THE REPLICATION OF VIRUSESVirology Lecture 2

Three lectures dealing with (1) replication of DNA viruses (2) the culture, growth and recognition of virus infections (3) the replication of RNA viruses

A growth curve for a virus in cell culture, based upon

the simultaneous infection of all cells in the culture

THE MODE OF REPLICATION OF VIRUSES -- WITH

PARTICULAR REFERENCE TO DNA VIRUSES There are a number of sequential steps in replication of

DNA viruses

1. Attachment or adsorption2. Penetration 3. Uncoating of virus and transport of genome to site of replication4. Early transcription (mRNA)5. Early translation and early proteins6. Viral DNA synthesis7. Late transcription of further mRNA8. Late translation and synthesis of structural proteins9. Assembly10.Release

These sequential steps in the replication of DNA

viruses can be considered in groups

1. Attachment or adsorption2. Penetration 3. Uncoating of virus .. transport of genome to site of replication4. Early transcription (mRNA)5. Early translation and early proteins6. Viral DNA synthesis7. Late transcription of further mRNA8. Late translation and synthesis of structural proteins9. Assembly10.Release

….. the red blood cells agglutinate (haemagglutination)

If the virus attaches to red blood cells ....and not all viruses will attach to red blood cells

Step 2. Penetration

• Fusion of the envelope of the virus with the plasma membrane of the cell and the direct release of the nucleocapsid into the cytoplasm. The fusion process is mediated by specific proteins or glycoproteins

OR

• Engulfment of the virus by the cell in a specific phagocytic process (adsorptive endocytosis) before the viral nucleic acid (genome) is released.

Step 3. Uncoating

• This is the general term applied to events after penetration which free the viral genome.

– It can occur in the cytoplasm, or in the cases of viruses that replicate in the nucleus (and this includes most DNA viruses) it occurs in the nucleus following transport of the virion through the cytoplasm to the nucleus.

• Different viruses have evolved different strategies.

– With poxviruses, which replicate in the cytoplasm, host factors induce the disruption of the virus.

– In herpesviruses, after fusion and deposition of the viral nucleocapsid ….in the cytoplasm of the cell …. nucleocapsid migrates to the cell nucleus along the microtubules ….to the nuclear pore, and … is released into the nucleus.

– With papillomaviruses, the virus is transported to the nucleus in vesicles

– Circoviruses gain entry to the nucleus during mitosis

• Next few slides demonstrate the initial stages in the replication of HIV

– attachment – penetration – uncoating and release of virus components into

the nucleus

CD4 antigen

Co-receptor

CD4+ cell

HIV binds to CD4-positive cells

HIV Gp120 (SU)

CD4 antigen

Co-receptor

gp41

(TM)

T4 cell

SU binds to CD4and SU changes conformation

CD4 antigen

Co-

receptor

Modified SU then binds to co-receptor

CD4 antigen

Co-

receptor

CD4 antigen

Co-

receptor

TM undergoes conformational change bringing membranes close together

TM is exposed and embeds in the membrane of the CD4 cell

HIV membrane and cell membrane begin to fuse

Membranes fuse

Nucleocapsid enters cytoplasm

Nuclear membrane

Cell

membrane

Nuclear

pore

Nucleocapsid moves toward nucleus

Nuclear membrane

Interacts with nuclear protein (nuclear pore?)

Reverse transcriptase

protease

integrase

RNA

Surface membrane

Nuclear membrane

tRNA primer

Components enter the nucleus

Steps 4 and 5. Early transcription and translation of viral proteins

The next step is the formation of a mRNA from the virus DNA (transcription) and formation of early viral‑coded proteins (translation).

DNA‑dependent RNA‑polymerase (cell origin, except poxviruses) Viral DNA Viral mRNA Nucleotide pool (ATP,TTP,CTP,GTP)

using cell ribosomes

Viral-coded proteins

What are the early coded proteins? Enzymes necessary for the next step in the process --DNA synthesis (eg. thymidine kinase and a DNA-dependent DNA polymerase) .

Where in the cell does this occur?

With most DNA viruses (but not poxviruses, which replicate in the cytoplasm), the transcriptional events occur in the nucleus.

From there mRNA is transported to the cytoplasm where translation of the coded proteins occurs.

The proteins are then transported back to the nucleus where they are needed before further steps in the replication process can proceed.

Uncoating and release of virus genome in nucleus

Transcription of mRNA from DNA

Translation of mRNA and production of early virus-coded proteins

Some of these virus-coded proteins re-enter the nucleus where they are needed for DNA replication, eg. The virus encodedDNA-dependent DNA polymerase

Step 6. Viral DNA synthesis (DNA replication)

Nucleotide pool in cell

Viral DNA Progeny viral

DNA DNA-dependent DNA polymerase

Progeny virus DNA

Steps 7 and 8. Late transcription and translation of proteins

• Late transcriptional events occur only after DNA synthesis has begun and does not occur if DNA synthesis is inhibited [by inhibitors such as IUDR]

• The late mRNAs which are transcribed are again transported to the cytoplasm and code for structural proteins which are later assembled to form the progeny virus particles.

Some of the structural proteins formed may be glycosylated on the trans-Golgi network before they are transported back to the nucleus, where assembly of the proteins into viral particles occurs.

Late transcription of mRNA

Late translation of virus proteins

Step 9. Assembly of viral particles

• After synthesis of viral nucleic acid and protein these are packaged together to form progeny virus.

• In assembly, the various proteins are assembled in a defined sequence. The inner `core' proteins are the first to associate with DNA, and the capsomers are laid down last.

• With the poxviruses and the iridoviruses ‑ which replicate in the cytoplasm of the cell ‑ progeny DNA and protein accumulate in a common cytoplasmic factory (inclusion) and there is little problem in assembly when the concentration of the two reach an adequate level.

• Other DNA viruses are assembled in the nucleus ‑ assembly occurs after structural proteins (synthesised in the cytoplasm) have migrated back to the nucleus where DNA replication and transcription have occurred.

Assembly

Progeny virus DNA

Structural virus proteins

Step 9. Assembly of viral particles

• After synthesis of viral nucleic acid and protein these are packaged together to form progeny virus.

• Most DNA viruses are assembled in the nucleus ‑ assembly occurs after structural proteins (synthesised in the cytoplasm) have migrated back to the nucleus where DNA replication and transcription have occurred.

• • Poxviruses replicate in the cytoplasm of the cell ‑ progeny DNA

and protein accumulate in a common cytoplasmic factory (inclusion) and assemble when the concentration of the two reach an adequate level.

Step 10. Release of the virus particles from the cell

• By autolysis of the cell - this is the method used by most DNA viruses

– Virus-induced programmed cell death (apoptosis) may be involved

• Herpesviruses mature by a more complex process during which the viruses acquire an envelope

Primary envelopement of capsids by budding at the inner leaflet of the nuclear membrane and translocation of capsids into the cytoplasm after loss of the primary envelope by fusion with the outer leaflet of the nuclear membrane

Tegumentation occurs in the cytoplasm

Capsids obtain final envelope by budding into vesicles of the trans-Golgi network

Final endocytosis from the plasma membrane and directional budding of virions results in release of virus from the cell.

Glycosylation of envelopeproteins and insertion into membrane of vesicles of the Golgi network