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ASSIGNMENT ON

DNA and RNA Viruses


Course name: Medicinal Chemistry III Course code: PHRM 412 Section 1 Semester: Fall 2012

SUBMITTED TO: Md. Anisur Rahman Lecturer Department of Pharmacy

SUBMITTED BY: Samiya Khondaker Rinta (ID: 2010-3-70-048)

East West University

Submission date: 10th November, 2013

INTRODUCTION
Virus is a Latin word meaning poison or toxin. Viruses are the smallest infectious microorganisms and can infect animals and plants and even bacteria (Crosta, 2009). Their diameter is approximately 20300 nm and in their genome they contain only one kind of nucleic acid (DNA or RNA). The genetic material is enclosed inside a capsid (protein coat). In some viruses the capsid in turn is surrounded by a lipid bilayer called the envelope (Mandal, 2013). A virus does not contain organelles or ribosomes and depends solely on a host cell for its replication. It uses the host cellular energy, biochemicals and processes for multiplication and survival (Hilleman, 2004). Based on whether a virus contains one or more DNA or RNA molecule(s), viruses are classified as DNA virus or RNA virus. They are divided in the following arbitrary groups: 1) dsDNA such as adenoviruses, herpesviruses; poxviruses, etc. 2) (+)ssDNA such as parvoviruses 3) dsRNA such as reoviruses, birnaviruses 4) (+)ssRNA such as picornaviruses, togaviruses, etc. 5) (-)ssRNA such as orthomyxoviruses, rhabdoviruses, etc. 6) (+)ssRNA with DNA intermediate in life-cycle such as retroviruses. 7) dsDNA with RNA intermediate such as hepadnaviruses.( Koonin et al., 2006) During replication all viruses need to penetrate the host cell then they carry out the replication process and then the new viruses leave the host cell to infect other healthy host cells of the body. However the detailed process for each of these three stages varies among the viruses and depends upon the nature of their genome (Boevink and Oparka, 2005).

Figure 1: Schematic diagram showing replication in simplified form for each class of virus (Microbiology Bytes, 2004).

ADENOVIRUS
Adenoviruses have a linear, non-segmented dsDNA genome inside an icoshedral structure and are nonenveloped. These viruses infect the mucoepithelial cells of the GI, GU and respiratory tracts. They enter via epithelium, replicate and spread to lymphoid tissues (Doerfler, 1996).

Figure 2: Adenovirus structure showing capsid proteins, core proteins and minor proteins (Microbiology Bytes, 2010).

Figure 3: Showing attachment, penetration and uncoating of virion and insertion of viral genome in host nucleus (Leppard, 1997) As show in the above figure the viral fiber binds to a host cell surface receptor (CAR). Then an interaction between a penton base viral protein with a host cell integrin molecule takes place. The

virus thus enters the host cell through endocytosis. Lowering of pH causes lysis of the endosome membrane and the capsid (viral core) enters the cytoplasm. Microtubules carry the capsid to the nucleus. The viral DNA is released into the nucleus at a nuclear pore (Latif, 2010).

Figure 4: Schematic diagram of linear adenovirus genome, showing the Early genes (E) and the Late genes (L) (Latif, 2010). Adenovirus DNA replication takes place in the nucleus and occurs in two phases which are: 1) Early phase: Here genes responsible for control of host and viral transcription and viral DNA replication are being transcribed using the host RNA polymerase. These genes express regulatory, non-structural proteins which activate other types of viral genes and even prevent host immune defense mechanisms like apoptosis and activity of interferon. 2) Late phase: Here genes express structural proteins required for virion structure (Hunt, 2010). After adequate amounts of structural proteins have been synthesized then, within the nucleus, the newly replicated viral DNAs are packed inside the immature protein shells, which undergo a process of maturation and released from the host cell through lysis (Hunt, 2010).

HERPES VIRUS
Herpesviruses have a large linear dsDNA inside an icosahedral capsid which is itself wrapped in a tegument (protein layer) containing the viral proteins and the viral mRNAs. The tegument is in turn enclosed in a lipid bilayer envelope (Hunt, 2011).

Figure 5: Herpes virus structure (Davidson College Biology Department, 2013)

Figure 6: A schematic diagram of herpesvirus replication (Stanford University, 1992). Each step of replication in the above figure is explained as follows: (1) The virus attaches to the host cell with viral glycoproteins present in its envelope onto the heparan sulfate moieties of the cellular proteoglycans. (2) Both membranes get fused together and the nucleocapsid enters the host cell. (3) The capsid then moves along the cytoskeleton and reaches the nuclear pore. Here viral DNA is released (Stanford University, 1992). (4) Cellular RNA polymerase II transcribes mRNA from viral. As shown in figure 7 the viral genes are divided into three classes: I. Immediate early genes (IE): Theses genes are transcribed and translated first and are responsible for expressing proteins responsible for initiating transcription of early genes by host cells RNA polymerase. II. III. Early genes: These genes serve to upregulate the expression late genes, which are the third set of genes. They code for proteins like DNA polymerase and thymidine kinase (Hunt, 2011). Late genes: These genes express structural proteins and serve to downregulate the expression early genes (Davidson College Biology Department, 2013). (5) After completion of transcription, all mRNAs move into the cytoplasm where they are translated to proteins. These proteins may remain in the cytoplasm, become parts of membrane bilayer or can go back to the nucleus. (6) Empty capsid proteins assemble in the cell nucleus. (7) Complete-length viral DNA is packed inside capsids and nucleocapsids are formed. (8) Nucleocapsids then associate with the segments of nuclear membrane in places where tegument and glycosylated envelope proteins have been bound. This stimulates envelope formation by budding through cellular nuclear membrane. (9) Then accumulation of the enveloped virions in endoplasmic reticulum occurs. (10) Virions unmdergo maturation and are released from the cell by exocytosis.

(11) Virus-specific proteins are left behind on the infected cells plasma membrane (Stanford University, 1992).

Figure 7: Three classes of herpesrvirus genes (Davidson College Biology Department, 2013).

PAPOVAVIRUS
Papovavirus belongs to the virus family Papovaviridae, which is divided into Papillomaviridae and Polyomaviridae in present taxonomy. They have circular dsDNA surrounded by icosahedral shaped capsid with no lipid envelope.

Figure 8: Common replication steps of Papovavirus (Resino, 2013)

POXVIRUS
This virus has an envelope of protein bilayer surrounding a complex-shaped capsid. It has a dsDNA inside the capsid.

Figure 9: Poxvirus structure The viral proteins attaches with the GAGs (glycosaminoglycans) present on the host cell membrane and this causes penetration of the viral cell inside the host cell via endocytosis. The virus loses the outer membrane as it passes through the host cell membrane. In the early phase, transcription of early genes takes place with the help of viral RNA polymerase. The virus is now further uncoated and the viral genome is released in the cell cytoplasm. Then the intermediate genes are being expressed which initiate DNA replication. Expression of late genes produces structural proteins. Newly synthesized virions assemble in the cytoplasm and are released from the infected cell by lysis or budding.

PICORNAVIRUS
The virus is taken up by endocytosis after attachment with the host cell surface receptors. Replications take place in the cytoplasm. A conformational change occurs in the capsid and a viral protein, VP4, is released which opens up a pore in the endosomal membrane of host cell and this allows the vRNA to enter the cell cytoplasm. The vRNA is then translated and the polyproteins formed are cleaved and folded to form the required proteins and enzymes. Replication takes place in the viral factories made up of the membrane vesicles of the endoplasmic reticulum and dsRNA genome is produced. From this dsRNA new viral mRNAs or new (+)ssRNA are transcribed. New viral RNA are wrapped by capsids and released from host cell by lysis.

REOVIRUS
Reovirus have a segmented, dsRNA inside an icosahedral nucleocapsid. It does not have an envelope. This virus mainly affects the respiratory tract and the GI (gastrointestinal tract).

Figure 10: Replication steps of reovirus (Connolly and Dermody, 2002) The virus is taken up by endocytosis after attachment with the host cell surface receptors. Replications take place in the cytoplasm. Partially uncoated viral particles penetrate the cytoplasm from the endosome. Inside this naked core (sub-viral particle) viral dsRNA undergoes early transcription by the viral polymerase; so that the dsRNA genome is never in a complete uncoated state and hence activation of antiviral response by host cell is prevented. Formation of mRNA templates from each dsRNA segment is carried out by translation. (+)RNAs are transcribed to give (-)RNA molecules and then they are base-paired to synthesize new dsRNA genomes. Capsids assemble in the cytoplasm and new virions are released from host cell by lysis, followed by host cell death (Connolly and Dermody, 2002). TOGAVIRUS This virus has a (+)ssRNA inside an icosahedral nucleocapsid surrounded by a lipoprotein envelope. Two thirds of the genome has encodes structural proteins and the remaining one third codes for nonstructural proteins.

Figure 11: Non-structural and structural part of togavirus (Stanford University, 2013)

The virus is taken up by endocytosis after attachment with the host cell surface receptors. Replications take place in the cytoplasm.

Figure 12: Formation of new genomes Here the (+)ssRNA is transcribed into mRNA. There are two start codons (AUG) in the (+)ssRNA. When the infection is in the early phase, translation occurs from the first start codon yielding a long polyprotein. This polyprotein is cleaved and folded to form proteins and enzymes required for replication of complementary RNA from the (+)ssRNA. The complementary strand is then copied from the second start codon producing a positive strand template, which does not have the first start codon. In the late phase the complementary strand is again translated starting from the second (AUG) codon to produce different viral proteins. Meanwhile repeated replication of the (-)RNA strand produces many (+)ssRNA genomes, which are packed inside capsids and then this virions leave the cells through budding (Stanford University, 2013). CALICIVIRUS This virus has a icosahedral nucleocapsid inside of which it has a non-segmented (+)ssRNA. It does not have an envelope.

Figure 13: Structure of calicivirus

Figure 14: Formation of new genomes After attachment with the host cell receptor the virus enters the cell through endocytosis. The viral RNA is released in the cytoplasm through uncoating of the capsid. From the vRNA (viral RNA) the protein, VPg, is removed and translation of the RNA takes place yielding a polyprotein (ORF1), which is cleaved to produce matured non-structural proteins. Replication takes place in the viral factories made up of the membrane vesicles of the endoplasmic reticulum and dsRNA genome is produced. From this dsRNA new viral mRNAs or new (+)ssRNA are transcribed. Translation of subgenomic RNA forms VP2 and capsid protein. New virions are assembled and released from host cell by lysis. ASTROVIRUS Astrovirus has non-segmented, linear ss(+)RNA as its genome enclosed in a icosahedral capsid and has no lipoprotein envelope (Brown et al., 2008). It carries out its replication in the host cell cytoplasm. In humans main cause of gastroenteritis is astrovirus (Dryden et al., 2012). Route of transmission for this virus is by the fecal-oral route from person to person. The viral replication takes place in the human intestinal epithelial cells (Mndez et al., 2004).

Figure 15: Genome of astrovirus (Swiss Institute of Bioinformatics, 2010) After attachment with the host cell receptor the virus enters the cell through endocytosis. The viral RNA is released in the cytoplasm through uncoating of the capsid. This virion RNA serves both as a genome and as a viral mRNA. The viral nucleic acid had three overlapping yet open frames for reading, which are ORF2, ORF1a, and ORF1b. ORF1a encodes viral protease and ORF1b encodes polymerase. ORF2 encodes capsid precursor protein, the VP90, and is translated from a subgenomic RNA. From viral RNA two large polypeptides, nsP1a and nsP1a/1b, are translated to form replication/non-structural proteins (Bass and Qiu, 2000). Replication takes place in the viral factories

made up of the membrane vesicles of the endoplasmic reticulum. The capsid matures and is released from the host cell without causing lysis (Swiss Institute of Bioinformatics, 2010). ORTHOMYXOVIRUS Orthomyxovirus has eight segments of ss(-)RNA enclosed inside a helical shaped capsid which in turn is surrounded by a lipoprotein envelope containing the viral glycoproteins neuraminidase and hemagglutinin. Any virus that belongs to orthomyxoviridae family is called orthomyxovirus. Thogotovirus, Influenzavirus A, Influenzavirus B, and Influenzavirus C are the four types of viruses that belong in this family (Encyclopedia Britannica, 2013).

Figure 16: Structure of orthomyxovirus (Hunt, 2010). Orthomyxovirus infects epithelial cells of respiratory tract and the virus is transmitted via tiny particle aerosols from person to person. Replication of this virus takes place in the host cell nucleus (Hunt, 2010).

Figure 17: Steps of replication of orthomyxovirus (National Center for Biotechnology Information, 2006).

After attaching with the host cell membrane via hemagglutinin (HA), the virus penetrates into the cytoplasm by endocytosis and forms an endosome (STEP 1). HA is cleaved into HA1 and HA2 by a cellular trypsin-type enzyme. HA2 initiates fusion of virus envelope with endosome membrane. M2 (minor viral envelope protein) increases the pH inside the virus causing dissociation of M1 (major viral envelope protein) and translocation of viral RNAs, RNA-dependent RNA polymerase and accessory proteins into the host cell nucleus (STEP 2). In the nucleus complementary (+)cRNA is being transcribed (STEP 3a) and replicated (STEP 3b). cRNA moves into the cytoplasm and is translated into new viral proteins. These viral proteins (neuraminidase and hemagglutinin) either move onto host cell surface by secretion via Golgi apparatus (step 5b) or migrate back to the nucleus and bind with newly formed vRNAs forming new particles of viral genome (step 5a). The freshly synthesized nucleocapsids move into the cytoplasm where they interact with a portion of cell membrane via M1. M2, NA and HA have been inserted in this region of membrane (STEP 6). Finally the newly synthesized virion is released from the infected cell by budding (STEP 7) (National Center for Biotechnology Information, 2006).

PARAMYXOVIRUS
These viruses are single-stranded (-)RNA viruses containing a helical shaped capsid and a lipoprotein envelope. They undergo replication in the cytoplasm of host cells and cause various types of respiratory diseases (Narayan and Horvath, 2009).

Figure 18: Structure of paramyxovirus After being attached to the cell surface receptors of host cells via G, H or HN glycoproteins, the membrane of virion fuses with the host plasma membrane and releases the ribonucleocapsid into the cytoplasm. Transcription and translation takes place using the RNA-dependent RNA polymerase provided by the virus and the first mRNA is synthesized. L and P proteins also carry out the polymerase function. Replication of the genome results in the formation of (+)RNA template with which (-)RNA is transcribed. Nucleocapsids are assembled and M proteins are associated with modified

cell membranes containing viral glycoprotein. Finally the newly synthesized matured virions are released from the infected cell by budding (Narayan and Horvath, 2009).

RETROVIRUS

Figure 19: Replication and structure of retrovirus Retrovirus is a (+)ssRNA virus. It has a icosahedral capsid and a lipoprotein envelope. The replication of retrovirus, such as HIV, is a multistage process. Infection of a suitable host cell such as a CD4 positive T lymphocyte is the first step. Entry of HIV into the cell requires the presence of certain cell surface receptors such as CD4 receptors and co-receptors such as CCR5 or CXCR4. These receptors interact with the protein complexes embedded in the viral envelope. These complexes are made up of two glycoproteins (extracellular gp120 and transmembrane gp41). When HIV approaches a target cell, gp120 binds to the CD4 receptors. This process is termed as attachment.

It promotes further binding to a co-receptor. Co-receptor binding results in a conformational change in gp120. This permits gp41 to unfold and this causes the fusion of viral membrane with host cell membrane. Thus the viral nucleocapsid penetrates the host cell and dissociates and two viral RNA strands and 3 essential replication enzymes (reverse transcriptase, protease and integrase) are released. Then the reverse transcriptase begins the reverse transcription of viral RNA. It has two catalytic domains, the ribonucleic H active site and the polymerase active site. In the polymerase active site single stranded viral RNA is transcribed into RNA-DNA double helix. Ribonucleic H breaks down the RNA-DNA double helix. The polymerase then completes the remaining DNA strand to form a DNA double helix. Now integrase cleaves the dinucleotide from each 3 end of the DNA creating two sticky ends. Integrase then takes the DNA into cell nucleus and facilitates its integration into the host cell genome. Now host cell genome contains genetic information of HIV. Activation of the cell induces transcription of proviral DNA into mRNA. The viral mRNA moves into the cytoplasm where building blocks for a new virus are synthesized. Longer proteins are then cleaved into smaller proteins by the viral protease. Two viral RNA strand and replication enzymes then come together and core proteins assemble around them forming the capsid. This immature viral particle leaves host cell acquiring a new envelope of host and viral proteins. The virus eventually matures and starts to infect other cells. HIV virus replicates millions of times per day and eventually causing disease progression and de stroys the hosts immune cells.

HEPADNAVIRUS
Hepadnavirus has an icosahedral capsid inside which there is a dsDNA that is non-segmented, open, circular and partially double-stranded. The virion also consists of a lipid bilayer membrane (Swiss Institute of Bioinformatics, 2010).

Figure 20: Structure of Hepadnavirus (Swiss Institute of Bioinformatics, 2010).

Figure 21: Replication steps of Hepadnavirus (Hunt, 2011) In the above figure the replication steps of this virus in elaborately explained. It can be seen that replication takes place in the host cell nucleus. Hepatitis B is a common disease caused by this virus (WHO, 2013).

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