mid-February Outbreak of respiratory illness in La Gloria, Veracruz, Mexico 31
12 April Mexican public health authorities report outbreak in Veracruz to the PAHO
15 April CDC identifies S-OIVs in the specimen of a boy from San Diego, California
17 April CDC identifies S-OIVs in the specimen of a girl from Imperial, California
21 April CDC alerts doctors to a new strain of H 1N1 influenza virus
23 April The Public Health Agency of Canada identifies S-OIVs in specimens from Mexico
24 April WHO issues Disease Outbreak Notice
27 April International spread and clusters of human-to-human transmission prompt WHO to raise the pandemic alert from phase3 to 4
29 April WHO raises the pandemic alert from phase 4 to 5 (human-to-human spread in at least two countries in one WHO region)
21 May 41 countries report 11,034 cases, including 85 deaths

77. The recent Ebola Outbreak
• The Ebola virus belongs to the class of
Filoviruses
• The family consists of the genera
Marburgvirus and Ebolavirus
• Filoviruses consist of a single-stranded
linear RNA genome
• After entry into the host cell it is
transcribed to generate polyadenylated
mRNA
• the gene has the following structure:
• np (nucleoprotein), virion protein 35,
30,24,40, (vp35,vp40,vp30,vp24),
glycoprotein (gp) and polymerase (L)
NP
3′ 5′
35 40 sGP/GP 30 24 L

79. Source: WHO

80. Feldmann H. N Engl J Med 2014;371:1375-1378.
NP
VP30
VP35 VP40/VP24
GP1,2
L

81. Source: WHO

82. The development of Ebola vaccines
• hAd3-ZEBOV, developed by GlaxoSmithKline (GSK) in collaboration with
the US National Institute of Allergy and Infectious Diseases (NIAID). hAd3 is
an attenuated version of a chimpanzee adenovirus (cAd3) that has been
genetically altered so that it is unable to replicate in humans.The cAd3
vector has a DNA fragment insert that encodes the Ebola virus
glycoprotein,
• VSV-EBOV, developed by NewLink Genetics and Merck Vaccines USA in
collaboration with the Public Health Agency of Canada . Currently, Phase
II and Phase III clinical trials for VSV-EBOV are underway in Guinea and
Sierra Leone. This vaccine is based on the vesicular stomatitis virus (rabies-
like) which was genetically modified to express a surface glycoprotein of
Zaire Ebola virus
Both vaccine candidates have been shown to be safe and well tolerated
in humans.
Source: WHO/Wikipedia
These include replication-deficient adenovirus vectors, replication-competent
vesicular stomatitis (VSV) and human parainfluenza (HPIV-3) vectors, and virus-like
nanoparticle preparations.

83. The development of Ebola drugs
Favipiravir, also known as T-705 or Avigan, is an experimental antiviral drug
with activity against many RNA viruses. The mechanism of its actions is
thought to be related to the selective inhibition of viral RNA-dependent RNA
polymerase
ZMapp is an experimental biopharmaceutical drug comprising three chimeric neutralizing
monoclonal antibodies under development as a treatment for Ebola virus disease.
Components:
Amino Lipid
Structural Lipid
PEG - Lipid
Nucleic Acid
40-140 nm diameter
TKM-Ebola siRNA cocktail adjusted to
Guinea strain, IV once daily infusion over
two hours
Brincidofovir (CMX001) is an experimental antiviral drug being developed
by Chimerix of Durham, NC, for the treatment of cytomegalovirus,
adenovirus, smallpox, and ebolavirus infections. Conjugated to a lipid, the
compound is designed to release cidofovir intracellularly, allowing for
higher intracellular and lower plasma concentrations of cidofovir,
effectively increasing its activity against dsDNA viruses, as well as oral
bioavailability. Source: Wikipedia

84. source: presentation Peter Horby, Oxford, UK

85. The Hepatitis viruses
Hepatitis implies injury to liver characterized by presence of inflammatory cells
in the liver tissue.
• Hepatitis A, (formerly known as infectious hepatitis), is caused by Hepatitis A
virus, which is most commonly transmitted by the fecal-oral route via
contaminated food or drinking water. Every year, approximately 10 million
people worldwide are infected with the virus.The Hepatitis virus (HAV) is a
Picornavirus; it is non-enveloped and contains a single-stranded RNA
packaged in a protein shell.The virus spreads in conditions of poor sanitation
and overcrowding.
•Hepatitis B virus is a DNA virus. Transmission results from exposure to infectious
blood or body fluids containing blood. Several vaccines have been developed
for the prevention of hepatitis B virus infection. These rely on the use of one of
the viral envelope proteins (hepatitis B surface antigen or HBsAg).
•The hepatitis C virus (HCV) is spread by blood-to-blood contact. No vaccine
against hepatitis C is available. An estimated 150-200 million people worldwide
are infected with hepatitis C. Current treatment is a combination of pegylated
interferon alpha (brand names Pegasys and PEG-Intron) and the antiviral drug
ribavirin.