Virus Transcription, Translation and Transport

 ssDNA (Baltimore class II) must be converted to dsDNA before transcription

 The primary transcript is capped and polyadenylated. This primary transcript undergoes
splicing.
 TATA box (25-30 bp upstream of Transcription Start Site) is present in both eukaryotic
and virus genes (HIV, HBV).
 Many enhancers are cell-type specific.
 Some viruses produce their own TFs. Tissue specific TFs are required by some viruses,
which explains why some viruses are tissue-specific.
 Classes III, IV, V carry out transcription in the cytoplasm and must carry their own
enzymes.
 RT uses both DNA and RNA as the template during the process of reverse transcription.
(Functions as both RDDP and DDDP)

 In eukaryotes, the cap is a 5’-5’ linkage rather than a normal 5’-3’ linkage. A methyl
group is added to the guanosine, and in some cases to the ribose groups of 1st and 2nd
nucleotide.
 Function of cap:
- Aid mRNA transport from nucleus to cytoplasm
- Protect mRNA from degradation by exonucleases.
- Initiate translation
 (-)RNA viruses with segmented genomes (influenza) have mechanisms to snatch caps
from mRNAs. The virus protein-RNA polymerase complex has endonuclease activity
and cleaves cellular mRNAs 10-20 nucleotides from the 5’ end. This works as the
primer for viral mRNA transcription.
 Function of poly-A tail:
- Initiate translation
- Stabilize the mRNA
 Some viruses use the TATA box as the polyadenylation signal.
 Mechanism of Polyadenylation:
- RNA polymerase proceeds beyond the poly-A signal and the poly-A site.
- A protein cleaves the mRNA at the polyadenylation site.
- Poly-A tail is added by the poly-A polymerase.
- Some viruses have alternative mechanisms for polyadenylation
 Eukaryotic mRNAs are monocistronic: one open reading frame. Some large ORFs code
for polyproteins, which are then cleaved to form 2 or more functional proteins.
 Eukaryotic Initiation Factors (eIFs), a 40s ribosomal subunit, and a methionine-tRNA
bind at the 5’-cap and scan for the start codon. A poly-A binding protein circularizes the
mRNA that stimulates translation. When the start codon is reached, the 60s ribosomal
subunit binds and initiation factors are released.
 Some viruses lack 5’ cap. They have a IRES (internal ribosomal entry site), a
secondary structure to which the 40s subunit and the initiation factors bind. IRES also
helps in circularization.
 Leaky scanning. 2 different start codons. Frameshifting may occur in bicistronic mRNAs.
Elongated protein is the product.
 Nuclear localization signals and importins are necessary for transport into the nucleus:
The Rev protein of HIV-1 has both a nuclear localization signal and nuclear export signal
for translocating the RNA to which it binds.
 The bacterial mRNA has a shine- Dalgarno sequence. The ribosome has an anti-SD
sequence on the 16srRNA of the 30s subunit.
 In bacteria, the initiator methionyl tRNA is formylated.

Virus Genome Replication

 Slide 3
 Most dsDNA viruses enter the nucleus. Transcription precedes replication if viral
proteins are involved in replication and transcription factors for immediate early genes.
Poxvirus is an exception as in it is a dsDNA virus that carries replication and
transcription in the cytoplasm.
 Adenovirus encodes a protein primer that primes its own polymerase
 ssDNA viruses primarily infect bacteria and plants. They are the smallest viruses and
completely dependent on host for replication and transcription.
 ssDNA viruses self-prime by forming hairpin structures.
 dsRNA viruses are all non-enveloped and icosahedral capsids. They are segmented.
 Carry own RdRp. It transcribes +mRNA.
 Partially uncoats to release RNA strand for translation. RdRp functions only once
inside the capsid during assembly to form the complementary negative strand.
 +RNA is the most abundant type of virus (evolutionary success).
 A common characteristic is that their genome encodes a polyprotein.
 Slide 13
 dsRNA must carry RdRP with them. Since +RNA can work as mRNA directly, they must
code for the RdRP which is translated at first upon entry. +ssRNA enough for infection.
 -RNA virus do not act as mRNAs and just like dsRNA virus, must carry RdRp within
virion.
 Enveloped helical capsids, segmented or not
 Transcription happens first.
 The antigenome (+RNA) is not the same as viral mRNA which must be capped and
polyadenylated.
 At some point, a switch from transcription to replication occurs.
 Class VI viruses the first step is NOT transcription/translation. For +RNA the first step is
translation. For dsRNA and -RNA, the first step is transcription.
 Must carry RT and integrase.
 RT has
- RdDp
- DdDp (without proofreading ability)
- RNase H
 Diploid
 Genome DOES NOT serve as mRNA like other +RNA virus
 The U3, R, U5 make the LTR.
 Initiation of reverse transcription requires a primer tRNA (lysine or proline) that the virus
obtained from the previous host cell for the negative sense strand (RdRp)
 The polypurine tract (PPT) of the RNA serves as the primer for +sense DNA strand
(DdDp)
 The RNA circularizes during reverse transcription which facilitates the transfer of the
strong stop cDNA to the other end of the strand.
 Insertion requires integrase and cellular DNA repair enzymes
 U3 can be bound by host cell transcription factors.
 Class VII viruses are NOT retroviruses. Retroviruses undergo reverse transcription first;
these undergo reverse transcription at the end of cycle.
 rcDNA is converted to cccDNA inside the nucleus. The cccDNA remains as an episome.
 The cccDNAs are transcribed by host RNA polymerase II into mRNAs and pregenomes
(pgRNA). pgRNA leaves the nucleus and enters cytoplasm.
 5’-DR1………………DR2, DR1-3’
 Epsilon secondary structure serves as initial starting point of reverse transcription.
 The pgRNA and recently translated P proteins (RT) are packaged into forming capsids.
P protein forms the rcDNA.

Viruses and Cancer

 Germline mutation affects every cell of the individual and his offspring have a 50%
chance of inheriting the mutations.
 Malignant cells have a range of ploidy and may have low to high mitotic count.
 Benign tumors do NOT lose their specialization.
 Benign dysplasia  CIS  malignant
 DNA damage is the primary cause of cancer. A deficiency in DNA repair would increase
chances of cancer.
 Majority of the cancers are non-hereditary or sporadic cancers in which epigenetic
alterations reduce DNA repair gene expression.
 Field defects are normal appearing tissues with multiple mutations and epigenetic
alterations, and are common precursors to cancers.


 Cancer originates in normal, healthy stem cells.
 Most cancers are diagnosed in older people.
 Slide 13: Proof that a suspected chemical is carcinogenic.
 Transformed cells rely less on the culture medium and produce their own autocrine
growth factors known as contact inhibition. Transformed cells acquire anchorage
independence: they are able to survive without attachment to ECM.
 Oncogenic viruses: HBV, HCV, HPV, Merkel-cell associated polyoma virus, Epstein-
Barr virus, Kaposi’s Sarcoma associated herpes virus, Human-T lymphotropic virus type
I,
 Cyclin expression increases as a response to external factors such as growth factors,
nutrients, or hormones. When cyclin level reaches a certain threshold level, it activates its
CDK.
 Malignant cells acquire mutations in cyclins, CDKs, or their regulatory proteins.
 Proto-oncogenes are normal cellular genes that promote cell division. May become
oncogene by mutation, insertional mutagenesis (how viruses work), chromosomal
translocation.
 Mutations of the tumor suppressor genes are usually recessive.
 Retinoblastoma protein inhibits the transition form G1-S by inactivating E2F and
inhibiting the chromatin remodeling that makes the promoter accessible to transcription
factors.
 When cell incurs DNA damage or stress, p53 (a transcription factor) stimulates the
expression of protein p21 which binds to CDK2 and 1, inhibiting them from binding to
their cyclins.
 Telomerases are active in gametes, stem, and tumor cells.
 Growth promoting properties of viruses have oncogenic consequences.
 Oncogenic retroviruses: acute-transforming retroviruses by carrying oncogene homologs;
non-acute(slow-transforming) retroviruses work by insertional mutagenesis affecting
oncogenes or tumor suppressor genes. HTLV-1 encodes viral gene tax that encodes a
transcriptional factor that phosphorylates pRB, activates cyclins and CDKs, inhibits p53.
 HCV is an RNA tumor virus. Has a cytoplasmic life. Chronic inflammation produced
ROS, leading to DNA damage. 4/10 proteins promote oncogenic state
 Activates cyclins and CDKs, enhances telomerase, inhibits apoptosis, blocks p53 and
pRB.
 Small DNA tumor viruses contain viral origin oncogenes.
 HBV contains protein X promotes cell proliferation and inhibits p53. Insertional
mutagenesis may affect proto-oncogenes and tumor suppressor genes.
 Inhibits pRB and p53, increases expression of E2F.
 HPV is the most common STD.
 Low-risk HPVs remain as episomes whereas high-risk ones integrate into hose genome.
 High-risk HPVs can cause cervical and head-neck cancers.
 HPV inhibits p53, increases telomerase production.