The document summarizes the replication process of DNA and viruses. It describes how DNA replicates semi-conservatively to produce two identical copies. Viruses like bacteriophage T4 and plant virus TMV are then discussed, with T4 exhibiting a lytic cycle where it hijacks the host cell to produce new virus particles that ultimately burst the cell, while TMV has an RNA genome and protein coat. The stages of viral replication including attachment, penetration, replication of the genome, and assembly of new virus particles are also outlined.

1. Replication (general account), DNA virus (T4-phage), Lytic and
Lysogenic cycle; RNA virus (TMV)
By
N. Sannigrahi, Associate Professor
Department of Botany
Nistarini College, Purulia (W.B) India

2.  Life is the outcome of millions of biochemical reactions and
the centre of the reactions are contributed by the genetic
material. Genetic material is always nucleic acid and it is always
DNA except some viruses(RNA or Both). DNA is the storehouse of
genetic information. This information is in the form of nucleotide
sequences called genetic code. This information is copied and
transcribed into RNA molecules. This information (genetic code) is
for specific sequence of amino acids. The non-genetic RNA then
synthesizes proteins, which are specific sequence of amino acids in
Ribosomes, by a process called translation. In 1956 Francis Crick
called this pathway of flow of genetic information as the Central
Dogma. Both transcription and translation are unidirectional.
Proteins never serve as template for RNA synthesis. But sometimes
RNA acts as a template for DNA synthesis (reverse transcription),
Example is RNA viruses (HIV virus).

5.  Genetic information present in double stranded DNA
molecule(most eukariotes) is transmitted from one cell to
another cell at the time of mitosis and from parent to progeny
by faithful replication of parental DNA molecules. DNA
molecule is coiled and twisted and has enormous size. This
imposes several restrictions on DNA replication. DNA
molecule must be uncoiled and the two strands must be
separated for the replication process. All genetically relevant
information of any DNA molecule is present in its sequence of
bases on two strands. Therefore, the main role of replication is
to duplicate the base sequence of parent DNA molecule. The
two strands have complementary base pairing. Adenine of one
strand pairs with thymine of the opposite strand and guanine
pairs with cytosine. This specific complementary base pairing
provides the mechanism for the replication.

7.  The two strands uncoil(Helicase) and permanently separate
from each other. Each strand functions as a template for the
new complementary daughter strand. The base sequence of
parent or old strand directs the base sequence of new or
daughter strand. If there is adenine(A) in the parent or old
strand, complementary thymine(T) will be added to the new
strand. Similarly, if there is cytosine(C) in the parent strand,
complementary guanine(G) will be copied into the new
daughter strand. Maintenance of integrity of genetic
information is the main feature of replication. The semi-
conservative mode of DNA replication with enzyme mediated
ensures the faithful multiplication of the genetic material and
this enables the multicellular organisms to grow in size along
with to enjoy the healing basis to overcome any sorts of
emergency.

8.  DNA replication is semi conservative &It is bidirectional
process with multi-enzyme controlled process,
 It proceed from a specific point called origin
 It proceed in 5’-3’ direction
 It occur with high degree of fidelity
 DNA replication occurs by mostly three steps
 1. Initiation: Initiation complex formation
 Closed complex formation
 Open complex formation
 2. Elongation: Leading strand synthesis, Lagging strand
synthesis
 3. Termination

9.  The first step in DNA replication is to ‘unzip’ the double helix
structure of the DNA molecule.
 This is carried out by an enzyme? called helicase which breaks
the hydrogen bonds? holding the complementary? bases? of
DNA together (A with T, C with G).
 The separation of the two single strands of DNA creates a ‘Y’
shape called a replication ‘fork’. The two separated strands
will act as templates for making the new strands of DNA.
 One of the strands is oriented in the 3’ to 5’ direction (towards
the replication fork), this is the leading strand?. The other
strand is oriented in the 5’ to 3’ direction (away from the
replication fork), this is the lagging strand?. As a result of their
different orientations, the two strands are replicated
differently:

11.  LEADING: A short piece of RNA ?called a primer? (produced
by an enzyme called primase) comes along and binds to the
end of the leading strand. The primer acts as the starting point
for DNA synthesis. DNA polymerase? binds to the leading
strand and then ‘walks’ along it, adding new complementary?
nucleotide? bases (A, C, G and T) to the strand of DNA in the
5’ to 3’ direction. This sort of replication is called continuous.
 LAGGING: Numerous RNA primers are made by the primase
enzyme and bind at various points along the lagging strand.
Chunks of DNA, called Okazaki fragments, are then added to
the lagging strand also in the 5’ to 3’ direction. This type of
replication is called discontinuous as the Okazaki fragments
will need to be joined up later in presence of Ligase.

12.  Once all of the bases are matched up (A with T, C with G), an
enzyme called exonuclease strips away the primer(s). The gaps
where the primer(s) were are then filled by yet more
complementary nucleotides.
 The new strand is proofread to make sure there are no
mistakes in the new DNA sequence.
 Finally, an enzyme called DNA ligase seals up the sequence of
DNA into two continuous double strands.
 The result of DNA replication is two DNA molecules
consisting of one new and one old chain of nucleotides. This is
why DNA replication is described as semi-conservative, half
of the chain is part of the original DNA molecule, half is brand
new.
 Following replication the new DNA automatically winds
up into a double helix.

14.  Bacteriophage T4 is the most well-studied member of
Myoviridae, the most complex family of tailed phages. T4
assembly is divided into three independent pathways: the
head, the tail and the long tail fibers. The prolate head
encapsidates a 172 kbp concatemeric dsDNA genome. The
925 Å-long tail is surrounded by the contractile sheath and
ends with a hexagonal baseplate. Six long tail fibers are
attached to the baseplate’s periphery and are the host cell’s
recognition sensors. The sheath and the baseplate undergo
large conformational changes during infection. X-ray
crystallography and cryo-electron microscopy have provided
structural information on protein–protein and protein–nucleic
acid interactions that regulate conformational changes during
assembly and infection of Escherichia coli cells.

16.  Bacteriophage T4 is a well-studied, obligately lytic phage of
E. coli. It is a member of the family Myoviridae, meaning that
it has a contractile tail as well as a large icosahedral head.
Phage T4 has a relatively large dsDNA genome of
approximately 170 000 bases, including modified bases
(glycosylated hydroxymethylcytosines) that allow the phage
genome to resist digestion by numerous bacterial restriction
endonucleases. As one of the best studied of phage types, the
biology of phage T4, like that of phage λ, may be considered
archetypical. In studies of phage evolution, phage T4 along
with phage T2 and T6 form the original members of the T-
even family of bacteriophage, which now includes many
members that infect many other species of bacteria besides E.
coli.

17.  The phage consists of a long helical tail which is connected to
the head with a connector having a collar with attached
whiskers. Around the tail, fibres remain folded and held at
midpoint by the whiskers. Size of the tail is 80 x 18 nm.
 It consists of an inner tubular core (having a hole of 25 A)
which is surrounded by a contractile sheath. Protein subunits
(144) arranged in 24 rings each containing 6 subunits,
constitute the sheath. The sheath connects the head at one end
and base plate at the other end.
 From the head at distal end, there is a hexagonal base plate
attached to an end of tail. The base plate contains six spikes or
tail fibers at its six corners. The spikes are 130 x 2 nm in size.
The spikes have two parts, the proximal half fibre, and the
distal half fibre. The former is attached to the base plate and

18.  the later helps in recognition of specific receptor sites present
on cell surface of the bacterial cell wall. A dsDNA molecule of
about 50 µm is tightly packed inside the head. The DNA is
about 1,000 times longer than the phage itself. It is circular
and terminally redundant. Unlike other DNA, all the T-even
phages contain 5-hydroxy-methyl-cytosine instead of cytosine,
so that synthesis of phage can occur easily.
 The replication of T4 genome acts as unit and then several
genomic units are combined end to end to form a long DNA
molecule called concatemer as told earlier .When the T4 DNA
is packaged into capsids, the concatemer is not cut at a specific
sequence but instead, linear segments of DNA just long
enough to fill a phage head are generated. This is called
headful packaging and is common among bacteriophages.

20.  TMV is a simple rod-shaped helical virus. consisting of
centrally located single- stranded RNA (5.6%) enveloped by a
protein coat (94.4%). The rod is considered to be 3,000 Å in
length and about 180 Å in diameter.
 The protein coat is technically called ‘Capsid’. R. Franklin
estimated 2,130 sub-units, namely, capsomeres in a complete
helical rod and 49 capsomeres on every three turns of the
helix; thus there would be about 130 turns per rod of TMV.
 The diameter of RNA helix is about 80 Å and the RNA
molecule lies about 50 Å inward from the outer-most surface
of the rod. The central core of the rod is about 40 Å in
diameter. Each capsomere is a grape like structure containing
about 158 amino acids and having a molecular weight of
17,000 Dalton as determined by Knight.

21.  The ssRNA is little more in length (about 3300 Å) slightly
protruding from one end of the rod. The RNA molecule
consists of about 7300 nucleotides; the molecular weight of
the RNA molecule being about 25,000 Dalton.
 The genomic RNA has the information to produce 3 important
polypeptides
 i. First one is 126. 183 Da and is an RNA dependent RNA
polymerase enzyme,
 Ii. The second one is 299 k Da product that acts as a
transporter protein which facilitates the movement of the viral
particles across the cell through the Plasmodesmata and
 Iii. The third one is 17.6 k Da product is the Capsid protein
which is located at the 3’ end of the genome.

22.  A virus must undergo the process of replication to create new,
infectious virions that are able to infect other cells of the body
or subsequent hosts. After gaining entry into the body, a virus
makes physical contact with and crosses the plasma membrane
of a target cell. Inside, it releases and replicates its genome
while facilitating the manufacture of its proteins by host
ribosomes. Virus particles are assembled from these newly
synthesized biological molecules and become infectious
virions. Finally, the virions are released from the cell to
continue the process of infection.
 The seven stages of virus replication are categorized as
follows:
 Attachment,Penetration,Uncoating,Replication,Assembly,Mat
urationRelease

24.  LYTIC CYCLE:
 It is exhibited by virulent phage which multiply in bacteria and
kill the cell by lysis. Soon after the entry of the viral genome,
the phage becomes eclipsed and no infectious particles are
found either inside or outside the host cell. This continues till
the matured phages are released-called eclipse period. The
phage nucleic acid after entering the bacterial cell takes over
the host cell biosynthetic machinery, utilizes the host enzymes,
ribosome etc for the synthesis of mRNA and proteins. The
entire process is done by three phases-
 Early phase-T4 virus changes the sigma factor of the host by
producing an anti stigma factor to hide the host promoters,
 Middle phase-Synthesis of nucleic acid
 Late phase-Structural proteins, heads, tails are produced.

25.  a) Adsorption: The interaction between the phage specific
organelle — the tail and the receptor site of the host cell is
called the adsorption. The adsorption is facilitated by the
negatively charged carboxyl groups on the host surface and
the positively charged amino-group of protein present at the
tip of the phage tail. The nature of the receptors may be
lipoproteins Pilli etc on the bacterial cell. In T-even phages,
the tip of the tail fibre first attaches to the cell surface. The tail
fibre then bends and allows the tail pins to attach on the host
surface that makes an irreversible attachment.
 Penetration: After adsorption, the phage particle secretes an
enzyme which hydrolyses the murin complex of the host cell
wall and forms a pore. The sheath of the tail then contracts and
pushes the central tubular part, i.e., core of the tail, into the

26.  UNCOATING: host wall, like an injection needle. The
nucleic acid of the phage then passes through the core and
enters the host bacterium. The empty protein shell of the phage
is called ghost, which may remain attached even after release
of nucleic acid. Once the bacterial cell receives the nucleic
acid of a phage, it becomes resistant to the other phages.
 Once the phage nucleic acid takes the entry inside the bacterial
cell, it suppresses the synthesis of bacterial protein and directs
to synthesize the proteins of the phage particle .
 REPLICATION OF VIRAL GENOME: The DNA of phage
replicates following the semi-conservative process. Majority
of the DNA acts as a template for its own synthesis and the
rest is used as template for the synthesis of viral specific m-
RNA by utilizing the DNA dependent RNA-polymerase of the
host.

28.  The replication of DNA in the virus takes place by three
methods:
 1. Bidirectional replication from a circular DNA either by
theta form (Papilloma virus) or via rolling circle mechanism
results in the concatemeric (head to tail) viral genome.
 2. Replication for a linear DNA: Synthesis of new DNA
strands is not simultaneous rather it occurs sequentially i.e
the first one strand is made entirely and then the next strand is
made ( Adeno virus).
 3. Replication via RNA intermediate-seen in Hepatitis B virus
, contain partially dsDNA genome which is converted into an
RNA form by virion enzyme reverse transcriptase.

29.  The newly formed m-RNA directs the host cell to synthesize
the proteins which are used to build up the protein coat of the
phage particle. Almost at the end of replication of phage
nucleic acid, a protein, the phage lysozyme, is synthesized.
 ASSEMBLY: The new phage particles are formed by the
assemblage of nucleic acid and protein. This process is called
maturation, which is controlled by viral genome . In this
process, initially the condensation of nucleic acid molecule
takes place.
 MATURATION: The protein sub-units then aggregate around
the nucleic acid molecule and form the head of the phage. By
this time the tail formation starts. Initially the core tube is
attached with the basal plate and then sheath becomes
assembled around the core tube. In this stage, the tail becomes
attached to the base of

30.  the head taking a collar in between. At last, the tail fibres are
attached to the basal plate.
 LYSIS: In a cycle of phage development, about 200 phages
are formed which take about 30-90 minutes. In the host cell,
the phage DNA secretes lysozyme (an enzyme) which causes
the lysis of host cell wall. As a result of lysis the phage
particles are liberated .
 During this process, initially the λ-phage gets attached to the
bacterium with the help of tail fibre. The λ-phage then injects
its DNA thread into the host bacterium (E. coli K12). After
entry, the dsDNA thread is converted into a circular DNA as
described earlier.

31.  In lambda phage(non- virulent bacteriophage), another kind of
multiplication of virus takes place where the host cell is not
lyses quickly, virus continues to live and reproduce as usual.
The viral genome gets integrated with the bacteria genome and
is transmitted to the daughter cells at each subsequent cell
division of the bacterial host. The integrated host is known as
prophage.
 The phage can revert to normal lytic cycle and kill the host
cell.
 The cell which harbors the prophage is known as lysogen and
the cycle is called lysogeny.
 It occurs on the following steps:
 Circularization of Phage DNA: lambda dsDNA has single
stranded region at 5’ end. These single stranded ends are
complementary and first form a circular molecular ligation of
these cohesive ends.

32.  B. Size specific recombination: Then the size specific circular
phage DNA recombines with the host DNA at a particular site
of the host chromosome. This is mediated by the phage coded
enzymes. This results in the complete integration of the phage
genome with the host genome and the phage genome in this
stage is called prophage. The attachment site of the bacterial
site is called ‘att’ that of lambda is called ‘alt’. The sequence
of the baterial att site is called alt B1 between the gal and bio
operons and consists B-o-b’, whereas the complementary
sequence in the circular phage genome is called as att P and
consists of the parts P-O-B’ and the phage genome now
becomes the part of the host genome.
 C. Maintenance of Lysogeny: The phage genome is
represented by a repressor called Cl which binds to a particular
site of the phage DNA. This shuts off the transcription of most

34.  Phage genes except this repressor and the phage genome does
not function. Each phage repressor represses only its own
genome, not other genomes. There is another repressor called
‘Cro’ which is opposite to ‘Cl’. The ‘Cro’ protein suppresses
Cl and the lysogeny will not establish or continue. The
concentration of both ‘Cl’ and ‘Cro’ determines the
maintenance of lysogeny.
 TERMINATION OF LYSOGENY:
 The lysogeny state is not permanent. The phage can revert to
lyric depending on the environmental condition. Conditions
such as exposure to UV or ionizing radiations , desiccation,
presence of mutagenic agents etc terminate the lysogenic state
and the process is called induction. Adverse conditions lead to
the production of protease (called rac A protein) which
destroys Cl repressors which lead to expression of the phage
genome and reversal of lysogeny.

35.  Later on, the phage genome now by the help of host
machinery produces its viral protein, replicates its genome,
and follow the similar processes like assembly, maturation and
lyses.
 SIGNIFICANCE: Lysogeny is genetically significant
because it helps in genetic recombination in bacteria by the
process of transduction. While reverting to lytic phage from
the lysogeny state, the viral genome brings some genes of
bacteria in exchange of few of its genes which multiplies in
the bacterial cell to form more lambda phages. When these
lambda phages are released, they infect the other bacterial
cells and by some process introduce its genes to the host cells.
 Lysogenic bacteria are resistant to super infection by the same
or related phages which is called as super infection immunity.

36.  Viruses infect all organisms, including Bacteria and Archaea,
and , collectively viruses represent the greatest repository of
the genetic diversity on the planet. Many bacterial viruses and
archaeal viruses have been isolated and characterized thus far.
These include both DNA and RNA phages, some with single
stranded and others with double- stranded genomes. For
Archaea, however, no RNA viruses are known. The
metagenomics approach to viral diversity revealed the RNA
archaeal viruses indeed exist. Hope, this presentation will help
you to explore viral diversity from genomic perspective along
with their dual nature of multiplication mode. Thus, virus
being biologist puzzle is a great relics of lineages of the
diverse life forms in this blue planet.

38.  Google for images
 Different websites for information
 Brock Biology for Microorganisms
 A text book of Microbiology- Chakraborty
 Microbiology- Pelzer, Chan & Krieg
 Microbiology & Phycology- Das & Mishra
 A textbook of Botany- Hait, Bhattacharya & Ghosh
 A textbook of Microbiology-Dubey & Maheswari
 **** This PPT has been prepared for UG students without any
financial interest, to be treated as open source of learning.