РИБАВИРИН

1.The first unique step in the de novo cellular synthesis of guanine nucleotides is catalysed by the
enzyme IMPDH (Figure 2). This reaction is NAD+-dependent and converts inosine monophosphate (IMP)
to xanthosine monophosphate (XMP). XMP can then be aminated to guanosine monophosphate (GMP)
by the enzyme GMP synthase. GMP is further converted to guanine metabolites such as GTP and dGTP,
which are essential as precursors for RNA and DNA synthesis, respectively. Additionally, GTP plays
important roles in energy storage, intracellular signalling, translation by ribosomes and glycoprotein
synthesis.

Due to its structural similarity to GMP, RMP is a potent competitive inhibitor of IMPDH. RMP binds in the
active site substrate pocket of IMPDH and is an excellent mimic of the natural substrate IMP. Inhibition
of IMPDH has been suggested as a possible mechanism for the antiviral properties of ribavirin. GTP pools
have been demonstrated to be reduced approximately two-fold in ribavirintreated cells [13]. GTP and
dGTP are essential for translation, transcription and RNA and DNA replication. As such, reduction in
available GTP was hypothesised to inhibit these integral processes of the viral life cycle, thus explaining
the antiviral properties of ribavirn. Since this is a non-specific, cellular effect, IMPDH inhibition could also
explain the broad-spectrum activity of ribavirin.

2. Ribavirin has also been postulated to act via another indirect antiviral mechanism, by enhancing the
host T-cell response. This conclusion stems from observations in HCV-infected patients that ribavirin can
reduce serum alanine aminotransferase (ALT) levels (a marker of liver damage) without significantly
reducing levels of circulating HCV RNA as determined via PCR [34]. Ribavirin has been suggested to act in
combination therapy by maintaining the response to interferon treatment. Ribavirin is thought to induce
a switch in T-helper cell phenotype from type 2 to type 1 [35]. The T-helper type 1 response is
associated with cellular immunity and is associated with expression of IL-2, gamma-interferon, and
tumour necrosis factor-alpha [36,37]. The T-helper 2 response promotes humoural immunity and is
associated with expression of IL-4, IL-5 and IL10. It has been suggested that an ineffective host immune
response is an important factor in chronic infections. A T-helper 2 response has been associated with
the development of chronic disease in HCV infection [38]. Ribavirin has indeed been shown to modulate
cytokine expression in human Action of ribavirin against distinct viruses Low levels (5–10 mM) of
ribavirin inhibited a Type-2 response and promoted a Type-1 response in both CD4 +and CD8 + human T-
cells in vitro.
3. The 5’ -end of most cellular RNAs and some viral RNAs contains a 7-methylguanosine cap structure
essential for RNA stability and translation. As a guanosine nucleotide analogue, ribavirin has the
potential to interact with enzymes responsible for ‘capping’ cellular mRNAs and viral genomic RNAs.
Generally, the cap structure is synthesised via three enzymatic reactions (Figure 3): (1) an RNA
triphosphatase cleaves the 5’ triphosphate of the RNA to a diphosphate; (2) an RNA guanylyltransferase
catalyses the addition of GMP to this 5’ terminus via a unique 5’ -to-5’ linkage; (3) an RNA (guanine-7-)
methyltransferase catalyses the addition of a methyl group from S-adenosylmethionine to the terminal
guanosine at the N-7 position (reviewed in [45]).

It has been suggested that the mechanism of action of ribavirin against vaccinia virus is due to inhibition
of the capping reaction [54]. RTP was found to be a potent inhibitor of the vaccinia virus mRNA
guanylyltransferase, suggesting that ribavirin acts by producing mRNAs that are not competent for
translation. Bougie and Bisallion investigated the interaction of ribavirin with the N-terminal fragment of
the vaccinia virus D1 protein, which has both triphosphatase and guanylyltranseferase activity [55]. RTP
was found to serve as a substrate for this enzyme, and formation of a covalent RMP-enzyme complex
was demonstrated. Transfer of RMP to RNA was also shown biochemically. RNAs capped with ribavirin
were more stable to degradation than uncapped RNAs. However, the 7-methyl group found on the
normal cap structure is necessary for translation by interaction with eIF4E. The vaccinia virus capping
machinery was unable to add 7-methyl group to ribavin; thus, RNAs capped by ribavirin were not
efficiently translated. Although ribavirin was an inefficient competitor to GTP in a guanylyltransferase
assay, IMPDH inhibition by ribavirin may potentiate this effect in vivo.

4.The primary intracellular metabolite of ribavirin isRTP. It is possible that this nucleotide can
interactwith viral polymerases and inhibit nucleic acidsynthesis. Accumulation of RTP in cells may
allowefficient competition with essential GTP or ATPpools. Eriksson and coworkers demonstrated inhi-
bition of the influenza virus RNA polymerase invitro with RTP [57]. Neither ribavirin nor RMPshowed any
inhibitory activity. RTP was shownto act as a competitive inhibitor with respect toboth ATP and GTP. RTP
has also been suggestedto specifically inhibit RNA synthesis by reovirus[17]. Inhibition was apparently
independent ofthe concentration of the natural nucleotidesincluded in an in vitro assay, suggesting
inhibitionwas not by a competitive mechanism. The authorssuggested that RTP might bind at a site close
to theactive site, changing the conformation.There has also been demonstration of reducedelongation
activity by the HCV polymerase in vitrowhen ribavirin was present in the template [58,59].Polymerase
inhibition has also been noted withvesicular stomatitis virus [60,61]

5. In 2000, Crotty and colleagues were able to pro-vide strong evidence for ribavirin incorporation into
RNA during virus replication. Ribavirin was incorporated slowly, atapproximately the rate of an incorrect
nucleotide,which should result in an average of one or two molecules incorporated per 7500-nucleotide
RNAgenome. Of greater importance was the discoverythat ribavirin was templated by either uridine or
cytidine with equal efficiency. Furthermore, the presence of ribavirin in the RNA template was able to
direct incorporation of either CMP or UMP. Thus, ribavirin is capable of ambiguous base pairing,
mimicking either of the natural purines (guanosine normally base-pairs with cytidine while adenosine
normally base-pairs with thymidine [or uracil]). This ambiguous basepairing capacity is likely due to
rotation of the carboxamidemoiety of the pseudobase, generating hydrogenbond acceptor/donor sites
favourable for interaction with either of the pyrimidine bases.

The fact that ribavirin can act as an ambiguouspurine base analogue suggested that it has thepotential
to be mutagenic to RNAs into which itis incorporated. The in vitro incorporation studiessuggested that
ribavirin should induce transitionmutations (A-to-G and C-to-U).

RNA viruses have an extraordinarily high muta-tion frequency (103to 105per replication
cycle),thought to be due to lack of a polymerase proofread-ing activity [62,63]. As a result, RNA viruses
havebeen hypothesised to exist as a quasispecies, aheterogeneous population which hovers around
aconsensus or master sequence [64,65]. The genomevariability present in such a population may
bebeneficial in allowing more rapid adaptation toenvironmental changes, including tropism,
immuneresponse or antiviral therapy. However, quasispe-cies theory predicts the existence of an upper
limitto genome variability, known as the error threshold,beyond which additional mutations would be
dele-terious to the population. The term lethal mutagen-esis has been coined to describe an antiviral
strategyin which the population would be forced beyondthe error threshold
Crotty and colleagues demonstrated the existenceof an error threshold by investigating the effect
ofmutation frequency on poliovirus viability [67].Poliovirus was grown in the presence of a rangeof
ribavirin concentrations, and the purified RNAwas sequenced and assayed for infectivity. The spe-cific
infectivity of poliovirus RNA was found todecrease precipitously when the number of muta-tions per
genome was increased only modestlybeyond that of untreated viral RNA (Figure 5).Natural poliovirus
populations have an averageof approximately 1.5 mutations per genome.When this was increased to
two mutations pergenome, RNA specific infectivity was reduced to50%. A four-fold increase resulted in
only 5% gen-ome infectivity. Furthermore, the antiviral effect ofribavirin was fully explainable by only
the reduc-tion in specific infectivity and a mild decrease inRNA synthesis
ОСЕЛТАМИВИР

Infl uenza (or fl u) is an airborne, respiratory disease caused

by an RNA virus which infects the epithelial cells of the

upper respiratory tract. It is a major cause of mortality,

especially among the elderly or among patients with weak

immune systems. The nucleocapsid of the flu virus contains (−)

ssRNA and a viral enzyme called RNA polymerase
(see Fig. 20.1 ). Surrounding the nucleocapsid there is
a membranous envelope derived from host cells which
contains two viral glycoproteins called neuraminidase
(NA) and haemagglutinin (HA). Th e latter acquired
its name because it can bind virions to red blood cells
and cause haemagglutination. Th e NA and HA glycoproteins
are spike-like objects which project about
10 nm from the surface and are crucial to the infectious
process.
In order to reach the epithelial host cells of the upper
respiratory tract, the virus has to negotiate a layer of protective
mucus and it is thought that the viral protein NA
is instrumental in achieving this. Th e mucosal secretions
are rich in glycoproteins and glycolipids which bear a
terminal sugar substituent called sialic acid (also called
N -acetylneuraminic acid ). NA (also called sialidase ) is
an enzyme which cleaves sialic acid from these glycoproteins
and glycolipids ( Fig. 20.39 ), thus degrading the
mucus layer and allowing the virus to reach the surface
of epithelial cells. Once the virus reaches the epithelial cell, adsorption

takes place whereby the virus binds to cellular glycoconjugates

that are present in the host cell membrane, and

which have a terminal sialic acid moiety. Th e viral protein

HA is crucial to this process. Like NA, it recognizes

sialic acid but, instead of catalysing the cleavage of sialic

acid from the glycoconjugate, HA binds to it ( Fig. 20.40 ).

Once the virion has been adsorbed, the cell membrane

bulges inwards taking the virion with it to form a vesicle

called an endosome —a process called receptor mediated

endocytosis . Th e pH in the endosome then decreases,

causing HA in the virus envelope to undergo a dramatic

conformational change whereby the hydrophobic ends

of the protein spring outward and extend towards the

endosomal membrane. Aft er contact, fusion occurs and

the RNA nucleocapsid is released into the cytoplasm of

the host cell. Disintegration of the nucleo capsid releases

viral RNA and viral RNA polymerase, which both enter

the cell nucleus.

Viral RNA polymerase now catalyses the copying of

(−) viral RNA to produce (+) viral RNA, which departs

the nucleus and acts as the mRNA required for the

translation of viral proteins. Copies of (−) viral RNA

are also produced in the nucleus, then exported into the

cytoplasm.

Capsid proteins spontaneously self-assemble in the

cytoplasm with incorporation of (−) RNA and newly

produced RNA polymerase to form new nucleocapsids.

Meanwhile, the freshly synthesized viral proteins HA and

NA are incorporated into the membrane of the host cell.

Newly formed nucleocapsids then move to the cell membrane

and attach to the inner surface. HA and NA move

through the cell membrane to concentrate at these areas

and host cell proteins are excluded. Budding then takes

place and a new virion is released. NA aids this release by

hydrolysing any interactions that take place between HA

on the virus and sialic acid conjugates on the host cell

membrane.

Th ere are three groups of fl u virus, classifi ed as A,

B, and C. Antigenic variation does not appear to take

place with infl uenza C and occurs slowly with infl uenza

B. With infl uenza A, however, variation occurs almost

yearly. If the variation is small, it is called antigenic drift .

If it is large, it is called antigenic shift and it is this that

can lead to the more serious epidemics and pandemics.

Th ere are two infl uenza A virus subtypes which are epidemic

in humans—those with H1N1 antigens and those

with H3N2 antigens (where H and N stand for HA and

NA respectively). A major aim in designing eff ective antiviral

drugs is to fi nd a drug that will be eff ective against

the infl uenza A virus and remain eff ective despite antigenic

variations. In general, vaccination is the preferred

method of preventing fl u, but antiviral drugs have a role

in the prevention and treatment of fl u when vaccination

proves unsuccessful.
ХИВ

HIV ( Fig. 20.11 ) is an example of a group of viruses known

as the retroviruses . Th ere are two variants of HIV: HIV-1

is responsible for AIDS in the USA, Europe, and Asia,

whereas HIV-2 occurs mainly in western Africa. HIV

has been studied extensively over the last 20 years and

a vast research eff ort has resulted in a variety of antiviral

drugs which have proved successful in slowing down the

disease, but not eradicating it. At present, most clinically

useful antiviral drugs act against two targets: the viral

enzymes reverse transcriptase and protease . Th ere is a

need to develop eff ective drugs against a third target and

a good knowledge of the life cycle of HIV is essential in

identifying suitable targets ( Fig. 20.11 ).

HIV is an RNA virus which contains two identical

strands of (+) ssRNA within its capsid. Also present are

the viral enzymes reverse transcriptase and integrase ,

as well as other proteins called p6 and p7 . Th e capsid

is made up of protein units known as p24 ; surrounding

the capsid there is a layer of matrix protein ( p17 ),

then a membranous envelope which originates from host

cells and which contains the viral glycoproteins gp120

and gp41 . Both of these proteins are crucial to the processes

of adsorption and penetration. Gp41 traverses the

envelope and is bound non-covalently to gp120, which

projects from the surface. When the virus approaches

the host cell, gp120 interacts and binds with a transmembrane

protein called CD4 , which is present on host

T-cells. Th e gp120 proteins then undergo a conformational

change which allows them to bind simultaneously

to chemokine receptors ( CCR5 and CXCR4 ) on the

host cell (not shown). Further conformational changes

peel away the gp120 protein allowing the viral protein

gp41 to reach the surface of the host cell and anchor the

virus to the surface. Th e gp41 then undergoes a conformational

change and pulls the virus and the cell together

so that their membranes can fuse.

Once fusion has taken place, the HIV nucleocapsid

enters the cell. Disintegration of the protein capsid

then takes place, probably aided by the action of a viral

enzyme called protease. Viral RNA and viral enzymes are

then released into the cell cytoplasm. Th e released viral

RNA is not capable of coding directly for viral proteins

or of self-replication. Instead, it is converted into DNA

and incorporated into the host cell DNA. Th e conversion

of RNA into DNA is not a process that occurs in human

cells, so there are no host enzymes to catalyse the process.

Th erefore, HIV carries its own enzyme— reverse

transcriptase —to do this. Th is enzyme is a member of a

family of enzymes known as the DNA polymerases, but

is unusual in that it can use a RNA strand as a template.

Th e enzyme fi rst catalyses the synthesis of a DNA strand

using viral RNA as a template. Th is leads to a (+)RNA–(−)

DNA hybrid. Reverse transcriptase catalyses the degradation

of the RNA strand then uses the remaining DNA

strand as a template to catalyse the synthesis of dsDNA

( proviral DNA ). Proviral DNA is now spliced into the

host cell’s DNA—a process catalysed by the viral protein

integrase . Once the proviral DNA has been incorporated

into host DNA, it is called the provirus and can

remain dormant in host cell DNA until activated by

cellular processes. When that occurs, transcription of

the viral genes env, gag , and pol takes place to produce

viral RNA, some of which will be incorporated into new

virions, and the rest of which is used in translation to

produce three large, non-functional polyproteins, one

derived from the env gene, one from the gag gene, and

the other from the gag–pol genes. Th e fi rst of these polyproteins

is cleaved by cellular proteinases and produces

the viral glycoproteins (gp120 and gp41), which are

incorporated into the cell membrane. Th e remaining two

polypeptides ( Pr55 and Pr160 ) remain intact and move

to the inner membrane surface. Th e viral glycoproteins

in the cell membrane also concentrate in this area and

cellular proteins are excluded. Budding then takes place

to produce an immature, membrane-bound virus particle.

During the budding process a viral enzyme called

protease is released from the gag–pol polypeptide. Th is

is achieved by the protease enzyme autocatalysing the

cleavage of susceptible peptide bonds linking it to the rest

of the polypeptide. Once released, the protease enzyme

dimerizes and cleaves the remaining polypeptide chains

to release reverse transcriptase, integrase, and viral structural

proteins. Th e capsid proteins now self-assemble to

form new nucleocapsids containing viral RNA, reverse

transcriptase, and integrase.

Fusion inhibitors bind to envelope glycoprotein gp41 and prevent viral fusion to the CD4 T-cells

Chemokine receptore antagonists (CCRP5 antagonists): selectively and reversibly block entry into the
CD4 T cells by preventing interaction between CD4 cells and the gp120 subunit of the viral envelope.

ХИВ-протеаза инхибитори

As an aspartic protease, the dimerized HIV-1 PR functions through the aspartyl group complex, in order
to perform hydrolysis. Of the two Asp25 residues on the combined catalytic active site of HIV-1 PR, one
is deprotonated while the other is protonated, due to pKa differences from the micro-environment.[16]

In a general aspartic protease mechanism, once the substrate is properly bound to the active site of the
enzyme, the deprotonated Asp25 catalytic amino acid undergoes base catalysis, rendering the incoming
water molecule a better nucleophile by deprotonating it. The resulting hydroxyl ion attacks the carbonyl
carbon of the peptide bond, forming an intermediate with a transient oxyanion, which is stabilized by
the initially protonated Asp25. The oxyanion re-forms a double bond, leading to the cleavage of the
peptide bond between the two amino acids, while the initially deprotonated Asp25 undergoes acid
catalysis to donate its proton to the amino group, making the amino group a better leaving group for
complete peptide bond cleavage and returning to its original deprotonated state.[2][17]

While HIV-1 PR shares many of the same characteristics as a non-viral aspartic protease, some evidence
has shown that HIV-1 PR catalyzes hydrolysis in a concerted manner; in other words, the nucleophilic
water molecule and the protonated Asp25 simultaneously attack the scissile peptide bond during
catalysis.[17][18]
ИНТЕРФЕРОНИ