VIROLOGY ASSIGNMENT

QURAT UL AIN ROLL # 07

JANUARY 15, 2020

Question 01:
How template independent replication occur in RNA virus:

A significant number of viruses that source human, animal and plant diseases comprise
RNA as their genetic material. The genomic RNA plays both templated and non-templated
functions during the viral replication cycle. In positive-strand RNA viruses, the genomic RNA
serves as a mRNA and as template for viral RNA replication. Likewise storing genetic information
and operating as the template for RNA replication, the viral genome contains RNA determinants
which perform non-templated functions that are facilitated by distinct RNA sequences and
structures perform in the noncoding and coding regions of the viral genome. These RNA sequences
and structures function as cis-active elements that participate in both RNA-RNA and protein-RNA
interactions and are essential for viral replication. These cis-active RNA elements recruit and
assemble trans-acting viral and cellular proteins to form membrane bound viral replication
complexes that are sites of viral RNA replication.
The universal 3'-terminal CCA sequence of tRNA is built or synthesized by the CCA-
adding enzyme. cytidine triphosphate or (ATP) tRNA nucleotidyl transferase. This RNA
polymerase has no nucleic acid template, but faithfully synthesizes the defined CCA sequence on
the 3'-terminus of t RNA at one time, using cytidine triphosphate and ATP as substrates. The
mystery of CCA-addition without a nucleic acid template by unique RNA polymerases has long
fascinated researchers in the field of RNA enzymology. the mechanisms of RNA polymerization
by the remarkable CCA-adding enzyme and its related enzymes are presented, based on their
structural features.
A). A schematic diagram showing the RNA structures in the poliovirus RNA genome
which perform non-templated RNA functions during viral replication. The 5’ cloverleaf (5’CL)
and the internal ribosome entry site (IRES) are present in the 5’ non-translated region (NTR) of
the genome. The open reading frame in the viral genome encodes a polyprotein which is cleaved
to generate the structural and replication proteins. The cre hairpin is present in the 2C coding
region and the 3’ NTR and poly(A) tail is at 3’ end of the genome.
(B). The secondary structure and nucleotide sequence of the 5’CL showing the binding
sites for the cellular poly(C) binding protein (PCBP) and viral protein, 3CD.

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Question 02:
How viruses obtain long terminal repeats?

Retroviruses (which have an RNA genome) face a problem that they copy their RNA to
DNA using a virus-specific polymerase called reverse transcriptase. But when new virus is made,
the DNA genome must be copied back to RNA using a host enzyme, RNA polymerase II. This
enzyme is used by the host cell to make messenger RNA and when mRNA is made, some of the
DNA gene is not copied to RNA (since that part of the genetic information is not required in coding
for a protein).

The non-copied parts include the control elements of the gene such as promotors,
enhancers, stop signals etc. Thus, RNA polymerase II is ill-matched to the exact copying of a
genome but the retrovirus has no other choice and must overcome this limitation. It does this in
the following way. The RNA form of the genome of a retrovirus consists of a unique sequence that
contains the three structural (protein coding) genes, gag, pol and env (black in the diagram below).

These genes code for the internal antigens (gag), the enveloped glycoproteins (env) and the
enzymes needed for viral replication and maturation (pol). Bordering this region are two unique

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sequences, U5 (blue), at the 5 prime end and U3 (purple) at the 3-prime end of the RNA. These do
not encode a protein. They encode controlling elements that are used later in the copying of the
DNA back to RNA. At the ends of the RNA form of the genome are two sequences that are very
similar. These are the repeat (R) sequences and are shown in red

In fact, things are a little more complex than this because of two additional regions at the U5/gag
and the env/U3 interface. These are the primer binding site (below in green) and the poly purine
tract (brown).

THE DNA PROVIRUS FORM FOUND IN THE HOST CELL

The DNA form of the retrovirus genome is larger than the RNA form and has an extra
sequence duplicated at each end. Sequencing has shown that the extra sequence at each end are
duplications of internal sequences in the RNA form of the virus. The U3 region (purple), an internal
region in the RNA form, has been duplicated and lies at the opposite end of the DNA strand. The
U5 region has also been duplicated and lies at the other end of the DNA strand. The DNA is, of
course, double stranded.

So, two internal regions of the RNA form of the genome are copied during reverse transcription
and come to lie elsewhere in the DNA genome. How does this happen?

1.All DNA polymerases require a primer and reverse transcriptase is no exception. The primer is
a transfer RNA (tRNA) of the host cell that is packaged into the virus particle. It does not bind to

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the end of the nucleic acid (in this case RNA) to be copied but, instead, it binds at the primer
binding site (green). Reverse transcriptase copies the RNA into DNA as far as the end of the RNA.

2. Now we have double-stranded nucleic acid at one end and an enzyme called RNase H can
degrade RNA that is in a double stranded form. RNase H now removes the RNA of the double
strand DNA/RNA hybrid

3. First jump: The new piece of DNA (which is no longer hybridized to a long strand of RNA),
together with the primer, now jumps to the other end of the RNA where the repeat (R) sequences
hybridize.

4. Reverse transcriptase now copies the remainder of the RNA to the far end

5. Again we have a lot of double-strand RNA/DNA hybrid and the remainder of the RNA in the
hybrid is digested by RNase H except for the poly purine tract (brown) that remains to act as a new
RNA primer for reverse transcriptase. The second strand is now extended left to right from the
poly purine RNA primer

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6. The RNA primers are now removed by RNase H.

7. Next the above structure forms a circle since the primer binding sites (green) can hybridize

8. Using the PBS region (green) as a primer, the reverse transcriptase now copies a second strand
of DNA

9. Now the reverse transcriptase displaces one of the DNA strands and copies further. At this stage
one of the long terminal repeats is complete

10. Second jump: The reverse transcriptase jumps to the other stand and completes it so that now
both long terminal repeats are complete.

Question 03:
Summary of HBV genome replication?
Hepadnaviruses, including human hepatitis B virus (HBV), replicate through reverse
transcription of an RNA intermediate, the pre genomic RNA (pgRNA). There are fundamental
differences beyond the fact that hepadnavirions contain DNA instead of RNA. Most peculiar is
the initiation of reverse transcription: it occurs by protein-priming, is strictly committed to using
an RNA hairpin on the pg RNA, ε, as template, and depends on cellular chaperones; moreover,
proper replication can apparently occur only in the specialized environment of intact
nucleocapsids. This complexity has hampered an in-depth mechanistic understanding.
[1]. Attachment: virus binds to the receptor on cell and gains entry through clathrin or
caveolin-1 mediated endocytosis. Receptors are sodium taurocolate co transporting
polypeptide and these receptors are mainly present in the membrane of liver cells
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 [2]. Penetration: It occurs through membrane fusion and nucleocapsid is released in
cytoplasm.
[3]. Uncoating: Capsid is transported on microtubules to nuclear pore. The core proteins
dissociate from the partially double stranded viral DNA, which is then made fully double
stranded DNA (by host DNA polymerases) and transformed into covalently closed circular
DNA that serves as a template for transcription of four viral mRNAs.
[4]. Replication: The largest mRNA, (which is longer than the viral genome), is used to make
the new copies of the genome and to make the capsid core protein and the viral RNA-
dependent-DNA polymerase.
[5]. Assembly: These four viral transcripts undergo additional processing and go on to form
progeny virions which are released from the cell or returned to the nucleus and re-cycled
to produce even more copies.
Release: The long mRNA is then transported back to the cytoplasm where the virion P
protein synthesizes DNA via its reverse transcriptase activity