Lectures 3-4: Viral Life Cycles in Cells

As discussed in lecture 1, there is a period between infection of a cell and the appearance of new infectious virus that is known as the latent period. During this time, several different stages in the virus life cycle are occurring. These are summarized below.

I: Steps in a "Model" Viral Life Cycle:

1) Attachment (Adsorption)
2) Penetration
3) Uncoating
4) Targeting
5) Gene expression.
        -synthesis of viral mRNA (transcription)
        -synthesis of viral proteins (translation)
6) Genome replication
7) Virion assembly/maturation
8) Release of new infectious virus
        -lysis : breakdown of cell membrane and release of virus
        -budding: viruses "bud" through cell membrane and are released without necessarily killing the cell. Viruses acquire            envelopes (membranes) during this process.

I: Attachment:

In most cases, specific attachment proteins on the surface of viruses bind to specific receptors on the surface of animal cells. Cellular receptors are usually either glycoproteins or glycolipids, and have other functions for the cell in addition to virus binding. The specific interaction between attachment proteins and cellular receptors is a major determinant of the host-range, or tropism of the virus. Some viruses have a very narrow host range, meaning that they can only infect one or a small number of cell types, while others have broad host ranges, meaning that they can infect a large number of different cell types. This is partially determined by whether the receptor for the virus is expressed on many or a limited number of cell types. Some examples of specific viruses and their known or probable cellular receptors are given in the following table.
 
Virus Viral Attachment Molecule Likely Cellular Receptor Target Cell Type
rabies virus

(Rhabdoviridae)

glycoprotein Acetylcholine receptor neuron
FIV

(Retroviridae)

gp120 CD9, leukocyte

differentiation Ag

T cell, macrophage
pseudorabies virus

Herpesviridae

gC Heparin sulfate proteoglycans many cell types
influenza A virus

Orthomyxoviridae

Hemagglutinin (HA) Sialic-acid containing glycoproteins respiratory epithelium 

Understanding these virus/cell interactions can be important in treating and/or preventing disease. For example, antibodies that bind to the viral attachment molecule or to the cellular receptor can disrupt the normal interactions and prevent the first steps of the viral life cycle, thereby preventing infection. This is an important consideration in the development of vaccines.

II: Penetration:

Once bound to the cell membrane, the virus, or at least its nucleic acid, must enter the cell. Animal viruses do this primarily by one of two mechanisms.

Endocytosis: Many viruses enter cells via receptor mediated endocytosis . In this pathway, viruses bind to receptors at coated pits. The coated pits pinch off to form coated vesicles, which are uncoated and then fuse with endocytic vesicles, and eventually with lysosomes. As they go through this process, the endosomes become more acidic (remember lysosomes are a very acidic environment where the breakdown of cellular macromolecules occurs). Viral genomes must therefore escape the endosome before they are destroyed by proteases, nucleases, etc. For enveloped viruses, this usually occurs by membrane fusion mediated by a fusion protein. One example of this is the influenza virus HA protein, which undergoes a conformational change induced by the acidic environment of the endosome. After undergoing this change, it then induces membrane fusion, releasing the nucleocapsid into the cytoplasm. The genomes of non-enveloped viruses must also somehow escape the endosome. Again, this is often initiated by a conformational change in a capsid protein induced by the acidic environment of the endosome. In the case of poliovirus (a picornavirus), the capsid proteins undergo a conformational change that allows a hydrophobic domain on VP4 to be exposed and inserted into the membrane, forming a channel through which the RNA enters the cytoplasm.

Direct Membrane Fusion: Some enveloped viruses directly fuse with the plasma membrane. In these cases the activity of a fusion protein is not dependent on pH change, but rather is induced in response to receptor binding.
 

III: Uncoating and Targeting

With some viruses, the genome is completely released from the capsid during or after penetration. This is known as "uncoating". In others, such as retroviruses and reoviruses, the first stages of the viral replication cycle (transcription, replication) actually occur inside the capsid. These capsids have undergone some conformational changes during infection that allow viral gene expression and/or replication to begin, and the resulting structures are sometimes known as partially uncoated particles. Since almost all DNA viruses replicate in the nucleus of infected cells, they must be targeted there. In many cases the entire nucleocapsid enters the nucleus, where uncoating then takes place.
 

IV: Gene Expression and Genome Replication

In order for new virus to be assembled, both new viral genomes and other virion components (proteins) must be produced. Exactly how this occurs varies greatly depending on the family (and Baltimore Class) of virus. Summaries of common schemes are given below, and several specific viruses will be discussed in detail later in the semester.

Viruses with DNA Genomes:

Almost all DNA viruses have genomes that are similar to the host cell; that is, they are composed of double stranded DNA, and are therefore able to utilize host enzymes to express viral genes and replicate viral DNA. Most DNA viruses replicate in the cell nucleus, which is where cellular replication and transcription proteins are localized. After infection, the nucleocapsid of DNA viruses is therefore usually delivered to the nucleus where uncoating occurs. An exception is poxviruses, which replicate in the cytoplasm of infected cells.

A) Viruses with Small, Double Stranded DNA genomes.

Some of the best studied virus families fall into this group, including the Papovaviridae (simian virus 40, murine polyoma virus, bovine papilloma virus). Because of their relatively small genome size (5 - 10 kb), the facts that they can grow in tissue culture and use mostly cellular molecules to transcribe and replicate their DNA, these viruses have been used as model systems to study both viral replication and mammalian gene expression and DNA replication. For these simple DNA viruses, the replication cycle can be broken down into the following steps.

1) Early gene expression. The first stage in the viral replication cycle is expression of the viral early genes. Transcription of these genes occurs using cellular RNA polymerase II and cellular transcription factors. These proteins bind to the viral DNA in regions called early promoters/enhancers, and promote synthesis of the early pre-mRNAs. The early RNAs are processed (capped, polyadenylated and spliced) in the nucleus, and are then transported to the cytoplasm where they are translated, giving rise to the early proteins. In many cases, one primary transcript can give rise to several different mature mRNAs via a process known as alternative splicing. These different mRNAs then encode different proteins, as in the example of mouse polyoma virus. The use of alternative splicing is one mechanism by which viruses maximize the coding potential of their genomes.

Early proteins typically play several roles in the viral life cycle, including the following.

a) They are required for replication of the viral genome. Although small DNA viruses use cellular DNA polymerases and other enzymes to replicate their DNA, one or a few viral gene products are usually also required. In the case of polyomaviruses, these viral proteins bind to the viral origin of replication, are required for the initiation of replication, and may also play a role in elongation.

b) Early proteins are also involved in the regulation of viral gene expression. Typically they activate late gene transcription, and may also down regulate (autoregulate) their own transcription. The late genes can only be activated after viral DNA replication has begun. Thus, there are 2 phases of gene expression; early (pre-DNA replication) and late (post-DNA replication). This is true for virtually all DNA viruses.

c) Early proteins also play a role in altering host-cell metabolism by activating pathways that induce cell entry into S phase. In animals, most cells are not actively dividing, and are arrested in the G1 phase of the cell cycle. Cellular DNA synthesis only occurs during the S phase of the cell cycle, and cellular replication enzymes are only present during S phase. Because small DNA viruses require these cellular enzymes to replicate the viral DNA, they have evolved ways to induce resting cells to enter S phase.

2) Viral DNA replication. Once the viral early genes have been expressed, and the cells have been induced to enter S phase, viral DNA is replicated. This occurs in the nucleus of infected cells, and gives rise to new viral genomes. Many hundreds or thousands of new viral genomes can be produced in the nucleus of a lytically infected cell.

3) Late gene expression. After viral DNA replication has begun, the late genes are transcribed and translated to give rise to late proteins. Late genes encode the structural proteins of the virus, including capsid proteins and, for enveloped viruses, the matrix and envelope proteins. In the case of the polyomaviruses there are 3 late proteins, VP1, VP2 and VP3. VP1 is the major structural protein of the capsid, with 360 molecules being present per capsid. VP2 and 3 are less abundant, with approximately 30-60 molecules per virion. Both late and early viral proteins are synthesized in the cytoplasm, but are often transported back to the nucleus where both viral replication and nucleocapsid assembly occurs. The accumulation of high levels of viral proteins can sometimes be observed as "inclusion bodies" in infected cells.

Summary: For these fairly simple DNA viruses, the replication cycle can be broken down into the following steps: 1) Early gene expression (using host cell RNA polymerase II and transcription factors). The early genes encode proteins that are required for viral DNA replication and for late gene expression. 2) Replication of the viral genome (using host cell DNA enzymes, plus a limited number of viral proteins. 3) Late gene expression. The late genes encode the viral structural proteins.

B. Viruses with Small, Single-Stranded DNA Genomes

The sole family in this class is the Parvoviridae, which includes a number of animal pathogens including feline panleukopenia virus and canine parvovirus. The genomes of these viruses are approximately 5 kb in length, and only the negative strand is encapsidated. Before any viral genes can be expressed, the single stranded genome must first be converted to double stranded DNA, which is then transcribed to give rise to the viral mRNAs. Replication takes place in the nucleus, and requires host S - phase functions.

C: Viruses with Medium and Large Double Stranded DNA Genomes

In contrast to the Papovaviridae (genome about 5-8 kb), families such as the Adenoviridae (canine adenovirus, equine adenovirus, etc) and the Herpesviridae (pseudorabies virus, many others) have large double stranded DNA genomes (30-40 kb for adenoviruses and 100-200 kb for herpesviruses). The replication scheme of these viruses is similar to that of the small DNA viruses, although since the genomes are larger they are able to encode additional proteins. These proteins can either be structural (capsid proteins, matrix proteins, envelope proteins, etc) or regulatory (transcription factors, polymerases, enzymes involved in nucleotide metabolism, etc). Thus, these viruses typically have a more complex structure than the small DNA viruses, and their replication may be more independent of cellular enzymes. The fact that these viruses encode some enzymes that are required for their gene expression and replication also makes them susceptible to anti-viral drug therapies. For example, if a virus encodes a unique enzyme (polymerase, etc) that is not used by the cell, then drugs that block or inhibit this enzyme may block viral replication without damaging host cells. If a virus uses exclusively cellular enzymes, then it is very difficult to develop drugs that will target the virus without also killing the host animal.

For the large DNA viruses, the replication cycle can be broken down into the following distinct steps.

1) Expression of Immediate Early Genes. These are genes that are the first expressed after infection, and typically encode transcription factors that activate the next phase of gene expression, that of the early genes. Synthesis of immediate early mRNAs does not require any prior viral gene expression.

2) Expression of early or delayed early genes. These are genes that are activated by the products of the immediate early genes. They typically encode polymerases and other proteins required for viral DNA replication, as well as transcription factors that activate the late genes.

3) Viral DNA replication.

4) Viral late gene expression. Once again, the late genes encode the viral structural proteins, and require both immediate early/early gene expression and viral DNA replication in order to be expressed.

A unique family of viruses with large double stranded DNA genomes is the Poxviridae (smallpox, cowpox vaccinia, etc), whose members are the largest, and probably most complex, animal viruses known. The genomes of poxviruses range from 130 - 375 kb. What distinguishes a poxvirus life cycle from all others is that they are the only DNA viruses that replicate totally in the cytoplasm of infected cells. They therefore encode all of the enzymes needed for their transcription and replication, since the cellular enzymes are in the nucleus.
 

Viruses with RNA Genomes

As discussed previously, many families of animal viruses have RNA as their genetic material. These RNA genomes can be single stranded (+ sense, - sense, ambisense) or double stranded. Each class of genome has a different replication / gene expression strategy, and there is considerable variation within each class. In this section, we will summarize some of the general principles, and will discuss selected viruses in more detail in later lectures.

Some General Points about Viruses with RNA Genomes:

- Most replicate entirely in the cytoplasm of infected cells. Exceptions are orthomyxoviruses and retroviruses.

- They generally have high mutation rates due to high error rates of RNA dependent RNA polymerases, which have no proofreading function.

- Show high levels of "recombination".

- In the case of viruses with segmented genomes, there can be "reassortment" of the segments in cells infected by more than one strain/variant of virus.
 

A) Replication and Gene Expression of + Strand Viruses

These viruses share the common feature that the genome itself can serve as mRNA and be directly translated to give rise to proteins. The naked RNA ( with no protein attached) of these viruses is infectious because the viral replicase, which is not present in uninfected cells, can be translated directly from the viral RNA.

The general replication scheme used by + strand viruses is the following.

1) Synthesis of viral proteins (including replicase) from the viral genomic RNA.

2) Synthesis of viral RNA (- strand, then new + strand) using the viral replicase.

3) Use of the new progeny + strand for 2 purposes

a) production of more viral proteins (including structural proteins)

b) to be packaged into virions as new virus genomes.
 

Like DNA viruses, many RNA viruses have both "early" and "late"phases of gene expression, with regulatory proteins being synthesized early and structural proteins late. In addition, the structural proteins are usually required in greater amounts, so there is a need to regulate the expression of viral genes both temporally and quantitatively. There are several general strategies by which RNA viruses produce and regulate the expression of proteins, and some many viruses utilize more than one strategy. Several examples are given below.

1) Use of Polyproteins: Some of the simplest RNA viruses are members of the Picornaviridae, where the best studied example is poliovirus. Picornavirus RNA is released from the virion and enters the cytoplasm with a single protein (Vpg) bound at its 5' end. The entire viral RNA is then translated into a single "polyprotein" using the host cell ribosomes. This polyprotein is then cleaved using 2 virus-encoded proteases, which are active as part of the polyprotein. There is a specific cleavage pattern that occurs in a certain order, giving rise to cleavage intermediates. In fact, the intact polyprotein is never really seen in cells because it is cleaved virtually as it is being synthesized. The final cleavage products include the viral replicase (polymerase, or pol), the Vpg protein that binds to the 5' end of the RNA (and is used as a primer for replication), the proteases, and the structural proteins VP1, 2, 3 and 4. As stated above, the polymerase can be used to synthesize - strands, which then serve as templates for new plus strands. These new plus strands can then be used either for protein synthesis or (late in infection) be packaged into virions.

2: Use of Sub-Genomic mRNAs: As mentioned above, many plus strand viruses undergo two distinct phases of gene expression, which can be termed "early" and "late". Examples of this are the Togaviridae and the Coronaviridae. In the first phase, the 5' end of the viral genome is translated directly to give rise to the non-structural proteins including the RNA dependent RNA polymerase. These genes are located at the 5' end of the genome, since translation in mammalian cells usually starts at the first AUG on the mRNA. The polymerase is then used to synthesize a - strand. This - strand can be used for 2 purposes. A) as a template for the synthesis of new, intact + strand genomes or B) as a template for the synthesis of subgenomic mRNAs. There are a variety of mechanisms by which these subgenomic mRNAs can be synthesized (including leader sequences, internal promoters, etc.)

The new intact + strands can be used to make more non-structural proteins or, late in infection, are packaged into virions as new viral genomes. The subgenomic mRNAs are used to synthesize the structural proteins of the virus, including both capsid and envelope proteins. Note that both the "early" and the "late" proteins can be made as polyproteins.

3: Translational Readthrough and Frameshifting: Many RNA viruses use additional strategies to control the timing and level of expression of proteins including translational read-through and frame-shifting. In read-through: an amino acid is inserted where a stop codon occurs, so translation continues. This results in a longer protein that is in frame with the shorter one. In frame-shifting, the ribosome shifts reading frame during translation. This also results in a longer protein, but the reading frame has been changed.
 

B) Replication and Gene Expression Strategies of (-) Strand RNA Viruses:

By definition, the genome of these viruses cannot be used as a template for protein synthesis, so an RNA replicase (RNA dependent RNA polymerase) cannot be synthesized directly as it is with the + sense viruses. Since cells do not contain such an activity, (-) strand viruses must contain a replicase/polymerase as part of the virion. The overall replication/gene expression strategy for - strand viruses is outlined below.

1) Synthesis of + strands using the virion associated polymerase. These + strands can either be full length or subgenomic mRNAs.

2) Translation of viral mRNAs (+ strand) to make viral proteins, including additional polymerase.

3) Production of new - strands from the intact + strands, which are then used either as templates for additional + strands, or packaged into virions as new genomes.

One of the best studied families of - strand viruses are the Orthomyxoviridae, a family of enveloped viruses made up of the influenza viruses. This family has several interesting characteristics, some of which are described below.

1) They have the ability to agglutinate red blood cells, due to the hemagglutinin (HA) protein on the envelope. We will use this property in one of the lab exercises.

2) They contain segmented genomes, where each segment encodes1-2 proteins. In order to be infectious, each virion must contain one copy of each segment - otherwise it will be missing genes. This genomic strategy gives these viruses the ability to evolve very rapidly, due to a process called antigenic shift. Antigenic shift occurs when two subtypes of virus (2 antigenically distinct viruses) infect the same cell. During the process of virus assembly, there may be mixing or reassortment of the segments so that virus A picks up one genome segment from virus B (or the opposite). This causes a rapid shift in the antigenicity of virus A. For example, if the new segment that was picked up encodes the HA protein, it may have several different epitopes (aa sequences) compared to the original. In contrast to antigenic shift, the gradual antigenic changes that occur in viruses, particularly in RNA viruses, due to mutations caused by errors in replication, are known as antigenic drift.

3) Influenza viruses have the ability to infect a broad range of host species. In general, a given subtype preferentially infects certain species. However, they sometimes jump from one to another. In addition, if one animal is infected with 2 different subtypes, antigenic shift may occur, giving rise to an "new" virus that is antigenically distinct from either parent. This new virus may then have the ability to grow in another species.

4) Influenza viruses replicate in the nucleus of the cell. They are the only pure RNA viruses to do so.
 

C) Replication and Expression of Viruses with Double Stranded RNA Genomes

Members of the Reoviridae and Birnaviridae families contain segmented, double stranded RNA genomes. This gives them the ability to undergo antigenic shift, as discussed for Orthomyxoviruses. The strategy for Reovirus gene expression is very interesting. Early transcription occurs in partially uncoated virions within the cytoplasm, using enzymes that are brought in with the virus. This gives rise to early mRNAs. These mRNAs then translated into protein. The final stages of viral replication and gene expression actually occur inside newly formed virions within the infected cell.

Replication and Gene Expression of Retroviral Genomes

Members of the Retroviridae family contain a single stranded, + sense RNA genome. However, since they replicate via a DNA intermediate, both their replication and gene expression strategies are quite different than other RNA viruses. They will be discussed in a separate lecture later in the course.

Gene Expression in RNA Viruses (summary).

RNA viruses have evolved several strategies for maximizing their coding potential, and for regulating the amounts of various products produced. In general, viruses need to produce greater amounts of structural proteins, such as capsid, matrix and envelope proteins, than they do of regulatory proteins such as polymerases. The basic gene expression strategies used by RNA viruses are summarized below. A single virus may use a combination of several of these strategies.

1) Synthesis of a polyprotein that is cleaved to give mature proteins. This provides limited opportunities for varying the amounts of different proteins produced, since they are all synthesized together. Still, the stability of the mature proteins might differ, or the efficiency specific cleavage events might differ.

2) Translational frame-shifting or read-through allows a virus to make downstream products in lower amounts.

3) Synthesis of subgenomic-mRNAs. Some RNAs may be synthesized at higher frequencies than others. They may have stronger promoters, or differential processing (cleavage) of RNAs may occur to give different amounts of specific transcripts.
 

V: Virus Assembly and Release

Once new viral genomes and proteins have been produced, they are assembled into new virions. This usually occurs in a very specific order. For example, for many viruses, the viral capsid is partially assembled (ie, the newly synthesized capsid proteins associate together into a capsid-like structure). The viral genome is then inserted into the capsid to form a nucleocapsid, which then undergoes some type of maturation that can include proteolytic cleavage of capsid proteins. In the case of non-enveloped viruses, these newly formed virions accumulate in the cell and are released by cell lysis.

In the case of enveloped viruses, the nucleocapsids often assemble on the surface of a cellular membrane (such as the plasma membrane, the nuclear envelope, the ER, etc.) in regions of the membrane where viral envelope proteins are concentrated. Matrix proteins, if present, are underlying this part of the membrane. The virus then "buds" through the membrane to give rise to enveloped viral particles. These particles can then go through additional maturation events to give rise to infectious virus. In the case of viruses that form on the plasma membrane, they can bud from the cell without causing cell lysis. Other enveloped viruses, however, are lytic.

For more information on viral replication cycles, see  Replication Lectures in Course 224.