1. VIRAL GENE EXPRESSION
DR.SOBIA MANZOOR
MV LECTURE 04

2. Events of Viral Life Cycle
Attachment
Uncoating
Penetration
Gene expression
Replication/Translation
Assembly
Egress

3. Virus Entry and Uncoating
Viral particles ; single mission:
1:Transport the viral genome from an infected host cell to a non-infected host cell
2: Delivering it into the cytoplasm or the nucleus in a replication-competent form. The target can be a neighboring cell or a cell in another organism.
3: The process starts in an infected cell with the packaging of the viral genome and accessory proteins into a new virus particle, and then releasing the particle into the extracellular space.

4. Virus contacts the surface of a new host cell, a complex series of events ensues, which is tightly coordinated in time and space. It involves
1: Binding to receptors and attachments factors
2: Endocytosis, intracellular processing
3: Vesicular trafficking, membrane penetration, cytosolic transport, and nuclear import.

5. THE ATTACHMENT
The first step: Contact of virus and the cell.
It is the attachment of the virus surface on the cell surface.
This attachment is made with the help of the capsid proteins for naked viruses and by the glycoproteins of envelope for the enveloped virus.
These proteins or glycoproteins attached to receptors located on the cytoplasmic membrane of the host cell

6. Polioviruses can infect man and experimentally, the monkeys, but not the birds, nor the chickens because the poliovirus cannot find receivers for their attachment on the cells of primates and not on the cells of chicken.
The human immunodeficiency virus (HIV) infects mainly T lymphocytes CD4 because their envelope can attach on the molecule CD4, specific receiver of these viruses. The structure of attachment of the HIV is the surface glycoprotein of the envelope, the gp120 (glycoprotein of daltons, 120 kDa of molecular weight).
Influenza viruses infect primarily the cells of the respiratory track because their envelope is fixed on the sialic acid receptor (receptors present on the surface of these cells)

7. Viruses with different family and specific receptors that aid in their attachment and entry

8. VIRUS RECEPTORS
Virus receptors: cell surface molecules that bind the incoming viruses to the cell, and, in addition promote entry by
1: Inducing conformational changes in the virus that lead to priming association with other receptors, membrane fusion, and penetration
2: Transmitting signals through the plasma membrane that lead to virus uptake or penetration, and prepare the cell for the invasion; or
guiding bound virus particles into a variety of endocytic pathways.

9. Viral Proteins Responsible for Attachment
In enveloped viruses:
it is the spike glycoproteins that are responsible for binding.
these are oligomeric, type 1 integral membrane proteins that have the bulk of their mass outside the membrane with the receptor-binding domain prominently exposed. Other domains in the same protein may be responsible for membrane fusion.

10. X-ray crystal structures of spike glycoprotein receptor complexes exist for several enveloped viruses.
influenza HA with bound sialic acid
gp120 of HIV-1 with bound CD4
gD of HSV-1 with bound HveA
gp42 of Epstein Barr virus with bound HLA-DR
Newcastle disease virus HN protein with the beta anomer of sialic acid .
In nonenveloped viruses, the structures that bind receptors are projections or indentations in the capsid surface. The receptor binding site can involve a single protein or a surface composed of several polypeptide chains.

11. Role of Carbohydrates IN VIRAL ENTRY
Glycoconjugates on the cell surface have an important role during entry of many viruses.
For some viruses, they are true receptors and essential for infection
for others, they are attachment factors that increase the efficiency of infection.
cell surface carbohydrates are involved was first recognized by the inhibition of infection observed after cell treatment with carbohydrate-destroying enzymes such as neuraminidase, heparinase, and heparitinase.
Glycoproteins and glycolipids, with terminal sialic-acid residues, serve as specific receptors for a variety of viruses, including orthomyxo- paramyxo-, and papovaviruses .

12. Co-receptors
Although receptor remains essential to the entry of the virus, other proteins are involved in this step of the viral cycle.
the co-receptors CCR5 and CXCR4 are essential to the entry and to the fusion of HIV.
The discovery of these co-receptors has enabled the re-classification of viral strains. On the basis of the use of a co-receptor rather than another one, a virus is said of tropism R5 or X4.
The main cellular targets of HIV (T lymphocytes CD4 and monocytes/macrophages) express the two co-receptors at once (CXCR4 and CCR5), but only one will be preferentially used during the attachment.

13. Virus-induced Signals
Many viruses use the host cell's signaling systems to promote entry and optimize infection. The signals are used to trigger access to coreceptors, to induce endocytic responses, to reprogram endocytic pathways, and to induce favorable intracellular conditions for infection.
Signaling starts at the plasma membrane after binding of the virus to receptors and formation of receptor clusters, but it can most likely continue in cytoplasmic organelles (e.g., endosomes and caveosomes)

14. Penetration and uncoating
For viruses that use membrane lysis or pore formation, it is probably essential to penetrate through the membranes of intracellular organelles to avoid damage to the plasma membrane.
The current situation is often confusing because viruses that require low pH activation to penetrate do not all use clathrin-coated pits to enter, and some viruses can make use of multiple routes .
The clathrin-mediated endocytic pathway is most commonly used by viruses .
It is a process that cells use to internalize a diverse collection of receptor-bound ligands, fluid, membrane proteins, and lipids .

15. Uncoating: is an integral part of the entry process; the virus particle is modified or destabilized, disassembled, and eventually the genome, which occurs in a protected and condensed form in the virion, is decondensed and exposed to become replication- and transcription-competent.
The progression of a virus particle through the entry program depends critically on cellular functions.

16. VIRAL ENTRY MECHANISMS
Three mechanisms for the entry of the virus into the interior of the cell, the more often by microphagocytosis for naked viruses (eg: poliovirus) and, for the enveloped virus, by fusion of the viral envelope and the cytoplasmic membrane in a unique membrane. Fusion is followed by lysis, by the formation of a pore (hole) which expands and leaves the capsid into the cytoplasm (eg: HIV virus). A third mechanism associated with it is the through endocytosis and fusion.

17. After attachment on its cellular receptor, the virus is internalized within a vesicle or endosome. An acidification of the virus within the endosome is induced by a proton pump (eg: protein M2 for the influenza virus type A). This induces a conformational modification of the hydrophobic proteins of viral envelope freeing regions that are usually hidden which react with the membrane lipids of the endosome. It follows a fusion of the membranes and a release of the viral capsid into the cytoplasm of the cell. This mechanism affects many enveloped viruses. (eg:Orthomyxoviridae, Rhabdoviridae. )

18. Enveloped Virus
The entry of all enveloped viruses involves the fusion of the viral envelope with one of the membranes of the host cell, in order to deliver the content of the viral particles in the cytoplasm. The membrane fusion can take place directly to the plasma membrane due to binding of the virus to its receptor ie in the case of HIVor by internalization of the virus particle through endocytosis

19. Endocytic pathways used by various viruses.

20. Endocytic Pathways of Infection
Whether viruses penetrate directly through the plasma membrane or after endocytosis has been a debatable issue.
although several virus families are able to penetrate the plasma membrane for productive infection, most make use of one or more of a diverse set of endocytosis pathways.
The endocytic uptake of viruses was originally called viropexis .
Early electron microscopy studies observed viruses in coated vesicles, and in a clear, cytoplasmic vacuoles near the cell surface  (probably early endosomes)

21. Depending on the virus, the receptors, and the host cells, initial binding to receptors can lead to activation of tyrosine or other kinases which, in turn, trigger cascades of downstream responses at the plasma membrane, in the cytoplasm, and, in some cases, also in the nucleus.
Virus-induced signaling depends on the usual panel of second messengers (phosphatidylinositides, diacylglycerides, and calcium), and on Rabs, Arfs, Rho-like small guanosine triphosphatases (GTPases) and numerous other regulators of membrane trafficking and actin dynamics.
Dynamin, clathrin, caveolin, and membrane-modifying factors are recruited to the plasma membrane, and signal-mediated changes in the cortical cytoskeleton allow transit of the newly formed vesicles through the cell cortex to the cytoplasm.

22. Non enveloped Viruses
The capsid of some naked viruses is able to reorganize itself after the fixing on its receptor, and to form a pore protein that crosses the cell membrane. The genome of the virus then goes into the cell cytoplasm. This process can take place directly at the plasma membrane, or may be triggered only after the endocytosis of the virus particle.
Among other naked viruses, the entire capsid cross the cell membrane. Most often, this happens after the internalization of viral particles in endosomes. Then, the protein capsid destabilize, and even destroy the endosomal membrane, thus allowing the entry of the capsid into the cytoplasm

23. Mechanism of HIV entry into a host cell starting from CD4 binding, followed by co receptor engagement and finally entry of a virus through fusion pore enlargement.

24. Receptors mediated Viral entry
 Specific interactions between virus particles and cell receptors leads to viral entry into host cell. Human rhinovirus 2 interaction with LDLR leads to receptor-mediated endocytosis . Four host factors are required for HCV to enter cell. The CD81 and SR-B1 are the receptor to which virus particle interact directly, however it indirectly interact with the tight linked components occludin and claudin-1. It is observed that the heteromers of claudin-1/CD81 are essential for initiating infection. Still more research studies are required to identify the mechanism through which HCV get itself bound to clathrin-coated vesicles.

25. Human rhinovirus 2 interaction with LDLR (A)
Human rhinovirus 2 interaction with LDLR (A). SR-B1 and CD81 interaction with HCV (B).CD4, CCR5 or CXCR4 interaction with HIV (C).

26. Virus attachment consists of specific binding of a virus-attachment protein with a cellular receptor molecule.
Many examples of virus receptors are now known. Target receptor molecules may be
- proteins (usually glycoproteins)
- carbohydrates (found on glycoproteins or glycolipids).
Virus attachment is usually an energy-independent process.
Carbohydrate receptors tend to be less specific than protein receptors. The same configuration of carbohydrate side-chains may occur on many different glycosylated membrane bound molecules.

27. Viral Penetartion
Cell penetration is generally an energy dependent process. the cell must be metabolically active for this to occur.
Three main mechanisms are involved:
1) Translocation of the entire virus particle across the plasma membrane.
-rare amongst viruses and poorly understood.
2) Endocytosis of the virus into intracellular vesicles (endosomes).
- very common mechanism, understood in great detail for some viruses
3) Fusion of the virus envelope with a cellular membrane.
- only applicable to enveloped viruses.
- requires the presence of a specific fusion protein in the virus envelope.
Two types of virus-driven membrane fusion:
- pH independent (can occur at cell surface or within an endosome)
- pH dependent (occurs within an acidified endosome)

28. Viral Penetration

29. Uncoating
Uncoating is a general term for the events that follow penetration, virus capsid is completely or partially removed and the virus genome is exposed.

30. Uncoating