We constructed an infectious molecular clone of acquired immunodeficiency syndrome-associated retrovirus. Upon transfection, this clone directed the production of infectious virus particles in a wide variety of cells in addition to human T4 cells. The progeny, infectious virions, were synthesized in mouse, mink, monkey, and several human non-T cell lines, indicating the absence of any intracellular obstacle to viral RNA or protein production or assembly. During the course of these studies, a human colon carcinoma cell line, exquisitely sensitive to DNA transfection, was identified.

https://jvi.asm.org/content/jvi/59/2/284.full.pdf

Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone.

Four consensus sequences are conserved with the same linear arrangement in RNA-dependent DNA polymerases encoded by retroid elements and in RNA-dependent RNA polymerases encoded by plus-, minus- and double-strand RNA viruses. One of these motifs corresponds to the YGDD span previously described by Kamer and Argos (1984). These consensus sequences altogether lead to 4 strictly and 18 conservatively maintained amino acids embedded in a large domain of 120 to 210 amino acids. As judged from secondary structure predictions, each of the 4 motifs, which may cooperate to form a well-ordered domain, places one invariant amino acid in or proximal to turn structures that may be crucial for their correct positioning in a catalytic process. We suggest that this domain may constitute a prerequisite 'polymerase module' implicated in template seating and polymerase activity. At the evolutionary level, the sequence similarities, gap distribution and distances between each motif strongly suggest that the ancestral polymerase module was encoded by an individual genetic element which was most closely related to the plus-strand RNA viruses and the non-viral retroposons. This polymerase module gene may have subsequently propagated in the viral kingdom by distinct gene set recombination events leading to the wide viral variety observed today.

Identification of four conserved motifs among the RNA-dependent polymerase encoding elements.

Despite the rapid mutational change that is typical of positive-strand RNA viruses, enzymes mediating the replication and expression of virus genomes contain arrays of conserved sequence motifs. Proteins with such motifs include RNA-dependent RNA polymerase, putative RNA helicase, chymotrypsin-like and papain-like proteases, and methyltransferases. The genes for these proteins form partially conserved modules in large subsets of viruses. A concept of the virus genome as a relatively evolutionarily stable "core" of housekeeping genes accompanied by a much more flexible "shell" consisting mostly of genes coding for virion components and various accessory proteins is discussed. Shuffling of the "shell" genes including genome reorganization and recombination between remote groups of viruses is considered to be one of the major factors of virus evolution. Multiple alignments for the conserved viral proteins were constructed and used to generate the respective phylogenetic trees. Based primarily on the tentative phylogeny for the RNA-dependent RNA polymerase, which is the only universally conserved protein of positive-strand RNA viruses, three large classes of viruses, each consisting of distinct smaller divisions, were delineated. A strong correlation was observed between this grouping and the tentative phylogenies for the other conserved proteins as well as the arrangement of genes encoding these proteins in the virus genome. A comparable correlation with the polymerase phylogeny was not found for genes encoding virion components or for genome expression strategies. It is surmised that several types of arrangement of the "shell" genes as well as basic mechanisms of expression could have evolved independently in different evolutionary lineages. The grouping revealed by phylogenetic analysis may provide the basis for revision of virus classification, and phylogenetic taxonomy of positive-strand RNA viruses is outlined. Some of the phylogenetically derived divisions of positive-strand RNA viruses also include double-stranded RNA viruses, indicating that in certain cases the type of genome nucleic acid may not be a reliable taxonomic criterion for viruses. Hypothetical evolutionary scenarios for positive-strand RNA viruses are proposed. It is hypothesized that all positive-strand RNA viruses and some related double-stranded RNA viruses could have evolved from a common ancestor virus that contained genes for RNA-dependent RNA polymerase, a chymotrypsin-related protease that also functioned as the capsid protein, and possibly an RNA helicase.

Evolution and Taxonomy of Positive-Strand RNA Viruses: Implications of Comparative Analysis of Amino Acid Sequences

Human T-lymphotropic virus type III (HTLV-III) encodes a trans-acting factor that activates the expression of genes linked to the HTLV-III long terminal repeat. By functional mapping of complementary DNA transcripts of viral messenger RNA's the major functional domain of the gene encoding this factor was localized to a region immediately before the env gene of the virus, a region previously thought to be noncoding. This newly identified gene consists of three exons, and its transcription into messenger RNA involves two splicing events bringing together sequences from the 5' part (287 base pairs), middle (268 base pairs), and 3'part (1258 base pairs) of the HTLV-III genome. A similar messenger RNA with a truncated second exon (70 base pairs) does not encode a trans-acting function. It is proposed that this second messenger RNA is the transcript of a gene (3'-orf) located after the env gene. Messenger RNA's were also identified for the env and gag-pol genes of HTLV-III.

http://science.sciencemag.org/content/sci/229/4708/69.full.pdf

Trans-activator gene of human T-lymphotropic virus type III (HTLV-III).

https://www.cell.com/cell/pdf/0092-8674(86)90778-6.pdf

Identification and characterization of conserved and variable regions in the envelope gene of HTLV-III/LAV, the retrovirus of AIDS

The RNA genome of human hepatitis A virus (HAV) was molecularly cloned. Recombinant DNA clones representing the entire HAV RNA were used to determine the primary structure of the viral genome. The length of the viral genome is 7478 nucleotides. An open reading frame starting at nucleotide 734 and terminating at nucleotide 7415 encodes a polyprotein of Mr 251,940. Comparison of the HAV nucleotide sequence with that of other picornaviruses has failed to reveal detectable areas of homology. However, a computer analysis of the putative amino acid sequence of HAV and poliovirus demonstrated the existence of short areas of homology in virion protein 3 (VP3) and throughout the carboxyl-terminal portion of the polyproteins. In addition, extensive protein structural homologies with poliovirus were detected.

https://www.pnas.org/content/pnas/82/9/2627.full.pdf

Primary structure and gene organization of human hepatitis A virus.

Retroviruses cause a wide variety of diseases in avian and mammalian species. Human acquired immune deficiency syndrome (AIDS) leads to collapse of the immune system and death by a wide variety of opportunistic infections; unusual forms of cancer are associated with this syndrome. Retroviruses have been recovered from tissues of AIDS patients and from patients with related conditions. These similar newly-isolated viruses are lymphadenopathy-associated virus (LAV)1, human T-cell lymphotropic virus (HTLV-III)2,3 and AIDS-associated retrovirus (ARV-2)4. We have identified a RNA genome of ∼9 kilobases (kb) in virions purified from the culture medium of a human T-cell tumour line infected with ARV-2. A cDNA probe made from viral RNA detected circular DNA molecules and proviral forms in infected cells. We prepared a library of infected cell DNA. Recombinant phage included those with a 9.5-kb proviral DNA and viral DNA permuted with respect to the single EcoRI site. Comparison of three ARV isolates from different AIDS patients revealed polymorphism of restriction endonuclease sites.

Molecular cloning of AIDS-associated retrovirus

HEPATITIS A VIRUS VACCINES Abstract The complete genome of the hepatitis A virus (HAV) is disclosed. With the entire genomic sequence available, epitopes are identified by analogy to known picornaviruses such as polio virus, synthesized chemically, and conjugated to carrier proteins to provide protective vaccines against HAV. Alternate methods of preparing vaccines include preparation of specified peptide sequences using recombinant techniques, preparation of deleted virions by modifying the genome to destroy the replicase and LTR regions, and preparation of particulate immunogenic materials containing an epitope from HAV. The foregoing alternatives for vaccine preparation are described, and proteins thus prepared used to raise antibodies against HAV.

Steven J. Potter

Papers

Primary structure and gene organization of human hepatitis A virus.

Molecular cloning of AIDS-associated retrovirus

Hepatitis A virus vaccines

Hepatitis-a-virus vaccine.