Virus-Host Cell Interaction, Viral Genetics, and Pathogenesis

Permissive Cell

Permits production of progeny virus particles and/or viral transformation

Non-permissive cell

Virus cannot replicate, but, it may be able to transform the cell

Abortive infection

No progeny virus particles are produced, however, the cell may die because early viral functions can occur

Persistent infection

Small number of virus particles are produced; little or no cytopathic effects (CPE)

Interference

• Infection by virus A will inhibit second infection with virus B

  • Transforation: Induces unregulated cellular growth; cells form tumors in susceptible animals

Latency

Viral genetic information remains in host cell without production of virus; may be activated at a later time to produce virus and/or transform the host cell

Cytipathic effects (CPE)

• Nucleus: Inclusion bodies, thickening of the nucleus, swelling, nucleolar charges

  • Cytoplasm: Inclusion bodies, vacuoles

  • Membranes: Cells round up, loss of adherence, cell fusion (syncytia)

  • Cellular: Lysis (disintegration)

One Step Growth Curve

• Cells are infected at high multiplicity of infection (MOI) to ensure that every cell is infected. The number of infectious virions inside and outside the cells are measured at various times

• Latent Phase: No virions outside the cells

• Eclipse Phase: No virions outside or inside the cells

• Early Phase: Synthetic phase, viral gene expression for genome replication

• Late Phase: Begins with synthesis of proteins necessary for construction of the new particles

• Burst Size: Number of infectious virions released per average cell. Range less than 10 to millions.

Immune Responses

• Humoral and cell-mediated

Interferons

• Small proteins produced by cells in response to virus infection, mitogens, or immune stimulus

• Three major types of interferon

  • IFN-alpha (leukocyte) virus induced

  • IFN-beta (fibroblasts) virus induced

  • IFN-gamma (T-cells) antigen or mitogen induced

• Interferon is activated by double stranded RNA upon viral infection. Both RNA and DNA viruses induce interferon production.

• Interferon activates

  • Production of an oligoadenylate synthetase which in turn, actiavtes production of ribonuclease that degrades mRNA

  • Production of a protein kinase that phosphorylates and inactivates one of the subunts of initiation factor (elF-2) necessary for protein synthesis

• The action of both enzymes require the presence of double stranded RNA

  • Thus, interfeon inhibits both viral and host protein synthesis

    • This results in the death of an infected cell and no spread of viral infection to uninfected cells

  • In addition, interferon prepares uninfected cells to fight viral infections.

    • Interferon induces ologosynthetase and protein kinase in uninfected cells so if they are infected by a virus, the dsRNA will activate these two enzymes and inhibit virus replication

  • Presence of interferon will only induce oligosynthetase and protein kinase but not activate because there is no dsRNA in uninfected cells.

    • Thus, interferon kills only infected cells but not uninfected cells

Viral Genetics - Mutations

• Change in one or a few nucleotides

• Causes subtle changes in a viral antigen

• Allows for a virus to escepe pre-existing antibody or primed cytotoxic T-lymphocytes

• Best example: antigenic drift in influenze A virus; Point mutations accumulate in influenza virus genes encoding for two envelope proteins, hemagglutinin and neuraminidase, resulting in changes in the antigenic structure of the virions

• The retroviruses also show high rates of variation because of the reverse transcription of RNA genome to DNA genome in infected cells (HIV-1)

• Spontaneous Mutation - mutation in the absence of any known mutagen

• Induced Mutation - mutation derived froom mutagen treated populations

• Types of mutations - Plaque Morphology mutations

  • Host Range Mutations

  • Temperature Sensitive Mutations

  • Nonsense (Amber) Mutations

  • Cold Sensitive Mutations

  • Deletion Mutations

Gene Interactions between Viruses

• Complementation

  • Interaction of viral gene products resulting in one or both parental mutants

  • Genotypes unchanged

• Recombination

  • Exchange of genetic information with a closely related co-infected virus

  • Both DNA and RNA viruses exhibit recombination

  • Good Example: antigenic shift of influenza A viruses due to either recombination or reassortment of segments of viral RNA. Leads to new viral strains, which have resulted in pandemics (its about time for a new one)

• Mechanisms of Recombination

  • Intramolecular Recombination

  • Copy-Choice Recombination

  • Ressortment

• Gentic Reactivation

• Recombination in Nature

Defective Interfering (DI) Particles

• DI Particles are smaller than the wild type virus

• DI Particles are missing a portion of their genome

• They are defective particles, and cannot replicate without complete virions (Helper virus)

• DI particles interfere with normal virus replication

• Von Magnus phenomenon: As the titer of DI particles increase, the titer of infectious particles decreases

Defective Viral Genomes

• Nature of Defective Virus Genomes

  • Integrated Defective Viral Genomes

  • Satellite Viruses and RNAs

  • Pseudovirions

  • Conditionally Defective Genomes

  • Helper-Virus-Dependent DI Virus Genomes

• Structure of DI Genomes: Mechanisms of Generation and Interference

  • RNA Viruses

    • Negative-Stranded RNA viruses

    • Positive-Stranded RNA viruses

  • DNA Viruses

• Biological Roles of Defective Genomes

  • Gene Transfer and Expression

    • Gene trasduction from host to host

    • Gene conversion by defective bacterial viruses

    • Conversion and transduction by genes of animal viruses

  • Modulation of Virulent Virus Lethality by DI Particles and Satellite Viruses

    • Construction of defective viral genomes as potential antiviral agents

  • Involvement of Defective Genomes in Virus and Host Evolution

Pathogenesis of Viral Infections

• Viral pathogenesis can be defined as the methods by which viruses produce disease in the host

• Spread in the Host

• Localized vs Systemic Infection

  • Some viruses produce the brunt of their injury in close proximity of their site of entry into the host

  • This involves the upper respiratory infections of: influenza virus, parainfluenza virus, rhinovirus, and coronaviruss

  • Gastrointestinal infections caused by papillomaviruses

  • The dermatological infections caused by papillomaviruses

  • Viruses rarely spreads beyond the epithelial cell layer, although in some cases involvement of regional lymph nodes and even systemic invasion can occur

  • Factors that act to confine the scope of these infections are unknown

• Polarized Infection of Epithelial Cells

  • Certain viruses preferentially bud from either the apical or basal surfaces of polarized epithelial cells

  • Preferential release of virus from a particular surface of polarized epithelial cells may influence whether the subsequent disease is localized or systemic

  • If this results exclusively in release of virus toward the luminal surface, it is called localized infection

  • Conversely, release of virus toward subepithelial tissude may facilitate muscosal invasion and the development of systemic infection

  • Viruses with polarized patterns of release from epithelial cells include: vaccinia, vesicular stomatitis virus (VSV), certain retroviruses, influenza, parainfluenza, SV40, polio, etc.

  • Not all viruses exhibit polalrized patterns of infection and release, and some, such as poliovirus, are capable of bidirectional entry into polarized epitelial cells

• Hematogeneous Spread

  • Viruses that produce systemic disease must spread from their site of entry into the host to their ultimate target tissue

  • Two major pathways

    • via the bloodstream

    • via the nerves

  • In the case of bunyavirus, if the inoculum size is sufficient, passive viremia may be adequate to deliver the virus to the CNS and initiate lethal encephalitis

  • Viruses such as flavivirus, measles, and polio replicate at the primary site of entry followed by spread to regional lymph nodes and further to the target tissues

  • In almost all cases, some degree of replication at the primary site or near the site of entry in the host seems to preced the onset of viremia (primary viremia)

  • Further replication at other sites such as with higher virus titer is called secondary viremia

  • Virus spread is through cell free virus or cell associated virus

  • Important sites of primary replication for viruses that spread through the lood steam include

    • Togaviruses - skeletal muscle

    • Flaviviruses - connective tissue, muscle, endothelial cells, and reticuloendothelial organs

    • Enteroviruses - brown fat

    • Phagocytic cells in the reticuloendothelial system (RES) and serum factors including complement and antibody act to aid in clearance of virus from the bloodstream

    • The nature of interaction between the virus and the macrophages of the RES seems to be an important determinant of the development of viremia

    • If the virus can avoid phagocytosis by macrophages, this will facilitate the maintenance of viremia

    • For some viruses, uptake by macrophages results in inactivation and the factors that inhibit phagocytosis can serve to amplify viremia

    • Certain viruses replicate in macrophages resulting in the amplification of viremia. Not all, but some togaviruses, poxviruses, lentiviruses, coronaviruses, arenavirruses, and reoviruses have been shown to replicate in macrophages

• Neural Spread

  • Important route of viral spread

  • Herpesviruses, polioviruses, certain arboviruses, rabies virus, reoviruses, coronaviruses spread via neural routes

  • Primary infection at peripheral sites, although not essential, may facilitate the spread process by amplifying the size of the initial inoculum

• Molecular and Genetic Determinants of Viral Spread

  • Viral genes are involved in determining the process of viral spread

  • In reovirus infection, T1Lang (T1L) spreads to the CNS via the bloodstream whereas T3 Dearing (T3D) spreads via nerves

  • Viral S1 gene encoding the outer capsid protein (omega 1) determines the capacity of these viruses to spread via these routes

  • Several genetic factors influence HSV neuroinvasiveness, neurovirulence, and latency determining the neuroinvasive capacity of certain HSV strains

  • In bunyavirus infectionn, neuroinvasive and nonneuroinvasive capacities are determined by G1 glycoprotein of the virus

Tropism

• Is the capacity of viruses to infect discrete population of cells within an organ during an acute infection-producing systemic illness

• Cell tropism is influenced by the interaction between host and viral factors

• In addition to virus-receptor interactions, a variety of other virus-host-interactions can play an important role in determining the tropism of a virus

• Presence of a functional viral receptor may be insufficient to allow viral infection of the target cells, as has been shown in the case of HIV, poliovirus, rotaviruses, and mouse hepatitis virus

• Both poliovirus and mouse hepatitis virus seem to require additional cellular factors after receptor binding

• Although CD4 is clearly an HIV receptor, many cells expressing CD4 remain resistant or allowing inefficient infection

• HIV has dual tropism, infecting both lymphocytes and monocytes-macrophages. The cell tropism is determined by the variable region 3 (V3) of the envelope gene. However, other regions such as V1 and V2 may be important in the virus spread within macrophages

Pathogenesis Steps

• I. Binding to the Target Cells and Penetration of Cell Membranes

  • Interaction between the virus and receptor play an important role in tropism

  • Viral receptors

  • Sialic acid residues are important components of the receptor for certain coronaviruses, orthomyxoviruses, and reoviruses

  • Sometimes several different viruses belonging to the same family utilize the same receptor, as seems to be the case with heparin sulfate molecules, which are used by HSV-1, CMV, and bovine herpes-1 for initial attachment

• II. Viral Gene Expression

  • Although the interaction betwene a virus and its receptor is a major determinant of viral tropism, other important factors such as viral regulatory elements, enhancers, and regulation of viral transcription play some role in determining the cell tropism

  • Bot SV40 and polyoma virus enhancer elements show some degree of cell type specificity

  • In JC virus, the causitive agent of progressive multifocal leukoencephalopathy (PML), the enhancer is active only in glial cells and not HeLa cells

  • In tcase of human papillomavirus 11, the enhancer may play an important role in tissue tropism, as it can be shown that the enhancer is specifically active in keratinocytes (mature skin cells)

  • Enhancer elements within the LTR of avian and murine retroviruses play a key role to produce both neoplastic and nonneoplastic diseases

  • Viral enhancers in HBV promoter is more active in hapatic cells compared to nonhepatic cells

• III. Site of Entry and Pathway of Spread

  • Tropism of the virus may depend on both the site of entry and its oathway of spread

  • For polyomavirus, the route of inoculation seems to determine the site of primary replication and the eventual site of persistent infection

  • For some viruses, the pathway of spread may vary depending on the site of inoculation, and this difference may in turn influence tropism

Inoculation

The process of introducing a pathogen, antigen, or modified microorganism into a living organism, substance, or growth medium.

Molecular and Genetic Determinants of Virulence

• Viral envelope, core, matrix, nonstructural proteins and noncoding regions influence pathogenesis and virulence

Naked Capsid Viruses

• Attenuated vaccine derivatives, mousse model of poliovirus infection, and transgenic mice expressing poliovirus have been useful tools for analyzing poliovirus virulence and pathogenesis

• Mutations in poliovirus outer capsid protein including VP1 (amino acid position 143) are determinants of attenuation in vitro and in vivo

• Outer capsid proteins are also determinants of virulence for other picornaviruses such as coxsackievirus B4 - VP1 (Threonine at position 129)
  In reoviruses T1 Lang and T3 Dearing Infection, S1 gene encoding the cell recognition protein (omega 1) determines the tropism and virulence

Enveloped Viruses

• In the case of togaviruses, mutations in either E1 or E2 envelope glycoprotein result in altered virulence and highly attenuated viral strains

• Differences in flavivirus virulence have also been related to changes in the envelope (E) protein (attenuated 17D vaccine strain)

• Genetic determinants of influenza virus virulence have been mapped mainly in the viral hemagglutinin and neurominidase proteins

• Mutations in HIV- env protein is directly related to virulence and cytopathogenicity

Viral Polymerases, Core, Matrix, and Nonstructural Proteins

• Mutations in the RNA polymerase of poliovirus 1 may be a determinant of neuroattenuation, although the effect is less pronounced than that of attenuating mutations in the 5’-noncoding regions

• Matrix proteins may also play a role in cytopathicity of many enveloped viruses

• Among the HIV genes and cis-acting sequences that have been suggested to play a role in cytopathicity are tat, rev, nef, vif, nef, vpr, TAR, RRE, and portions of LTR

• HSV polymerase and nonstructural proteins have been shown to affect neurovirulence

Noncoding Regions of the Viral Genome

• Noncoding regions of viral genomes may be important determinants of virulence and pathogenicity by influencing replicatoin and transcriptioin

• The 5’-noncoding region of poliovirus (750 bp) has extensive RNA secondary structure including multiple stem loop and cloverleaf structure. This region has been shown to be vital for poliovirus expression and cytopathic effects.

• A substitution of U at position 472 in polio strains results in weaker interaction between poliovirus RNA and the host cell translational initiation factor. The P/Leon vaccine strain contains a C to U at position 472 that seems critical for its attenuated phenotype.

• Non coding regions play important role in the pathogenicity of several other viruses such as picornaviruses, togaviruses, flaviviruses, influenza viruses, and retroviruses.

Transmission of Viral Infections

• Virus transmission typically begins with shedding from the infected host through respiratory, enteric, or genitourinary secretions

• Arbovirus infections typically involve the ingestion by an arthopod vector

• Transmission of HIV, HBV, HCV, HTLV< CMV, EBV can occur when contaminated tissues or blood products are transplanted or transfuded into a susceptible host

• Several factors, including the titer of virus in the blood, the duration of viremic state, the amount of material transmitted, and the route of transmission influence the subsequent likelihood of infection.

Host Factors

• Play an important role in the pathogenicity of viral infections

• During epidemics of viral diseases, we see a range of outcomes varying from asymptomatic infection to fatal disease

• Inoculation of HBV contaminated lots o yellow fever virus vaccine into 45,000 military personnel resulted in the development of clinical hepatitis in only 2% of those vaccinated

• People infected with HIV don’t develop AIDS at the same time after infection

• Host factors may include immune status, genetic background, age, and nutrition

• Host immune response and genetic factors are the most important factors influencing the outcome of viral infection

• In general, several host factors that support viral replication have been identified and characterized

• In several human viral infections, there is a correlation between the age of the host and the severity of viral infection

• Some viruses tend to produce less severe infection in infants (e.g. varicella, mumps, polio, EBV, hepatitis A), whereas other more sever (e.g. rotaviruses, RSV)

• Hormones can also influence the outcome of viral infection

• Several viral infections including polio, hepatitis A and B, and smallpox are commonly more severe during pregnancy

Viral Transformation

• Many DNA viruses and some retroviruses (oncogenic viruses) can convert noraml cultured cells into abnormal cells called tumors (malignant or benign)

  • The process is called viral transformation

• Malignant tumor cells exhibit the following properties

  • Cell morphology altered

  • Fail to grow in normal cells

  • Grow to much higher cell densities than normal cells

  • Lower nutritional requirements than normal cells

  • Grow indefinitely in cell culture

• The process that converts normal cells to become abnormal caused by viral infectio and possess the above properties is called malignant transformation

• All known DNA animal viruses with the exception of parvoviruses are capable of causing aberrant cell proliferation under some conditions

• Viral transformation is the result of integration into the chromosome (papovavirus, adenovirus,, retrovirus) and continual expression of one or more viral or cellular genes

  • Some papillomaviruses and herpesviruses are also found as extrachromosomal DNA

Oncogenecity of Viruses

• DNA Viruses

  • Parvoviruses

    • Tumor in host: No

    • Tumor in other species: No

    • Transform cells in culture: No

  • Polyomaviruses

    • Tumor in host: No

    • Tumor in other species: Yes

    • Transform cells in culture: Yes

  • Papillomaviruses

    • Tumor in host: Yes, often benign

    • Tumor in other species: ?

    • Transform cells in culture: Yes

  • Hepatitis B Virus

    • Tumor in host: Yes

    • Tumor in other species: ?

    • Transform cells in culture: Yes

  • Human Adenoviruses

    • Tumor in host: No

    • Tumor in other species: Yes

    • Transform cells in culture: Yes

  • Human Herpesviruses

    • Tumor in host: Yes

    • Tumor in other species: Yes

    • Transform cells in culture: Yes

  • Poxvirus

    • Tumor in host: Ocassionally, usually benign

    • Tumor in other species: Yes

    • Transform cells in culture: No

• RNA Viruses

  • Human Retroviruses (HTLV-I, II)

    • Tumor in host: Yes

    • Tumor in other species: ?

    • Transform cells in culture: Yes

  • Hepatitis C Virus

    • Tumor in host: Yes/?

    • Tumor in other species: ?

    • Transform cells in culture: Yes

Mechanisms of Cell Transformation by the RNA Tumor Viruses

• Three distinct mechanisms of retroviral transformation

  • Retroviruses that carry an oncogene within their genomes, called transducing retroviruses

  • Retroviruses that lack cellular information but transform by integrating in the vicinity of a cellular oncogene, called cis-activating retroviruses

  • Nonstructural regulatory proteins that function to enhance transcrirption from the viral LTR, but that may also interfere with the transcriptional control of specific cellular genes and thus induce tumors. These viruses are called trans-activating retroviruses.

Transduced Oncogenes

• Oncogene as components of cellular regulatory systems that control growth and differentiation

The Transforming Potential of Ocogenes: Mechanisms of Activation

• Proto-oncogenes expressed in the appropriate cell type under normal cellular control are not oncogenic

• Structural and functional changes activate the latent oncogenic potential

• The cellular src gene overexpressed in a retrovirus vector, does not induce oncogenic transformation

• Proto-oncogene (mos), if overexpressed, can transform cells

Cellular Transformation by Retroviral Transduction of an Oncogene

• Sis: Growth factors and Autocrine Transformation

• ErbB and fms: Altered Receptors and Constitutive Mitotic Signals

• Src: Membrane-Bound Non-Receptor Tyrosine Kinases

• Ras: Growth Regulatory GTPase

• Mos and raf: Cytoplasmic Serine/Threonine Kinases

• Jun, myc, and erb A: Oncogenes Coding for Transcriptional Regulators

Mechanisms of Cell Transformation by the DNA Tumor Viruses

• The oncogenic properties of the DNA tumor viruses are associated with productive viral infection

• While the oncogenes of retroviruses are cellular genes that have been acquired, the DNA tumor virus oncogenes are essential viral genes that bear little or no relationship to cellular counterparts

• Permissive cells can’t lead to cellular transformation

• Under non-permissive circumstances, in which viral replicatoin process is aborted, a transformation event can be observed

• Lack of a specific mechanism for integration

• Oncogenis events meidated by DNA tumor virus oncoproteins

• Ability of the viruses to stimulate a quiescent, non-growing cell to enter cell cycle

DNA Tumor Virus Oncogenes

• Early and late phase of viral infection

• Early gene or at least a subset of early products responsible for oncogenic transformation

• Adenovirus

  • Two transcription units, E1A and E1B, each encode two mRNAs through alternative splicing of two primary transcripts

  • E1A encodes two identical proteins of 289 and 243 amino acids, the later lacking 43 amino acids

  • Distinct domains for transcriptional activation and transformation

  • Interact with cellular proteins such as retinoblastoma gene product (Rb) resulting in transformation

  • E1B encodes two distinct proteins of 495 and 175 amino acids

  • The 55-Kd (495 aa) E1B protein interacts with the cellular protein p53 (tumor suppressor) resulting in transformation

• Polyomavirus

  • Mouse polyomavirus and the monkey Sv40 virus

  • Both viruses encode multifunctional protein termed large T antigen

  • This protein is directly involved in DNA replication through specific binding to the origin or replication

  • Transforming abilities are associated with binding of this protein to a variety of cellular protein

    • Mouse and SV40 - Rb

    • SV40 - p53

  • While both viruses encode a small T antigen, polyomavirus encodes an addition protein called middle antigen

  • Small T antigen contributes to transformation efficiency in conjunction with large T antigen

  • Polyomavirus middle T antigen is responsible for the principal neutralizing transforming activity of the viru

  • It binds to and activates src family tyrosine kinases

• Papillomavirus

  • E5 gene of BPV has been shown to encode a major transforming activity

  • BPV E6 and E7 gene products also contribute to transformation

  • In contrast, E6 and E7 gene products of HPV are primarily responsible for transforming activity

  • E6 and E7 are always expressed in tumor cells