A retroviral RNA secondary structure required for efficient initiation of reverse transcription

Genetic evidence is presented which suggests the existence of an important structural element in the 5' noncoding region of avian retrovirus RNA. The proposed structure, which we term the U5-leader stem, is composed of sequences in the middle of U5 and in the leader, flanking the primer-binding site. U5 and leader mutations which would disrupt this structure caused a partial replication defect. However, nucleotide substitutions in the leader, which would structurally compensate for a U5 deletion mutation, restored normal replication. Analysis of replication intermediates of viruses with the above mutations suggests that the U5-leader stem is required for efficient DNA synthesis in vivo and for initiation of DNA synthesis from the tRNA(Trp) primer in melittin-activated virions. However, this structure does not appear to be required for binding of the tRNA(Trp) primer to viral RNA. These results support a role for the U5-leader stem structure, independent of its primary sequence, in the initiation of retroviral replication.     

Virion packaging determinants and reverse transcription of SRP RNA in HIV-1 particles

Diverse retroviruses have been shown to package host SRP (7SL) RNA. However, little is known about the viral determinants of 7SL RNA packaging. Here we demonstrate that 7SL RNA is more selectively packaged into HIV-1 virions than are other abundant Pol-III-transcribed RNAs, including Y RNAs, 7SK RNA, U6 snRNA and cellular mRNAs. The majority of the virion-packaged 7SL RNAs were associated with the viral core structures and could be reverse-transcribed in HIV-1 virions and in virus-infected cells. Viral Pol proteins influenced tRNAlys,3 packaging but had little influence on virion packaging of 7SL RNA. The N-terminal basic region and the basic linker region of HIV-1 NCp7 were found to be important for efficient 7SL RNA packaging. Although Alu RNAs are derived from 7SL RNA and share the Alu RNA domain with 7SL RNA, the packaging of Alu RNAs was at least 50-fold less efficient than that of 7SL RNA. Thus, 7SL RNAs are selectively packaged into HIV-1 virions through mechanisms distinct from those for viral genomic RNA or primer tRNAlys,3. Virion packaging of both human cytidine deaminase APOBEC3G and cellular 7SL RNA are mapped to the same regions in HIV-1 NC domain.     

NMR structure of the mature dimer initiation complex of HIV-1 genomic RNA

The two identical genomic RNA strands inside each HIV-1 viral particle are linked through homodimerization of an RNA stem-loop, termed SL1, near their 5' ends. SL1 first dimerizes through a palindromic sequence in its loop, forming a transient kissing-loop complex which then refolds to a mature, linear duplex. We previously reported the NMR structure of a 23-base truncate of SLI in kissing-dimer form, and here report the high-resolution structure of its linear isoform. This structure comprises three short duplex regions--derived from the central palindrome and two stem regions of each strand, respectively--separated by two bulges that each encompass three unpaired adenines flanking the palindromes. The stacking pattern of these adenines differs from that seen in the kissing-loop complex, and leads to greater colinear base stacking overall. Moreover, the mechanical distortion of the palindrome helix is reduced, and base pairs ruptured during formation of the kissing-loop complex are re-established, so that all potential Watson-Crick pairs are intact. These features together likely account for the greater thermodynamic stability of the mature dimer as compared to its kissing-loop precursor.