10.6: Animal Virus Life Cycles - Biology

10.6: Animal Virus Life Cycles - Biology

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Viruses that infect animal cells replicate by what is called the productive life cycle. The productive life cycle is also often referred to as the lytic life cycle, even though not all viruses cause lysis of their host cell during their replication. Some viruses, such as HIV and the herpes viruses are able to become latent in certain cell types. A few viruses increase the risk of certain cancers. We will now look at the life cycles of viruses that infect animal cells.

6.2 The Viral Life Cycle

All viruses depend on cells for reproduction and metabolic processes. By themselves, viruses do not encode for all of the enzymes necessary for viral replication. But within a host cell, a virus can commandeer cellular machinery to produce more viral particles. Bacteriophages replicate only in the cytoplasm, since prokaryotic cells do not have a nucleus or organelles. In eukaryotic cells, most DNA viruses can replicate inside the nucleus, with an exception observed in the large DNA viruses, such as the poxviruses, that can replicate in the cytoplasm. With a few exceptions, RNA viruses that infect animal cells replicate in the cytoplasm. An important exception that will be highlighted later is Influenza virus.

The Life Cycle of Viruses with Prokaryote Hosts

The life cycle of bacteriophages has been a good model for understanding how viruses affect the cells they infect, since similar processes have been observed for eukaryotic viruses, which can cause immediate death of the cell or establish a latent or chronic infection. Virulent phages typically lead to the death of the cell through cell lysis. Temperate phages , on the other hand, can become part of a host chromosome and are replicated with the cell genome until such time as they are induced to make newly assembled viruses, or progeny virus es.

The Lytic Cycle

During the lytic cycle of virulent phage, the bacteriophage takes over the cell, reproduces new phages, and destroys the cell. T-even phage is a good example of a well-characterized class of virulent phages. There are five stages in the bacteriophage lytic cycle (see Figure 6.7). Attachment is the first stage in the infection process in which the phage interacts with specific bacterial surface receptors (e.g., lipopolysaccharides and OmpC protein on host surfaces). Most phages have a narrow host range and may infect one species of bacteria or one strain within a species. This unique recognition can be exploited for targeted treatment of bacterial infection by phage therapy or for phage typing to identify unique bacterial subspecies or strains. The second stage of infection is entry or penetration . This occurs through contraction of the tail sheath, which acts like a hypodermic needle to inject the viral genome through the cell wall and membrane. The phage head and remaining components remain outside the bacteria.

The third stage of infection is biosynthesis of new viral components. After entering the host cell, the virus synthesizes virus-encoded endonucleases to degrade the bacterial chromosome. It then hijacks the host cell to replicate, transcribe, and translate the necessary viral components (capsomeres, sheath, base plates, tail fibers, and viral enzymes) for the assembly of new viruses. Polymerase genes are usually expressed early in the cycle, while capsid and tail proteins are expressed later. During the maturation phase, new virions are created. To liberate free phages, the bacterial cell wall is disrupted by phage proteins such as holin or lysozyme. The final stage is release. Mature viruses burst out of the host cell in a process called lysis and the progeny viruses are liberated into the environment to infect new cells.

The Lysogenic Cycle

In a lysogenic cycle , the phage genome also enters the cell through attachment and penetration. A prime example of a phage with this type of life cycle is the lambda phage. During the lysogenic cycle, instead of killing the host, the phage genome integrates into the bacterial chromosome and becomes part of the host. The integrated phage genome is called a prophage . A bacterial host with a prophage is called a lysogen . The process in which a bacterium is infected by a temperate phage is called lysogeny . It is typical of temperate phages to be latent or inactive within the cell. As the bacterium replicates its chromosome, it also replicates the phage’s DNA and passes it on to new daughter cells during reproduction. The presence of the phage may alter the phenotype of the bacterium, since it can bring in extra genes (e.g., toxin genes that can increase bacterial virulence). This change in the host phenotype is called lysogenic conversion or phage conversion . Some bacteria, such as Vibrio cholerae and Clostridium botulinum, are less virulent in the absence of the prophage. The phages infecting these bacteria carry the toxin genes in their genome and enhance the virulence of the host when the toxin genes are expressed. In the case of V. cholera, phage encoded toxin can cause severe diarrhea in C. botulinum, the toxin can cause paralysis. During lysogeny, the prophage will persist in the host chromosome until induction , which results in the excision of the viral genome from the host chromosome. After induction has occurred the temperate phage can proceed through a lytic cycle and then undergo lysogeny in a newly infected cell (see Figure 6.8).

Link to Learning

This video illustrates the stages of the lysogenic life cycle of a bacteriophage and the transition to a lytic phase.

5 Main Stages of Animal Viruses | Microbiology

Animal viruses differ from stages in mechanism of entering the host cell. This is due to differences in host cell i.e. one is prokaryotic and the other eukaryotic in nature.

It is accomplished into the following stages:

1. Attachment:

Animal viruses like bacteriophages posses the attachment sites with the help of which it attaches to the receptor sites present on host cell surface. The receptor sites are the proteins or glycoproteins present on membrane surface of the host cell.

The attachment sites of one group of viruses differ from the others. Distribution of these proteins plays a key role in tissue and host specificity of animal viruses. For example, poliovirus receptors are found only in human nasopharynx, gut and cells of spinal cord.

While receptors of measles virus are present in most tissues. Differences in nature of polio and measles can be explained through the dissimilarities in the distribution of receptor proteins of host cells to which viruses get adsorbed.

In some naked viruses (e.g. adenoviruses) the attachment sites are small fibres at the comers of icosahedron. In enveloped viruses (e.g. myxo-viruses) the attachment sites are the spikes present on the surface of envelope.

For example, influenza virus has two types of spikes: H (haemagglutinin) spikes and N (neuraminidase) spikes. The H spikes attach to the host cell receptor site and recognise siatic acid (N-acetyl neuraminic acid). Influenza neuraminidase helps the virus in penetrating the nasal and respiratory tract secretions by degrading the mucosal polysaccharides. However, the receptor sites also vary from person to person.

2. Penetration:

After the attachment, virus penetrates the host cell. In enveloped animal viruses, penetration occurs by endocytosis, a process of bringing nutrients into the cell.

If a virion attaches to a small out folding i.e. microvillus on plasma membrane of a host cell, it will unfold the virion into a fold or plasma membrane forming a vesicle. When the virion is enclosed within a vesicle, its envelope is disintegrated and the capsid is digested resulting in release of nucleic acid in cytoplasm.

The detailed mechanism of entry of virus in not clear. However, the following three modes of entry of viruses occur:

(a) Direct penetration:

Some naked viruses (e.g. poliovirus) undergo a major change in capsid structure after adsorption to plasma membrane. This change facilitates the release of nucleic acids into cytoplasm (Fig. 17.1A).

(b) Fusion with plasma membrane:

The envelop of enveloped viruses (e.g. paramyxoviruses) fuses directly with host plasma membrane and nucleocapsid is deposited in cytoplasmic matrix where uncoating is done (Fig. 17.1B). When virus is within the capsid, a virus polymerase attached to nucleocapsid transcribes the virus RNA.

Most enveloped viruses enter the host cell through engulfment by receptor-mediated endocytosis and form coated vesicles. Virions attached to coated pits with the protein clathrin. Lysosomes help in uncoating of virion inside the cytoplasm. (Fig.17.1C).

3. Uncoating:

It is a process of separation of viral nucleic acid from the protein coat. This process is not fully understood. In some viruses uncoating is done by lysosomal enzymes of the host cell which degrade protein coat and make the nucleic acid free in cytoplasm. In poxviruses, the viral DNA synthesizes a specific protein after infection. Thus, it varies with virus groups.

4. Replication:

The replication process of DNA viruses differs from that of RNA viruses. However, in some DNA viruses multiplication occurs in cytoplasm (e.g. poxviruses) and in some others replication occurs in the nucleus of host (e.g. parvovirases, papovaviruses, adenoviruses, herpes viruses).

Multiplication of RNA viruses is more or less the same as in DNA viruses except the mechanism of formation of mRNA among the different group (Fig. 17.2).

5. Assembly and Release:

After replication of genetic material and synthesis of viral proteins assembly of viral particles occurs inside the host cell. Thereafter, they are released from the host cell.

10.6: Animal Virus Life Cycles - Biology

For a typical virus, the lifecycle can be divided into five broad steps: attachment, entry, genome replication and gene expression, assembly, release.

In everyday life, we tend to think of a viral infection as the nasty collection of symptoms we get when we catch a virus, such as the flu or the chickenpox. At the microscopic scale, a viral infection means that many viruses are using your cells to make more copies of themselves. The viral lifecycle is the set of steps in which a virus recognizes and enters a host cell, “reprograms” the host by providing instructions in the form of viral DNA or RNA, and uses the host’s resources to make more virus particles (the output of the viral “program”).

For a typical virus, their lifecycle can be divided into five broad steps (though the details of these steps will be different for each virus):

  1. Attachment: The virus recognizes and binds to a host cell via a receptor molecule on the cell surface.
  2. Entry: The virus or its genetic material enters the cell.
  3. Genome replication and gene expression: The viral genome is copied, and its genes are expressed to make viral proteins.
  4. Assembly: New viral particles are assembled from the genome copies and viral proteins using the host cell ribosomes
  5. Release: Completed viral particles exit the cell and can infect other cells.

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• For a typical virus, the lifecycle can be divided into five broad steps: attachment, entry, genome replication and gene expression, assembly, release.

Genome: the whole of an organisms hereditary information encoded in its DNA

Ribosomes: organelles that carry out protein synthesis

RNA: is a single-stranded RNA molecule that is read by a ribosome to produce a protein

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Transcription and replication of the viral genome

The influenza viral genome is made up of negative sense strands of RNA. In order for the genome to be transcribed, it first must be converted into a positive sense RNA to serve as a template for the production of viral RNAs.

Replication of the genome does not require a primer instead, the viral RNA dependent RNA polymerase (RdRp) initiates RNA synthesis internally on viral RNA. This is possible, as the extreme 5’ and 3’ ends of the genome exhibit partial inverse complementarity and, hence, are able to base pair with one another to form various corkscrew configurations. It appears that a great number of di-nucleotide base pairs form, although the full mechanism of viral genome replication is still yet to be understood [13-16].

Given that the influenza A virus only encodes for 11 proteins, it has generated many sophisticated methods of utilizing the host cell’s machinery for its own purposes. Through understanding viral transcription, we have learned of a unique mechanism whereby the virus hijacks the host’s transcription machinery for its own benefits.

Mature cellular messenger RNAs (mRNAs) have a 5’ methylated cap and a poly(A) tail. It is known that the vRNPs have poly(A) tails but no 5’ caps. It was confusing when the influenza community discovered that the viral mRNAs did have a 5’ methylated cap and a poly(A) tail, but the 5’ cap was not found in the viral genome [17,18]. Much study went into this problem, and soon it was determined that the 5’ methylated caps of the viral mRNAs actually belonged to the cellular mRNAs. That discovery lead to the formulation of the �p-snatching” mechanism [19-26]. The viral RdRp is made up of three viral proteins: PB1, PB2, and PA. PB2 has endonuclease activity. It binds to the 5’ methylated caps of cellular mRNAs and cleaves the cellular mRNAs’ 10 to 15 nucleotides 3’ to the cap structure. This cellular capped RNA fragment is used by the viral RdRp to prime viral transcription [27].

Cellular RNA Polymerase II (Pol II) binds to DNA and starts transcription. During transcription initiation, serine 5 on the C-terminal repeat domain (CTD) of Pol II is phosphorylated, leading to the activation of cellular cap synthesis complex. The influenza RdRp has been shown to bind preferentially to this form of Pol II, indicating that this could be the point at which �p snatching” could occur [28].

Six but two of the viral segments encode for one protein. Segments 7 and 8 encode for two proteins each due to splicing. Segment 7 encodes for M1 and M2 whereas, segment 8 encodes for NS1 and NEP. M2 and NEP are the spliced products and generally are found in much lower abundance than NS1 and M1 [29]. The virus uses the host cell’s splicing machinery to express both of these proteins [30]. Despite influenza’s need for the cellular splicing machinery, it prevents the host cell from using its own splicing machinery for processing the host cell mRNAs. NS1 binds to U6 small nuclear RNAs (snRNAs) [31,32] and other splicing components, causing them to re-localize to the nucleus of infected cells [33]. In this way, influenza is able to inhibit splicing of cellular mRNAs. It also has been shown to bind to a novel protein called NS1 binding protein (NS1-BP), causing it to re-localize to the nucleus in infected cells. The function of NS1-BP is unknown, although it is predicted to be involved in splicing given its co-localization with SC35, a spliceosome assembly factor [34]. NP also has been shown to interact with UAP56, a splicing factor involved in spliceosomal formation and mRNA nuclear export, although the importance of NP’s binding to UAP56 is yet to be established [35].

The mechanism of polyadenylation of viral mRNAs is very unusual. Cellular mRNAs are polyadenylated through cleavage at the polyadenylation signal (AAUAAA) by cleavage and polyadenylation specificity factor (CPSF) and subsequent addition of a poly(A) tail at the 3’ end of the mRNA. Viral mRNAs do not contain this sequence instead, the viral RdRp remains bound to the 5’ end of the template viral RNA, leading to steric blockage at the end of viral RNA synthesis [36,37]. Each viral segment has a stretch of five to seven U residues approximately 17 nucleotides from the 5’ end, and this forms the basis of the viral polyadenylation signal [38]. Therefore, polyadenylation of the viral mRNAs occurs due to a stuttering mechanism, whereby the RdRp moves back and forth over this stretch of U residues, leading to the formation of a poly(A) tail [39,40]. Interestingly, NS1 inhibits the nuclear export of cellular mRNAs by preventing cellular mRNAs from being cleaved at the polyadenylation cleavage site [41]. It does this by binding to the CPSF [42] and poly(A) binding protein II (PABPII), which is involved in stimulating poly(A) polymerase to add the poly(A) tail onto newly cleaved mRNAs [43].

Replication Cycle of Rabies Viruses | Microbiology

In this article we will discuss about the replication cycle of rabies viruses.

The infection process is initiated with adsorption of virus on the host cell. Interaction takes place between spike G protein and specific cell surface receptors. It results in fusion of the rabies virus envelope with the host cell membrane. After adsorption, the virus penetrates the host cell and enters the cytoplasm by pinocytosis through clathrin-coated pits.

The virions aggregate in the large endosomes i.e. cytoplasmic vesicles. The viral membranes fuse to the endosomal membranes, and viral uncoating is carried out. This causes the release of viral RNP into the cytoplasm. Because the rabies virus consists of a linear single- stranded (-) sense RNA genome. Soon the genomic RNA is transcribed into mRNAs so that virus replication must be started.

An L gene of the virus encodes an enzyme, polymerase which transcribes the genomic strand of rabies RNA into leader RNA and five capped and polyadenylated mRNAs. The latter are translated into proteins. Translation occurs on free ribosomes in the cytoplasm, which synthesizes the N, P, M, G and L proteins.

Synthesis of the G protein is initiated on free ribosomes, but its complete synthesis and glycosylation (i.e. processing of the glycoprotein) process occurs in the endoplamsic reticulum (ER) and Golgi bodies. The switch from transcription to replication is regulated by the intracellular ratio of leader RNA to N protein.

Replication of viral genome begins upon activated of this switch. The first step of replication is the synthesis of full-length copies of (+) sense ssRNA. When replication is switched on, RNA transcription takes place uninterrupted even stop codons are ignored.

The viral polymerase enters a single site on the 3′ end of the genome, and proceeds to synthesize full-length copies of the genome. These (+) sense ssRNA of rabies virus acts as templates for synthesis of full-length negative strands of the viral genome.

At the time of viral assembly, N-P-L complex is formed. I This complex encapsulates negative- stranded genomic RNA to form the RNP core. The M protein forms a capsule or matrix around the RNP. The RNP-M complex migrates to an area of the plasma membrane containing glycoprotein inserts and the M-protein initiates coiling.

The RNP-M complex binds to glycoprotein, and the B complete virus buds from the plasma membrane. There is preferential viral budding from plasma membranes within the central nervous system (CNS). In the salivary glands, virus buds from the cell membrane into the acinar lumen. Viral budding into the salivary gland and virus-induced aggressive biting-behaviour in the host animal maximize chances of viral infection of a new host.

Viral entry

Once bound to cell surface receptors, viruses enter eukaryotic cells through two main pathways. Some viruses gain entry to cells by directly fusing with or penetrating the cell membrane, but most viruses traffic into a cell via endocytosis (coronaviruses are able to enter both ways) (Figure 1) [5,6]. Most often this endocytosis occurs via clathrin-coated pits, whose cargo is shuttled to increasingly acidic organelles, from early endosomes to late endosomes to lysosomes [7]. For example, coronaviruses can undergo fusion at different stages of endocytosis: MERS coronavirus fuses with early endosomes, whereas SARS coronavirus fuses with late endosomes [4]. Viruses that require an even more acidic pH will not undergo fusion until they reach the lysosome [2]. These endocytic and lysosomal vesicles have low pH and abundant proteases that can trigger conformational changes in a virus, leading to uncoating, fusion, or penetration into the cytoplasm. Even those viruses without a low-pH requirement exploit endocytosis as a convenient route to rapidly cross the plasma membrane and transit through the cytoplasm to their sites of replication [2]. Labeling virus particles with pH-sensitive dyes such as the pHrodo IFL Green STP ester dye can enable visualization of virus entry [8].

Figure 1. Pathways of virus entry into cells. (A) Enveloped viruses can bind to cell surface receptors and directly fuse with the plasma membrane. Virus particles can also be internalized via endocytosis, with escape to the cytosol occurring either from the (B) early endosome or (C) late endosome and lysosome. The acidic environment and proteolytic enzymes in these compartments are required for fusion and cytosol entry by different viruses.

Virological perspectives


The Hepadnaviridae family, with HBV as the prototype, is comprised of a group of hepatotropic DNA viruses which on the whole are species-specific and are divided into two genera. The Orthohepadnavirus genus includes members that infect mammals (woodchucks, ground squirrels, bats and primates) and have about 70% nucleotide homology between them. The Avihepadnavirus genus infects birds such as ducks (duck hepatitis B virus, DHBV), herons, storks, geese and parrots with about 80% homology between them. The homology, however, between the two genera is around 40%, but nevertheless they share a common genomic organisation [18].

Nucleotide sequencing studies of human HBV isolates from around the world have established, based on sequence divergence of > 8%, eight genotypes designated A–H, with characteristic geographical distribution. Genotypes A and D are frequently found in Africa, Europe, and India, genotypes B and C in Asia, genotype E is restricted to West Africa, and genotype F in Central and South America. Genotype G and H distribution is less clear, but isolates have been reported from Central America and southern Europe [18, 19], while possibly two new ones, genotype I from Vietnam, Laos and Eastern India appears to be an intergenotypic recombinant between genotypes A, C, and G [20], and genotype J isolated from a Japanese man who lived in Borneo, and which appears to be a recombinant between genotype C and gibbon HBV [21]. Genotypes A, B, C, D, F and I can be further subdivided based on nucleotide divergence of 4% into at least 44 sub-genotypes A1–6, B1–9, C1–16, D1–7, F1–4 and I1–2 [19, 22]. In addition, other than the above-mentioned recombinants, B/C and C/D recombinants represent the majority of such isolates, while other intergenotypic recombinants that occur less frequently involve most of the other genotypes [23].

Virion structure and genome organisation

The infectious virion or Dane particle of 42 nm in diameter is comprised of an outer envelope made of HBsAg in a lipid bilayer [9], wrapped around the nucleocapsid core of the virus, which in turn encloses the viral genome and a copy of its polymerase [9, 24]. In addition, there is an abundance of sub-viral particles circulating in serum consisting entirely of HBsAg and devoid of any nucleic acid containing cores. These are the 25-nm spheres and the 22-nm diameter filaments, which outnumber infectious virions by 100- to 10,000-fold [25].

The partially double-stranded DNA genome of the virus is a relaxed circle of 3.2 kb in length (rcDNA) [26, 27]. In view of this, it is the smallest among DNA viruses and one of the most compact as the 4 open reading frames (ORFs) that the genome contains are either wholly or partially overlapping. Thus, every nucleotide of the genome forms part of an ORF. In addition, all regulatory elements such as the two enhancers (Enh1, Enh2), the four promoters (core, S1, S2 and X), polyadenylation, encapsidation (ε) and replication (DR1, DR2) signals lie within these ORFs (Fig. 1).

The cccDNA, depicted here in linear outline, is the transcriptionally active form of HBV. The enhancers and promoters involved in transcript synthesis are also shown as are the transcripts themselves, the ORFs which they contain and the proteins which they encode, their lengths and their co-terminal nature at a common polyadenylation signal (An). Modified from Baltayiannis and Karayiannis [28]

RNA transcription and protein translation

The four ORFs are those of the surface (PreS/S), core (C), polymerase (P) and X genes, which encode in total 7 proteins translated from six co-terminal, unspliced and capped mRNAs ending at a common polyadenylation signal, which is situated in the core ORF (Fig. 1, [29]). The aforementioned promoters and enhancers direct the synthesis of the mRNA transcripts through the recruitment of transcription factors which are particularly enriched in hepatocytes (reviewed in [30]). This in part also explains the liver tropism of the virus.

The core promoter is responsible for the synthesis of two longer than genome length mRNAs (3.5 kb) which differ with respect to the start of their 5′ end. The longer of the two by a small number of ribonucleotides is the precore mRNA containing the initiation codon for synthesis of the precore protein, which is the precursor for hepatitis B e antigen (HBeAg). The protein undergoes proteolytic processing at its N-terminus for the removal of a signal peptide of 19 amino-acids in length, which targets the protein to the ER, and a furin cleavage for the removal of an arginine rich domain at its C-terminus [31,32,33]. What is left is the 15-kD HBeAg which is secreted and dispensable protein for replication that appears to act as a tolerogen in newborns and chronic infection [34]. The other transcript is the pregenomic RNA (pgRNA) which is bicistronic in nature and encodes for the core (21kD) or hepatitis B core antigen (HBcAg) and the polymerase (90kD) proteins. The pgRNA in addition constitutes the template for reverse transcription during the replication of the viral genome, as explained below. The core has the capacity to dimerise and form nucleocapsids by self-assembly consisting of 240 copies (120 dimers) of the protein [35], while the polymerase is a multifunctional protein which acts as a reverse transcriptase (rt), as DNA polymerase and has RNase H activity also [36]. Regulation of production of these two proteins is such as to favour the generation of the numbers mentioned above of core molecules required for nucleocapsid formation per single molecule of polymerase packaged with the pgRNA.

The Pre-S/S ORF of the genome encodes for the three envelope glycoproteins produced by differential initiation of translation at each of three in-frame initiation codons. These are known as the large (L), middle (M) and small (S) HBsAgs, with the latter being the more abundant constituent of the viral envelope. Two transcripts of 2.4 and 2.1 kb are involved, the synthesis of which is controlled by the S1 and S2 promoters respectively. L-HBsAg is translated from the 2.4 kb transcript while the M- and S-HBsAgs are translated from the 2.1 kb transcript, the latter through leaky ribosome scanning. The S-HBsAg is thus the smallest of the three co-terminal proteins and its 226 amino-acids are shared by the other two at their C-terminus. The M protein has an additional 55 amino-acids at its N-terminus encoded by the Pre-S2 region, while the L protein includes in addition another 107–118 amino-acids (depending on genotype) from the Pre-S1 region [29]. The first 48 N-terminal amino-acids of Pre-S1, as originally thought (see later), contain the region responsible for attachment of the virus to its hepatocyte receptor [37, 38]. Moreover, myristylation of L-HBsAg appears to be essential for infectivity [39]. All three proteins are glycosylated while the L and S proteins may also be present in an unglycosylated form in particles. They are synthesised at the ER and maintain a transmembrane configuration that enables budding of the virus through the ER during maturation [40].

The envelopes of the Dane particles and of the two types of sub-viral particles contain all three HBsAgs, but their relative ratios are not identical. The S protein represents the majority in Dane particles with equal amounts of the M and L proteins, whereas the spheres contain mainly S and M proteins, with trace amounts of the L protein. Filaments have S-HBsAg as the majority protein with M and L proteins being present in equal amounts, which, however, are not as high as those in the Dane particle [41].

The fourth and smallest ORF encodes for the 17kD HBx protein which is translated from the shortest transcript of 0.7 kb in length. It consists of 154 amino-acids and appears to modulate host-cell signal transduction, acts as a gene transactivator under experimental conditions, can activate transcription factors and therefore is implicated in binding the covalent closed circular DNA (cccDNA) minichromosome (reviewed in [42, 43]).

Viral life cycle


It is now clear that species-specificity and hepatotropism are determined by the requirement of transcription factors enriched in hepatocytes as mentioned above and the expression on human hepatocyte cells of the recently described sodium taurocholate co-transporting peptide (NTCP) which constitutes the HBV receptor (Fig. 2a, [44]). NTCP is a bile acid transporter expressed at the basolateral membrane of hepatocytes. The receptor binds the N-terminal end of L-HBsAg as described above, and in fact the region involved may include the first 75 amino-acids [45]. In addition, subsequent studies have indicated that heparin sulfate proteoglycans may be involved in the initial stages of binding [46], as well as glypican 5 [47], thus suggesting co-operative binding in the process of attachment and uptake.

Diagrammatic representation of hepatocyte infection with HBV. a The various stages of the life cycle of the virus from attachment to release, as explained in the text and numbered as: 1 attachment 2 endocytosis 3 capsid release 4 rcDNA entry into the nucleus 5 cccDNA synthesis 6 transcription 7 mRNA transfer to the cytoplasm 8 encapsidation 9 (−)-DNA strand synthesis by reverse transcription 10 (+)-DNA strand synthesis 11 budding of virions into the ER lumen 12 virus release through multivesicular body (MVB) transfer to hepatocyte surface. b Basolateral release of the virus for cell-to-cell spread. Part modified from Baltayiannis and Karayiannis [28]

Penetration and uncoating

Evidence suggests that, following binding, the virion is internalised through clathrin-mediated endocytosis [48]. However, information on the removal of the viral envelope and the trafficking of the nucleocapsid to the nuclear pores is still lacking. Transport factors such as importin alpha and beta and component nucleoporin 153 ensure nucleocapsid delivery to the nuclear basket [49, 50]. Disassembly of nucleocapsids ensues leading to the release into the nucleoplasm of the rcDNA genome of the virus with its covalently attached polymerase.


The rcDNA is converted into the cccDNA form in a process involving a number of stages whereby the viral polymerase covalently attached to the 5′ end of the negative (−)-DNA strand and the short RNA oligomer from the 5′ end of the positive (+)-DNA strand which is used to prime (+)-DNA strand synthesis are removed, the variable positive strand is completed and finally the ends of the two now complete strands are ligated together [29, 51]. This process likely involves the use of specific cellular factors that are presently unknown. In this form, cccDNA is quite stable and behaves as a minichromosome. Moreover, a number of epigenetic factors which are recruited onto the cccDNA, such as histones H3 and H4, transcription factors that include CREB, ATF, STAT1 and STAT2, chromatin-modifying enzymes, histone acetyltransferases and deacetylases, and the HBc and HBx proteins [52,53,54] appear to modulate cccDNA transcriptional activity. Thus, the cccDNA constitutes the template for viral transcript synthesis by the host RNA polymerase II. The synthesised transcripts are then exported to the cytoplasm where they are translated into the various viral proteins described above.


The pgRNA, in addition to being the transcript for core and polymerase synthesis, also serves as the template for DNA synthesis by reverse transcription in the first instance. Being longer than genome length, it has a terminal redundancy which is the result of readthrough, passing the start of the transcript synthesis by about 120 nt, terminating with the poly-A tail. This redundancy contains a second copy each of the direct repeat 1 (DR1) and the encapsidation signal ε, a secondary RNA structure that encompasses the precore nucleotide sequence [55, 56].

Encapsidation of the pgRNA into the nucleocapsid is the next step in the virus life cycle and involves a series of events employing both viral and host factors. The polymerase has three functional domains, each one of which is in turn involved in DNA priming (terminal protein), reverse transcription (rt) and pgRNA degradation (RNAse H). The terminal protein is separated from the rt domain by a spacer region of unknown function. The polymerase engages the ε at the 5′ end of the pgRNA, a process that triggers encapsidation of the complex by the core protein (Fig. 3). It appears that the cap structure is also involved in this process [58], as well as eIF4E and heat shock proteins, which are thought to be instrumental in aiding encapsidation, stabilisation and activation of the polymerase [59, 60]. The C-terminus of the core protein, which as mentioned before is arginine-rich and in addition certain of its amino-acids are phosphorylated, is involved in pgRNA binding thus facilitating encapsidation. The core is also involved in reverse transcription initiation, nucleocapsid envelopment and fulfils other roles, as recently reviewed [61].

Replication strategy of HBV. a Primer synthesis b translocation and binding to DR1 c synthesis of the (-)-DNA strand with concurrent degradation of the pgRNA d circularisation of the (−)-DNA strand though covalent attachment of the polymerase at its 5′ end and preservation of the RNA primer (DR1) from the 5′ end of the pgRNA e hybridisation of the DR1 RNA primer with DR2 at the 5′ end of the (−)-DNA strand and extension for (+)-DNA strand synthesis. Modified from Karayiannis [57]

The subsequent steps in virus nucleic acid synthesis then take place within the nucleocapsid. The ε encapsidation structure consists of a lower stem, an upper stem, a side bulge and an apical loop, formed through base pairing of palindromic nucleotide sequences. Part of the side bulge of ε serves as a template for the synthesis of a 4-nucleotide-long DNA primer [62], covalently attached to the terminal protein domain of the polymerase through a phosphodiester linkage between dTTP and the hydroxyl group of a tyrosine residue in the terminal protein (position 63) [63, 64]. The polymerase–primer complex next translocates to the 5′ of the pgRNA, where it hybridises with part of the DR1 region with which the primer is homologous. This translocation event to the correct site, i.e. DR1 at the 3′ end of the pgRNA, is likely assisted by two other elements, ϕ and ω (Fig. 3), which interact with ε [65, 66]. (−)-DNA strand synthesis is thus initiated by reverse transcription as the complex advances towards the 5′ end of the pgRNA, having a terminal redundancy of about 10 nucleotides. The RNA template is concurrently degraded by the RNAse H activity of the polymerase, except for the final 11–16 or so ribonucleotides which encompass the DR1 region of the 5′ of the pgRNA. This ribonucleotide fragment serves as the primer for (+)-DNA strand synthesis [67]. It hybridises with the homologous to it DR2 at the 5′ end of the newly synthesised (−)-DNA strand necessitating a second translocation event. (+)-DNA strand synthesis then proceeds to the 5′ end of the (−)-DNA strand. Circularisation facilitated by the short terminal redundancy of the (−)-DNA strand allows template exchange and continuation of (+)-DNA strand synthesis along the 3′ end of the (−)-DNA strand [68]. Synthesis of both DNA strands occurs within the nucleocapsid and this is facilitated through pores in the capsid that allow passage of nucleotides. However, once the maturing nucleocapsid is enveloped by budding through the endoplasmic reticulum membrane, the nucleotide pool within the capsid is depleted leaving an incomplete (+)-DNA strand, hence the partially double stranded nature of the HBV genome [69].


Mature nucleocapsids, meaning that they contain the newly synthesised partially double-stranded rcDNA with the polymerase still bound to the 5′ end of the (−)-DNA strand, can follow one of two pathways. Early on infection and until sufficient amounts of HBsAg accumulate, nucleocapsids are shuttled back to the nucleus in order to replenish the cccDNA pool [52, 70]. In the final stages of morphogenesis, virions bud through the ER membrane where HBsAg proteins are already localised into the lumen, acquiring in the process their outer envelope [71]. A crucial determinant of these processes is the topology and conformational arrangement of L-HBsAg. Studies have established that about half of the protein lies with its N-terminus on the cytosolic side of the ER, whence it favors binding to the nucleocapsid and therefore budding. L-HBsAg with its terminus in the lumen allows its exposure on the surface of the virion and therefore easy access to the NTCP receptor during binding to hepatocytes [41].


It was generally assumed that, as HBsAg proteins accumulate in the ER-Golgi intermediate compartment, virions will accumulate in the lumen and follow the secretory pathway during cell exit, akin to the route that SVPs follow. It is now known that virions exit by using a different pathway which relies on proteins associated with the endosomal sorting complex required for transport, which form multivesicular bodies [72].

Infection through cell-to-cell spread

The life cycle described above is initiated following receptor-mediated attachment of a cell-free virus which moves from the sinusoidal lumen filled with blood into the Space of Disse. In view of the large size of the liver, the mode of viral spread must be efficient and of course dependent on the size of the inoculum, both of which determine the length of the incubation period. Clusters of virus-infected cells are frequently observed following immunohistochemical staining of liver sections from patients and experimentally infected animals [73]. Such observations suggest that HBV may also spread through cell-to-cell infection. Thus, movement of infectious virions to remotely situated hepatocytes possible only through their secretion into the extracellular milieu may not be the only means of virus spread.

It is generally accepted that HBV is not directly cytopathic, but that the histological pathology observed is the result of activation of the adaptive immune response and, in particular, the production of virus specific cytotoxic T-cells [74,75,76]. Lysis of infected hepatocytes evident by a rise in transaminase levels during acute infection or persistently raised levels in chronic infection entails their replacement through regeneration. Hepatocytes although terminally differentiated retain their capacity for substantial proliferation in response to liver injury. Their normal lifespan is longer than 6 months [77]. Replacement of lost hepatocytes may also occur through stem cell differentiation, as these cells may be resident in the region of portal tracts [78]. These events allow the liver to maintain its mass, but at the same time have implications when one considers which determinants are needed which facilitate cell-to-cell spread of HBV.

As described above, chronic HBV infection is maintained by the presence of the cccDNA minichomosome which persists throughout the lifespan of the infected hepatocyte. During liver regeneration as a result of immune-mediated hepatocyte lysis and hepatocyte proliferation to compensate for this, cell division may result in cccDNA decline and generation of cccDNA-free cells. Indeed, a recent study using the urokinase-type plasminogen activator/severe combined immune-deficiency mouse model with transplanted tupaia hepatocytes infected with woolly monkey HBV, indicated that, although there was increased hepatocyte proliferation, this was accompanied by a 75% reduction in virion production, as well as reduced pgRNA synthesis and core protein production [79]. It appears therefore, that during cell division, the cccDNA pool is reduced through loss of cccDNA, and that only a fraction of daughter cells carry cccDNA. Therefore, this mechanism only accounts for limited cell-to-cell spread of infection.

Cell-to-cell spread employed by other viruses often involves complex inter-cellular adhesion and is safeguarded by the presence of the appropriate receptors cellular polarity which determines whether virus is released into the extracellular compartment or basolaterally may contribute to pathogenicity, and finally intra-cellular trafficking (Fig. 2b, [80]). What is more, cell-to-cell spread is favored by viruses which are released from the infected cell through budding from the cell membrane or use an exocytic route such as that of multivesicular bodies described above for hepadnaviruses. This mode of virus spread/transmission avoids immune attack, in particular antibody-mediated blocking of receptor binding. In the case of HBV, experimental evidence suggests that cell-to-cell spread is favored by polarised release of virions [73, 81]. In fact, hepatocytes traffic and export HBV basolaterally by polarity-dependent mechanisms, which in the case of DHBV is associated with sphingolipid structures [82].

Exosomes are extracellular vesicles that originate from multivesicular bodies, 40–150 nm in diameter, which are produced by most cell types. They have been implicated in a number of processes, and, following their secretion into the extracellular space, they can mediate indirect cell-to-cell communication through the transfer of macromolecules, miRNAs and other RNAs, but also viruses [83]. Cell-to-cell spread of hepatitis C virus is well documented [84] and, indeed, exosomes appear to transmit the virus to hepatocytes in a receptor-independent manner [85].

The impact of cell-to-cell spread in HBV infection and persistence are not well understood at all. However, mathematical modelling has recently been employed in an attempt to shed light on these events and study the effect on outcome following potential actions by the immune system that can result in clearance, non-clearance or fulminant hepatitis of acute HBV infection. Cell-to-cell transmission, although not impacting establishment of infection, it appeared to hinder its clearance and suggested that it might be a factor in causing fulminant hepatitis. The model showed that it is the combination of cell-to-cell transmission strength, cytokine production and the T cell clearance number that decides the fate of HBV acute infection [86]. Clearly, further work is needed to shed more light on the mechanisms involved in cell-to-cell spread of HBV.

Viral Growth Curve

Unlike the growth curve for a bacterial population, the growth curve for a virus population over its life cycle does not follow a sigmoidal curve. During the initial stage, an inoculum of virus causes infection. In the eclipse phase, viruses bind and penetrate the cells with no virions detected in the medium. The chief difference that next appears in the viral growth curve compared to a bacterial growth curve occurs when virions are released from the lysed host cell at the same time. Such an occurrence is called a burst, and the number of virions per bacterium released is described as the burst size. In a one-step multiplication curve for bacteriophage, the host cells lyse, releasing many viral particles to the medium, which leads to a very steep rise in viral titer (the number of virions per unit volume). If no viable host cells remain, the viral particles begin to degrade during the decline of the culture (see Figure 8).

Figure 8. The one-step multiplication curve for a bacteriophage population follows three steps: 1) inoculation, during which the virions attach to host cells 2) eclipse, during which entry of the viral genome occurs and 3) burst, when sufficient numbers of new virions are produced and emerge from the host cell. The burst size is the maximum number of virions produced per bacterium.

Think about It

Unregistered Treatments

Ebola is incurable and deadly. The outbreak in West Africa in 2014 was unprecedented, dwarfing other human Ebola epidemics in the level of mortality. Of 24,666 suspected or confirmed cases reported, 10,179 people died. [1]

No approved treatments or vaccines for Ebola are available. While some drugs have shown potential in laboratory studies and animal models, they have not been tested in humans for safety and effectiveness. Not only are these drugs untested or unregistered but they are also in short supply.

Given the great suffering and high mortality rates, it is fair to ask whether unregistered and untested medications are better than none at all. Should such drugs be dispensed and, if so, who should receive them, in light of their extremely limited supplies? Is it ethical to treat untested drugs on patients with Ebola? On the other hand, is it ethical to withhold potentially life-saving drugs from dying patients? Or should the drugs perhaps be reserved for health-care providers working to contain the disease?

In August 2014, two infected US aid workers and a Spanish priest were treated with ZMapp, an unregistered drug that had been tested in monkeys but not in humans. The two American aid workers recovered, but the priest died. Later that month, the WHO released a report on the ethics of treating patients with the drug. Since Ebola is often fatal, the panel reasoned that it is ethical to give the unregistered drugs and unethical to withhold them for safety concerns. This situation is an example of “compassionate use” outside the well-established system of regulation and governance of therapies.

Ebola in the US

On September 24, 2014, Thomas Eric Duncan arrived at the Texas Health Presbyterian Hospital in Dallas complaining of a fever, headache, vomiting, and diarrhea—symptoms commonly observed in patients with the cold or the flu. After examination, an emergency department doctor diagnosed him with sinusitis, prescribed some antibiotics, and sent him home. Two days later, Duncan returned to the hospital by ambulance. His condition had deteriorated and additional blood tests confirmed that he has been infected with the Ebola virus.

Further investigations revealed that Duncan had just returned from Liberia, one of the countries in the midst of a severe Ebola epidemic. On September 15, nine days before he showed up at the hospital in Dallas, Duncan had helped transport an Ebola-stricken neighbor to a hospital in Liberia. The hospital continued to treat Duncan, but he died several days after being admitted.

Figure 9. Researchers working with Ebola virus use layers of defenses against accidental infection, including protective clothing, breathing systems, and negative air-pressure cabinets for bench work. (credit: modification of work by Randal J. Schoepp)

The timeline of the Duncan case is indicative of the life cycle of the Ebola virus. The incubation time for Ebola ranges from 2 days to 21 days. Nine days passed between Duncan’s exposure to the virus infection and the appearance of his symptoms. This corresponds, in part, to the eclipse period in the growth of the virus population. During the eclipse phase, Duncan would have been unable to transmit the disease to others. However, once an infected individual begins exhibiting symptoms, the disease becomes very contagious. Ebola virus is transmitted through direct contact with droplets of bodily fluids such as saliva, blood, and vomit. Duncan could conceivably have transmitted the disease to others at any time after he began having symptoms, presumably some time before his arrival at the hospital in Dallas. Once a hospital realizes a patient like Duncan is infected with Ebola virus, the patient is immediately quarantined, and public health officials initiate a back trace to identify everyone with whom a patient like Duncan might have interacted during the period in which he was showing symptoms.

Public health officials were able to track down 10 high-risk individuals (family members of Duncan) and 50 low-risk individuals to monitor them for signs of infection. None contracted the disease. However, one of the nurses charged with Duncan’s care did become infected. This, along with Duncan’s initial misdiagnosis, made it clear that US hospitals needed to provide additional training to medical personnel to prevent a possible Ebola outbreak in the US.

  • What types of training can prepare health professionals to contain emerging epidemics like the Ebola outbreak of 2014?
  • What is the difference between a contagious pathogen and an infectious pathogen?

Key Concepts and Summary

  • Many viruses target specific hosts or tissues. Some may have more than one host.
  • Many viruses follow several stages to infect host cells. These stages include attachment, penetration, uncoating, biosynthesis, maturation, and release.
  • Bacteriophages have a lytic or lysogeniccycle. The lytic cycle leads to the death of the host, whereas the lysogenic cycle leads to integration of phage into the host genome.
  • Bacteriophages inject DNA into the host cell, whereas animal viruses enter by endocytosis or membrane fusion.
  • Animal viruses can undergo latency, similar to lysogeny for a bacteriophage.
  • The majority of plant viruses are positive-strand ssRNA and can undergo latency, chronic, or lytic infection, as observed for animal viruses.
  • The growth curve of bacteriophage populations is a one-step multiplication curve and not a sigmoidal curve, as compared to the bacterial growth curve.
  • Bacteriophages transfer genetic information between hosts using either generalized or specialized transduction.

Multiple Choice

Which of the following leads to the destruction of the host cells?

[reveal-answer q=�″]Show Answer[/reveal-answer]
[hidden-answer a=�″]Answer b. The lytic cycle leads to the destruction of the host cells.[/hidden-answer]

A virus obtains its envelope during which of the following phases?

[reveal-answer q=�″]Show Answer[/reveal-answer]
[hidden-answer a=�″]Answer d. A virus obtains its envelope during release.[/hidden-answer]

Which of the following components is brought into a cell by HIV?

  1. a DNA-dependent DNA polymerase
  2. RNA polymerase
  3. ribosome
  4. reverse transcriptase

[reveal-answer q=�″]Show Answer[/reveal-answer]
[hidden-answer a=�″]Answer d. Reverse transcriptase is brought into a cell by HIV.[/hidden-answer]

A positive-strand RNA virus:

  1. must first be converted to a mRNA before it can be translated.
  2. can be used directly to translate viral proteins.
  3. will be degraded by host enzymes.
  4. is not recognized by host ribosomes.

[reveal-answer q=�″]Show Answer[/reveal-answer]
[hidden-answer a=�″]Answer b. A positive-strand RNA virus can be used directly to translate viral proteins.[/hidden-answer]

What is the name for the transfer of genetic information from one bacterium to another bacterium by a phage?

[reveal-answer q=�″]Show Answer[/reveal-answer]
[hidden-answer a=�″]Answer a. Transduction is the name for the transfer of genetic information from one bacterium to another bacterium by a phage.[/hidden-answer]

Fill in the Blank

An enzyme from HIV that can make a copy of DNA from RNA is called _________________.

[reveal-answer q=�″]Show Answer[/reveal-answer]
[hidden-answer a=�″]An enzyme from HIV that can make a copy of DNA from RNA is called reverse transcriptase.[/hidden-answer]

For lytic viruses, _________________ is a phase during a viral growth curve when the virus is not detected.

[reveal-answer q=�″]Show Answer[/reveal-answer]
[hidden-answer a=�″]For lytic viruses, eclipse is a phase during a viral growth curve when the virus is not detected.[/hidden-answer]

Watch the video: Virus Life Cycles Explained (January 2023).