1)   TYPES OF VIRUSES  (RNA and DNA)

    2)   VIRUS ASSEMBLY  (TMV, T4 phage)

    3)   RNA VIRUSES:REPLICATION/INFECTION  STRATEGIES    (poliovirus)

    4)   MEMBRANE VIRUSES  (Semliki Forest Virus, Influenza)

    5)   RETROVIRUSES
               RNA tumor viruses and oncogenes
               HIV (human immunodeficiency virus) and AIDS

Reading Assignment: Class notes and text where indicated (most figure and page numbers refer to Stryer text, 3rd Ed.)

OVERVIEW:

• Viruses (virions) are highly evolved, specialized, and efficient self-reproducing intracellular parasites. They are "packets" of infectious nucleic acid (RNA or DNA) surrounded by protective coats of protein (capsids) and/or cell-derived membranes (envelopes).

 
• Virus gene expression and virion assembly requires sequential, orderly expression of genes and assembly of macromolecular complexes. As such, they are models for helping to understand cellular development.

 
• Viruses can cause fatal(AIDS) as well as life-threatening diseases (polio, influenza, smallpox, etc) and minor ailments.

 
• Viruses can cause cancer (particularly retroviruses whose RNA genome is replicated as DNA and integrated into host chromosomal DNA). Viral-related cancers can be due to expression of a single gene (called an oncogene) which, for any given virus, is an altered form of normal cellular genes which control cell growth.

 

A)  TYPES OF VIRUSES

• RNA and DNA viruses can have either single or double-stranded genomes with from 4 to several hundred genes. [Fig Vir-2]

 
• Viruses are rod-shaped, icosahedral, or with membraneous coats (envelope) and have variable symmetry properties.

 
• Viral coats are made of one (TMV) or more (T4) proteins - can be one or many copies of each protein per virion.

 

• TMV coat protein associates into double discs ("life-savers") of 34 subunits which dislocate into a "lock-washer" helical structure upon interaction with a TMV-RNA. [Figs. Vir-3]

 
• Assembly process: The nucleation site for TMV assembly on the TMV-RNA (6390 bases) is 1000 bases from the 3'-end at a specific sequence which can form a base-paired hairpin structure. Coat protein addition (as lock-washers) continues to build the rod while the TMV-RNA is threaded through the core of the growing virion until the 5'-end is fully encapsidated. Then the 3'-end is coated to complete virion assembly. [Figs.Vir-4]

 

• T4 has 135 genes [Fig. Vir-5].

 
• Virus assembly proceeds by a strict sequentially ordered pathway of interaction between virus proteins. The head, tail and tail fibers are assembled independently from proteins coded by 40 different genes [Fig. Vir-6]. Head and tail combine only after each is fully assembled, then tail fibers are attached. 13 scaffold proteins and proteases are required for virus assembly and are coded by T4 DNA.

 
• T4 DNA is inserted into preformed phage heads. Concatameric T4 DNA is cut after a single genome length is packed into a phage head.
   

• Several classes of RNA viruses [Fig. Vir-7] have either a mRNA (+)-like RNA in the virus or the complementary (-) strand which must be copied into a (+) mRNA for virus gene expression. Retroviruses have (+)-sense RNA which is expressed only after reverse transcription into DNA.

• Poliovirus and TMV are +-strand viruses which replicate similarly

• Poliovirus is a (+)-stranded RNA virus. It's RNA translated as one large (over 2,000 amino acids) polyprotein which is cleaved into several coat proteins (4), an RNA replicase, a protease and other proteins. The coat proteins are derived from VP1 after proteolytic cleavages. [Figs. Vir-8, Vir-9]

 
• POLYPROTEIN PROCESSING IS A COMMON THEME FOR EUCARYOTIC VIRUSES. WHY???

 
• EUCARYOTIC MEMBRANE-ENVELOPED RNA VIRUSES: INFECTION CYCLE

F)  INFLUENZA A VIRUS

• A (-)-stranded RNA virus whose genome consists of 8 different RNA molecules. The virus has a lipid bilayer membrane which fuses with the cellular membrane after receptor-mediated endocytosis by respiratory tract lining cells. One RNA component codes for a major antigenic protein (hemagglutinin) which can rapidly mutate, yielding new strains of virus which are not recognized by the host's immune system [Fig. Vir-13, Vir-14, Vir-15]. An additional source of variation is reassortment with genes from infected animals.

• WHY NEW INFLUENZA STRAINS EVERY YEAR???

 
• Frequent mutations fixed in population of virus

 
• Reassortment of chromosomes (influenza RNAs) with other animals.

 

• Reverse transcription of retrovirus RNA to DNA initiates with a tRNA base-pairing to the virus RNA and proceeds by a very complex mechanism which is characterized by, among other things, repositioning partially-synthesized DNA fragments along the virus RNA template.

 

• Retrovirus genomes are integrated into the host genome as DNA which only then is transcribed [Fig. Vir-16, Vir-17]. Retroviruses may have been derived from retrotransposons (such as the yeast Ty elements)- DNA elements which jump from one chromosome to another via a RNA intermediate which is converted into a DNA copy by cellular reverse transcriptase activities [Fig. Vir-18, Vir-19, Vir-20]

 

• Some retroviruses [avian sarcoma virus (ASV), Rous sarcoma virus (RSV)] can cause cancer. The RNA tumor causing viruses are all retroviruses. ASV, for instance, has a 10Kb genome which contains only 4 major protein coding genes (gag, pol, env, and src). [Fig. Vir-21]

 
• Tumor viruses cause alterations of normal cellular processes which lead to uncontrolled cell growth in "transformed" cells.

 
• Oncogenes in retroviruses cause cancer and are derived from proto-oncogenes which are normal cellular genes (v-src of ASV is a tyrosine kinase which is related to c-src, a normal cellular tyrosine kinase). Many oncogenes have been identified [Fig Vir-22]. In addition to tyrosine kinases, growth factors (sis), growth factor receptors (erbB), guanyl-nucleotide binding proteins (ras), and nuclear proteins (fos, myb, myc) are among the known oncogene coded polypeptides. The mechanism whereby an oncogenic tyrosine kinase can cause deregulation of cell growth involves a cascade of effects on growth controlling proteins [Fig. Vir-23]

• AIDS is caused by HIV (human immunodeficiency virus) - a retrovirus with a genome of about 10Kb. which is more complex than that of ASV [Figs. 34-44,45]. HIV is a difficult virus against which to develop a vaccine, since the env gene (coding for gp120) mutates rapidly (even within an infected individual) - similar to the problems with emergence of new strains of influenza but much worse (more rapid)!

Reverse Transcriptase has 3 enzyme activities:
        · RNA-directed DNA synthesis (5'-3') - makes first DNA strand
        · RNase H (ribonuclease) - removes viral RNA
        · DNA-directed DNA polymerase (5'-3') - makes second DNA strand

Final product of process:
    · Double-stranded DNA copy of viral genome
    · Integrates into chromosome of infected cell