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Viral Pathogens

Classification of Viruses

Viruses are "obligatory intracellular parasites" that subvert the molecular machinery of host cells to replicate. Classification of viruses is based on several criteria:

Morphology- Viruses consist of a core of nucleic acid material which constitutes their genome and a protein-based coating or capsid.  An additionally lipoprotein bilayer surrounds some viruses and is the basis for their classification as enveloped and non-enveloped viruses. Based on capsid shape, viruses may be classified as filamentous or helical (e.g., plant viruses-TMV and animal viruses such as rabies virus), icosahedral (e.g., human pathogens such as adenoviruses and Hepatitis A virus), and complex such as “head and tail viruses” (e.g., bacteriophages) and pleomorphic viruses (e.g., Ebola virus).

Genomic composition- The viral genome together with surrounding proteins constitutes the nucleocapsid. Viral genetic material may be encoded by RNA or DNA which may be single (ss) or double stranded (ds). The genomic material in ssRNA viruses may consist of a positive sense or negative sense strand.

Replication Mechanism- Depending on the nature of their genome, viruses rely on different mechanisms for mRNA synthesis. The Baltimore classification, conceived by and named after virologist David Baltimore, classifies viruses based on their genome composition and replication mechanisms.  Seven different groups are recognized under this system:  Group 1- Double stranded DNA, Group 2- Single stranded (+) sense DNA, Group 3- Double stranded RNA, Group 4- Single stranded (+) sense RNA, Group 5- Single stranded (-) sense stranded RNA-, Group 6- Single stranded (+) sense RNA with DNA intermediate- (Retro-transcribing), Group 7- Double stranded DNA with RNA intermediate (Retro-transcribing).


Viral Entry

Mechanisms of cell entry differ between enveloped and non-enveloped viruses. For cell entry, the lipid bilayer of enveloped viruses fuses directly with the host cell’s plasma membrane or endosomal membrane. This process is mediated by the interaction of envelope viral proteins with key cellular receptors. Fusion via the endocytic compartment is a common mechanism for host cell entry used by a variety of viruses including alphaviruses (e.g., mosquito-borne chikungunya virus), flaviviruses (e.g., dengue and zika viruses), orthomyxoviruses (e.g., influenza viruses), and rhabdoviruses (e.g., rabies virus). In contrast, non-enveloped viruses rely on capsid proteins which bridge the plasma or endosomal membrane (e.g., pore formation) allowing viral genome entry. In addition to endosomal entry, non-enveloped viruses may bridge other internal cellular membranes including the Golgi (e.g., papillomavirus) and ER (e.g., SV40).


Non-enveloped

Poliovirus

Papillomavirus

Hepatitis A

Adenovirus

Mechanisms of cell entry are different for enveloped and non-enveloped viruses: enveloped viruses directly fuse via their lipid membrane to the host’s cell membranes, while non-enveloped viruses use capsid proteins to bridge cell membranes.

Enveloped

HIV

Influenza

Varicella Zoster

Mumps

HHV

Mechanisms of cell entry are different for enveloped and non-enveloped viruses: enveloped viruses directly fuse via their lipid membrane to the host’s cell membranes.


Immunocytochemical detection of adenovirus infected cells with anti-adenovirus mouse monoclonal antibody.

Immunocytochemical detection of HHV8 infected cells with anti-ORF73/HHV8 rat monoclonal antibody.

Immunocytochemical staining of cells infected with adenovirus using Adenovirus Mouse Monoclonal Antibody (M58 + M73) [NB120-3648]. Immunocytochemical staining of cells infected with HHV8 using an ORF73/HHV8 Rat Monoclonal Antibody (LN53) [NBP1-22767].

Viral Attachment Factors and Entry Receptors

Initial viral-cell contact or attachment is mediated by electrostatic interactions between viral proteins and host surface carbohydrate groups (e.g., glycosaminoglycan groups such as heparan sulphate and sialic acid). Binding to specific receptors follows initial weaker interactions with the host cell membrane and leads to viral protein processing and cellular signaling. The receptors engaged by a specific virus determine its internalization path.


Viruses gain access into host cells through different endocytic pathways including clathrin-mediated, caveolae/raft-dependent, clathrin/caveolin-independent, and micropinocytosis (SARS-CoV, Influenza A, and HIV).

Viruses engage different cellular mechanisms to gain entry into host cells including direct entry at the cell membrane and through endocytic pathways such as clathrin-mediated, caveolae/raft-dependent, clathrin/caveolin-independent, and macropinocytosis. However, examples abound of viruses which exploit more than one mechanism for cell entry, and entry mechanisms may differ based on the host cell type. For SARS coronavirus, a clathrin-dependent entry pathway has been identified, nevertheless SARS coronavirus cell entry may also occur through a clathrin/caveolin-independent pathway.
* HIV entry mechanisms are cell type dependent and include clathrin-mediated, dynamin-dependent endocytosis, and macropinocytosis.


Find Endosomal Marker Antibodies


Entry Receptors for Major Human Viral Pathogens

Viral host cell entry is a multistep process involving a number of cellular attachment factors, receptors, and proteases. For some viruses, a predominant entry receptor has been identified (e.g., SARS coronavirus ACE-2 receptor). However, identification of a unique entry receptor remains challenging for important human viral pathogens including Hepatitis A Virus (HAV) and Chikungunya virus (CHIKV).

Virus Family Virus Species Enveloped (E) or Non-Enveloped (N) Genome Entry Receptors Main Endocytic Pathways

Adenovirus

Adenovirus 2

N

dsDNA CXADR, alpha 5 integrin Clathrin-dependent

Adenovirus

Adenovirus 3

N

dsDNA

CD46, alpha 5 integrin

Macropinocytosis

Coronavirus

MERS-CoV

E

ssRNA+ DiPeptidyl Peptidae-4 (DPP4) Clathrin-dependent

Coronavirus

SARS-CoV

E

ssRNA+ ACE-2 Clathrin-dependent, Clathrin /caveolin-independent

Filovirus

EBOV

ssRNA- TIM-1, Axl, NPC-1 Macropinocytosis
Flavivirus Dengue E ssRNA+ DC-SIGN, Mannose receptor (MMR), CD14, HSP-70, HSP-90, GRP78 Clathrin-dependent
Flavivirus HCV E ssRNA+ CD81, SR-BI, LDLR, Claudin-1, Occludin Clathrin-dependent
Flavivirus Zika E ssRNA+ DC-SIGN, TIM-1, TAM (TYRO3, AXL, MER) Clathrin-dependent, Mucolipin-2-dependent
Orthomyxovirus Influenza E ssRNA- Sialic acid Clathrin-dependent, Clathrin /caveolin-independent
Papovavirus HPV E dsDNA syndecan-1, laminin-5, integrin alpha 6 Clathrin-dependent
Picornavirus HAV N
eHAV-Quasi-enveloped**
ssRNA+ TIM-1, ALIX, Integrin beta 1** Clathrin-dependent, Dynamin-dependent
Picornavirus Rhinovirus N ssRNA+ ICAM-1, LDLR, p-cadherin Clathrin-dependent, Clathrin independent, Macropinocytosis
Retrovirus HIV E ssRNA+ CD4, CCR5, CXCR4, CCR2 Clathrin-dependent, Dynamin-dependent, Macropinocytosis
*Togavirus Chikungunya E ssRNA+ TIM-1, DC-SIGN Clathrin-dependent

*Yeast two-hybrid screens and loss of function screens have identified other interacting proteins including Actin gamma 1, collagen type I-alpha-2, Tyrosine phosphatase non-receptor 2 (PTPN2), Fuzzy homolog, Tspan9, PHB-1 (prohibitin 1), Mxra-8. The role of these proteins in viral entry remains inconclusive.
**Quasi-enveloped HAV virions are secreted from infected cells through a non-lytical pathway and are surrounded by host cell derived membranes. A specific HAV cell entry receptor remains elusive, proteins listed participate in viral attachment and endosomal trafficking.


Identifying Viral Entry Mechanisms

Elucidating viral entry mechanisms often relies on the use of small molecule inhibitors targeting specific molecules or events in the endocytic pathway. Small molecule inhibitors present various advantages including ease of acute application and potential reversibility of effects. A main disadvantage to chemical manipulation of endocytic pathways is the potential for generalized disruption of endocytosis or other cellular processes. Therefore, combined use of chemical inhibitors with other approaches such as targeted genetic manipulation of endocytic relevant targets is recommended.

Endocytic Pathway Inhibitors Activities Notes

Clathrin-mediated endocytosis

Chlorpromazine

Induces assembly of adaptor proteins and clathrin on endosomal membranes, depletes clathrin from the plasma membrane

Inhibits receptor recycling and clathrin independent endocytosis

Dynasore

Inhibitor of dynamin GTPase activity

Affects cholesterol homeostasis, disrupts lipid rafts, and destabilizes actin

Cytochalasin B

Inhibitor of actin polymerization

Affects several endocytic pathways

Caveolin-dependent endocytosis

Filipin III

Binds sterols with high affinity, complexes with membrane cholesterol

Affects cholesterol dependent processes and is cytotoxic, inhibits clathrin-mediated endocytosis

Okadaic acid

Inhibitor of phosphatase 1 and 2A, promotes removal of caveolae from plasma membrane

Inhibits clathrin-mediated endocytosis

Phorbol 12-myristate 13-acetate

PKC activator, inhibits caveolae dependent entry of Ebola virus

Low toxicity, interferes with endocytic traffic

Macropinocytosis Amiloride

Inhibitor of Na+/H+ exchange at cell membrane

Blocks clathrin-mediated and lipid raft-dependent endocytosis
Cytochalasin D Inhibitor of actin polymerization Blocks phagocytosis, affects several endocytic pathways
Latrunculin A, Latrunculin B Inhibitor of actin polymerization Blocks phagocytosis, affects several endocytic pathways

What are Neutralizing Antibodies?

During an adaptive immune response against viral pathogens or following vaccination, generation of neutralizing antibodies represents the best indicator of protection. Neutralizing antibodies by definition are those able to target specific surface viral antigens and block the virus replication cycle at an early stage, prior to transcription and translation events. Viral neutralization may occur through different mechanisms including inhibition of attachment, receptor or co-receptor binding, and blockade of viral-cellular membrane fusion or cell membrane penetration. Neutralization events may occur by inhibition of interactions at the host cell surface or within the endosomal compartment. Additionally, some neutralizing antibodies may induce viral aggregation leading to reduced infectivity.

Neutralizing Antibody Role of Targeted Protein

Chikungunya (CHIKV) (DDX9100P-100, DDX9102P-100, DDX9103P-100DDX9104P-100)

Envelope glycoproteins E1 (interacts with cellular receptor to mediate viral entry via membrane fusion) and E2 (involved in attachment to receptor) are encoded by a large polyprotein (capsid, E3, E2, 6K,E1).

Flavivirus group antigen (NBP2-52666)

E protein (Dengue virus, West Nile Virus, Japanese Encephalitis, Yellow Fever Virus, Zika virus) is an envelope glycoprotein with three main domains (DI-DIII). DII domain mediates membrane fusion and DIII interacts with cellular receptors.

HIV gp41 (DDX1306, DDX1304, DDX1305)

gp41 is a unit of the viral envelope glycoprotein complex (gp120/gp41) involved in membrane fusion.

Influenza A Hemagglutinin H7N7 (NBP2-89790)

HA is one of two envelope glycoprotein spikes that functions in receptor binding and membrane fusion.

MERS-CoV NCoV Spike (NBP2-90336)

Spike is an envelope glycoprotein that forms trimers on the viral surface, binds to cellular receptors, and mediates membrane fusion.


View Antiviral Small Molecules


Select References

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