Autophagy is a cytoplasmic degradative pathway that can participate in biosynthetic processes, as in the yeast Cvt pathway, but is more commonly known for its functions in removing damaged or surplus organelles and macromolecular complexes. Here, we find that autophagy intersects with human immunodeficiency virus (HIV) biogenesis, mirroring the above dichotomy. Early, nondegradative stages of autophagy promoted HIV yields. HIV Gag-derived proteins colocalized and interacted with the autophagy factor LC3, and autophagy promoted productive Gag processing. Nevertheless, when autophagy progressed through maturation stages, HIV was degraded. This, however, does not occur, as the HIV protein Nef acts as an antiautophagic maturation factor through interactions with the autophagy regulatory factor Beclin 1, thus protecting HIV from degradation. The dual interaction of HIV with the autophagy pathway enhances viral yields by using the early stages while inhibiting the late stages of autophagy. The role of Nef in the latter process enhances yields of infectious HIV and may be of significance for progression to clinical AIDS.

Several domains of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein have been identified that are involved in HIV-1-mediated membrane fusion. One domain that is involved in membrane fusion is the hydrophobic amino terminus of the HIV-1 transmembrane glycoprotein gp41. Here we show that a polar substitution at gp41 amino acid 2 (the 41.2 mutation) results in an envelope glycoprotein that dominantly interferes with both syncytium formation and infection mediated by the wild-type HIV-1 envelope glycoprotein. The interference by the 41.2 mutant is not a result of aberrant envelope glycoprotein synthesis, processing, or transport. The 41.2 mutant elicits a dominant interfering effect even in the presence of excess wild-type glycoprotein, suggesting that a higher-order envelope glycoprotein complex is involved in membrane fusion. These results shed light on the process by which the HIV-1 envelope glycoproteins induce membrane fusion reactions and present a possible approach to anti-HIV therapy.

The minus strand and ambisense segmented RNA viruses include multiple important human pathogens and are divided into three families, the Orthomyxoviridae, the Bunyaviridae, and the Arenaviridae. These viruses all initiate viral transcription through the process of ''cap-snatching,'' which involves the acquisition of capped 5 oligonucleotides from cellular mRNA. Hantaviruses are emerging pathogenic viruses of the Bunyaviridae family that replicate in the cytoplasm of infected cells. Cellular mRNAs can be actively translated in polysomes or physically sequestered in cytoplasmic processing bodies (P bodies) where they are degraded or stored for subsequent translation. Here we show that the hantavirus nucleocapsid protein binds with high affinity to the 5 cap of cellular mRNAs, protecting the 5 cap from degradation. We also show that the hantavirus nucleocapsid protein accumulates in P bodies, where it sequesters protected 5 caps. P bodies then serve as a pool of primers during the initiation of viral mRNA synthesis by the viral polymerase. We propose that minus strand segmented viruses replicating in the cytoplasm have co-opted the normal degradation machinery of P bodies for storage of cellular caps. Our data also indicate that modification of the cap-snatching model is warranted to include a role for the nucleocapsid protein in cap acquisition and storage.bunyavirus ͉ minus strand RNA virus ͉ RNA degradation ͉ viral transcription ͉ RNA translation T he paradigm for transcription initiation involving cap-snatching is based on the orthomyxovirus influenza and posits that the heterotrimeric viral RNA-dependent RNA polymerase (RdRp) acquires 5Ј caps through the endonuclease activity of the PB1 subunit of the influenza RdRp (1, 2). This general mechanism of cap-snatching has been assumed for all minus strand segmented RNA viruses including the bunyaviruses and arenaviruses. However, one rather than three genes encode the RdRp of bunyaviruses and arenaviruses, and RdRp-associated endonuclease activity has yet to be established. Moreover, whereas influenza viruses carry out cap-snatching and transcription in the nucleus of infected cells, bunyavirus and arenavirus transcription and genome replication is cytoplasmic (3-8).Cellular mRNA degradation begins with removal of the poly(A) tail. Two alternative pathways that are both dependent on prior deadenylation then further degrade mRNA (9-11). mRNA can undergo 3Ј to 5Ј exonucleolytic decay, catalyzed by cytoplasmic exosomes under the control of peptides of the SKI complex. Alternatively, the 5Ј mRNA cap can be removed by the decapping enzyme DCP2/DCP1, rendering the mRNA susceptible to 5Ј to 3Ј digestion by the exonuclease XRN1. Decapping and XRN1-dependent 5Ј to 3Ј degradation is the predominant pathway for turnover of cellular mRNAs. Moreover, the components of the 5Ј to 3Ј decay machinery, including DCP2/DCP1 and XRN1, as well as a host of other peptides that function in RNA degradation and RNA regulation, are located in discreet cytoplasmic foci called processing bodie...

We analyzed the leader region of human immunodeficiency virus type 1 (HIV-1) RNA to decipher the nature of the cis-acting E/⌿ element required for encapsidation of viral RNA into virus particles. Our data indicate that, for RNA encapsidation, there are at least two functional subregions in the leader region. One subregion is located at a position immediately proximal to the major splice donor, and the second is located between the splice donor and the beginning of the gag gene. This suggests that at least two discrete cis-acting elements are recognition signals for encapsidation. To determine whether specific putative RNA secondary structures serve as the signal(s) for encapsidation, we constructed primary base substitution mutations that would be expected to destabilize these potential structures and second-site compensatory mutations that would restore secondary structure. Analysis of these mutants allowed the identification of two discrete hairpins that facilitate RNA encapsidation in vivo. Thus, the HIV-1 E/⌿ region is a multipartite element composed of specific and functional RNA secondary structures. Compensation of the primary mutations by the second-site mutations could not be attained in trans. This indicates that interstrand base pairing between these two stem regions within the hairpins does not appear to be the basis for HIV-1 RNA dimer formation. Comparison of the hypothetical RNA secondary structures from 10 replication-competent HIV-1 strains suggests that a subset of the hydrogen-bonded base pairs within the stems of the hairpins is likely to be required for function in cis.

Retroviruses contain two copies of the plus stranded viral RNA genome. As a means of determining whether both of these RNA's are used in the reverse transcription reaction, cells were infected with heterozygous virus particles that varied in nucleotide sequence at two separate locations at the RNA termini. The DNA proviruses formed from a single cycle of reverse transcription were then examined. Of the 12 proviruses that were characterized, all exhibited long terminal repeats (LTR's) that would be expected to arise only if both RNA templates were used for the generation of minus strand DNA. In contrast, only a single minus strand DNA appeared to be used as template for the plus strand DNA in the generation of fully double-stranded viral DNA. These results indicate that the first strand transfer step in reverse transcription is an intermolecular event while that of the second transfer is intramolecular. Thus, retroviruses contain two functionally active RNA's, and both may be required for the generation of a single linear DNA molecule. Formation of heterozygotes during retrovirus infection would be expected to result in the efficient generation of LTR recombinants.