Retroviruses have various characteristics that make them unique as gene delivery vehicles. Their life cycle includes an integrated state in the DNA of the host chromosome. Retroviruses are the only animal viruses that integrate into the host cell’s genome during the normal growth cycle. Retroviral genomes are non-segmented, of positive polarity, and typically between 7 to 12 kb long in size. All retroviral genomes consist of at.

., Ariko Miyake,and. Department of Microbiology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, JapanRetroviruses can cause diseases such as AIDS, leukemia, and tumors, but are also used as vectors for human gene therapy. All retroviruses, except foamy viruses, package two copies of unspliced genomic RNA into their progeny viruses. Understanding the molecular mechanisms of retroviral genome packaging will aid the design of new anti-retroviral drugs targeting the packaging process and improve the efficacy of retroviral vectors. Retroviral genomes have to be specifically recognized by the cognate nucleocapsid domain of the Gag polyprotein from among an excess of cellular and spliced viral mRNA.

Extensive virological and structural studies have revealed how retroviral genomic RNA is selectively packaged into the viral particles. The genomic area responsible for the packaging is generally located in the 5′ untranslated region (5′ UTR), and contains dimerization site(s). Recent studies have shown that retroviral genome packaging is modulated by structural changes of RNA at the 5′ UTR accompanied by the dimerization. In this review, we focus on three representative retroviruses, Moloney murine leukemia virus, human immunodeficiency virus type 1 and 2, and describe the molecular mechanism of retroviral genome packaging. IntroductionRetroviruses belong to a diverse family of RNA viruses causing various diseases, such as leukemia, tumors, demyelinization disease, and AIDS. One unique feature of retroviruses is their integration of reverse transcribed genome into the host chromosome as a provirus. Some retroviruses have been engineered to function as vectors for the delivery of corrective human genes, and vectors derived from the Moloney murine leukemia virus (MoMLV) have been used for the treatment of severe combined immunodeficiency.

An understanding of the molecular mechanism of retroviral replication is needed for the development of anti-retroviral therapies as well as retroviral vectors.All retroviruses, except foamy viruses, package two copies of full-length genomic RNA into progeny viruses, the genomic RNA having to be specifically selected from among a large amount of spliced viral and cellular RNA (Figure; ). Virological and genetic studies have shown that the specific packaging of retroviral genomic RNA is mediated via interaction with the nucleocapsid (NC) domain of the Gag polyprotein (;;;;; ). Retroviral NC domains are generally highly basic and contain one or two zinc knuckle motifs composed of C–C–H–C arrays (;;;; ). The zinc knuckles form a metal-coordinating “reverse turn” stabilized by NH–S hydrogen bonds. Most retroviral zinc knuckles contain a hydrophobic cleft on the surface of the mini globular domain, recognizing specific structures of RNA or DNA.

The basic N- and C-terminal tails of NC domains are conformationally labile. Illustration showing retroviral genome packaging events.

The retroviral genomic RNA is exported from the nucleus to cytoplasm and used as the mRNA for ribosomal synthesis of the Gag and Gag–Pol polyproteins. (A) The retroviral genomic RNA promotes conversion of the dimeric form of genomic RNA via the NC domain of Gag. (B) The Gag and retroviral genomic RNA complex is transported to the plasma membrane, where further assembly and budding occur, mediated by the myristoylated MA domain of Gag. (C) In contrast, spliced viral RNA or cellular RNA is not bound to the NC domain of Gag or may be bound to a small number of Gag polyproteins insufficient for trafficking to the plasma membrane. Retroviral genomes are known to be non-covalently dimerized in progeny virions. The region responsible for retroviral genome packaging is generally located between the splice donor (SD) site and the gag start codon in the 5′ leader region (;;;;;; ).

Interestingly, the packaging signal generally overlaps with the site of dimerization (,;; ), implying that the packaging event is coupled with genome dimerization. Inhibition of genome dimerization by deletion or insertion mutations at dimer initiation sites (DIS) causes a drastic reduction in genome packaging (;;; ). Moreover, studies with mutant viruses containing two 5′ untranslated region (UTR) packaged monomeric genome, indicate that genome packaging is achieved by the interaction of two 5′ UTRs (, ).

Experiments with MoMLV have indicated that the conformational change induced by genome dimerization causes the exposure of NC-binding sites. A recent study also indicated that human immunodeficiency virus type 1 (HIV-1) employs a similar strategy for genome packaging. In addition, several structures have been determined among complexes of NC and RNA fragments functioning in genome packaging, which provide the molecular mechanism for retroviral genome recognition of NC at the atomic level. In this review, we describe the molecular mechanisms of retroviral genome packaging. Moloney Murine Leukemia VirusMoloney murine leukemia virus is a simple prototypical retrovirus, with a single splicing event producing a spliced RNA for synthesizing Env during its life cycle. MoMLV is one of the most extensively studied retroviruses.

Nucleotides 215–565 of the 5′ UTR have been identified as a responsible site for genome packaging (Ψ-site; ). The secondary structure of the 5′ UTR was determined by RNase protection assays using cross-linking reagents combined with computational analyses such as phylogenetic and free-energy calculations (; ).

The monomeric Ψ-site is composed of a series of RNA stem-loops. Differences in RNase protection patterns were observed for the dimeric Ψ-site (;; ). It was reported that a dimerized RNA fragment containing the entire Ψ-site was bound to a significant number of NCs. Meanwhile, a mutant RNA fragment that inhibited dimerization was bound to a few NCs. Thus, dimerization-dependent genome packaging is strongly indicated in MoMLV.The minimum region sufficient for genome packaging is referred to as the core encapsidation signal (Ψ CES), though a virus containing only Ψ CES exhibits less efficient packaging than a virus containing the entire Ψ-site (;;;; ). Ψ CES consists of three RNA stem-loops (DIS-2, SL-C, and SL-D, see Figures A,B). DIS-2 harbors a palindromic sequence and is able to convert heterologous extended dimers.

The structure of NC in a complex with a mutant RNA of Ψ CES mimicking the dimer-like conformation was determined by nuclear magnetic resonance (NMR) spectroscopy (Figures C,D; ). UAUCUG residues sequestered by base-pairing in the monomeric conformation are exposed as a linker by dimerization. NC recognizes the UCUG sequence. NC is a highly basic protein, consisting of a zinc knuckle motif and labile tails in both the N- and C-terminus. Biophysical study indicated that NC specifically recognizes RNA fragments including a Py (C or U) – Py-Py-G sequence.

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The interface of NC–UCUG is complementary in both shape and charge (; ). The guanosine base attaches to the deep hydrophobic pocket of the zinc knuckle via hydrophobic and hydrogen bonds. The three upstream nucleotides make contact with hydrophobic residues on the surface of the zinc knuckle. (A) Genomic organization of MoMLV.

(B) Secondary structure of the core encapsidation signal (Ψ CES). Dimerization of DIS-2 induces a frame shift, exposing a UCUG element as a linker. (C) Secondary structure of mutant Ψ CES that mimics the dimer-like conformation. (D) The complex of NC and mutant Ψ CES. (E) Hypothetical mechanism for the genome packaging of MoMuLV. Upon dimerization, SL–CD exhibits a cross-kissing interaction, promoting the proximity of NC-binding sites.

The SL–CD dimer functions as a scaffold that facilitates the Gag–Gag interaction. The ribonucleoprotein complex is transported to the plasma membrane. Reprinted with permission from (A–D):; (E):. (A) Genomic organization of HIV-1. (B) The latest model for HIV-1 genome packaging. Conformational change of RNA in the 5′ leader region regulates genome dimerization and genome packaging. The stem-loop structure of AUG causes sequestering of DIS via long range interaction with the residues of U5, resulting in the inhibition of HIV-1 genome dimerization (left).

In this conformation of the 5′ leader RNA, a small number of NCs are capable of binding the 5′ leader RNA. In contrast, the interaction of AUG with U5 residues promotes genome dimerization via exposure of DIS (center). In this conformation of the 5′ leader RNA, a greater number of NCs are capable of binding the 5′ leader RNA. The different RNA elements in the 5′ leader region are color coded: DIS (red), SD (blue), Ψ (orange), and AUG (green). RNA fragments of DIS, SD, Ψ, and AUG have been studied extensively since the region from DIS to AUG was initially identified as a Ψ-site (;;;;;;,; ). The RNA fragments of SD and Ψ are bound to NC with high affinity (;,), whereas the RNA fragments of DIS and AUG are bound to NC with relatively low affinity (;; ). The RNA fragment of Ψ is bound to NC with the highest affinity ( K d = 100 nM) among these four RNA fragments.

Deletion or destabilization of the stem of Ψ results in a significant reduction in genome packaging efficiency (; ). This indicates that the stem-loop structure of Ψ is important for genome packaging. Ψ consists of a GGAG tetra-loop and a stem. The structure of the ribonucleoprotein complex NC–Ψ has been determined by NMR spectroscopy. Guanosine residues of the GGAG tetra-loop are inserted into the hydrophobic clefts of both the N- and C-terminal zinc knuckles. The adenosine residue of the loop packs against the N-terminal zinc knuckle and an N-terminal alpha-helix domain binds to the major groove of the RNA stem.

Thus, overall, NC residues are involved in binding with Ψ RNA, by which the tight binding of NC–Ψ is achieved. In addition, the intra-molecular interaction of two zinc knuckles may help to stabilize the complex of NC and Ψ RNA.The RNA fragment of SD is also bound to NC with high affinity though the binding is slightly weaker than that of Ψ. SD RNA contains a GGUG tetra-loop and a stem containing a characteristic AUA triple base-pairing structure. All guanosine residues of the loop inserted into the hydrophobic clefts of zinc knuckles as observed in the complex of NC and Ψ RNA.

A major structural difference between the NC and SD RNA complex and the NC and Ψ RNA complex is the orientation of the N-terminal alpha-helix domain of NC. The domain does not stick into SD RNA and is exposed outside of SD RNA. The N-terminal zinc knuckle interacts with an AUA triple base-pairing motif in the minor groove of the SD RNA stem. A mutant virus with a disrupted lower stem structure of SD exhibited a 20% reduction in genome packaging compared with the wild-type virus, despite the robust binding of Ψ RNA and NC. Further study will be needed to clarify the link between the tight binding of SD RNA with NC and its biological relevance.Dimer initiation site contains a GC-rich palindromic sequence and an inner bulge (G-AGG) in the middle of the stem. This inner bulge is recognized by NC.

As described above, guanosine residues not in base-pairs possibly play an important role in the binding to NC. However, the binding affinity of NC–DIS is relatively weak (approximately five times weaker than that of NC–SD/ Ψ; ). NC catalyzes the conversion of DIS from a kissing dimer to a more stable extended duplex conformation (; ). Virological studies have suggested that interaction between the two 5′ UTR is required for genome packaging (, ). A very recent study indicated that genome dimerization is controlled by a unique strategy (Figure B). RNA containing the entire 5′ UTR and the first 21 residues of the gag coding region forms two alternative conformations.

In one conformation, DIS is sequestered via a long range interaction with U5, which inhibits genome dimerization. In the other conformation, DIS is exposed by release from the interaction with U5, which promotes genome dimerization.AUG was believed to form a stem-loop structure, consisting of a GAGA tetra-loop and an unstable short stem containing two wobble G–U base-pairs. The GAGA tetra-loop is a type of GNRA (N is A, C, G, or U; R is A or G) tetra-loop, which is frequently observed in ribosomal RNA. GNRA tetra-loop helps fold stem-loops, and one Watson–Crick base-pair is enough to close off the RNA fragment. However, it has been suggested that AUG does not form a stem-loop structure and a portion of the residues of AUG may have a long range interaction with the residues of U5 as indicated in the BMH structure (;;;; ). Studies using deletion mutants indicate that the entire 5′ UTR and gag coding regions are important for genome packaging (;;;; ). Evidence of the importance of long range interactions for genome packaging was reported recently (Figure B; ).

It shows that AUG regulates genome dimerization. AUG forms two alternative conformations. One is a stem-loop conformation as initially suggested.

The other is a complex with the residues of U5. The interaction of AUG with U5 leads to the exposure of DIS, promoting genome dimerization.

Of note, when the residues of AUG form a stem-loop structure, in which the dimerization of the 5′ leader RNA is inhibited, a small number of NCs can bind the 5′ leader RNA (Figure B). On the other hand, when the residues of AUG interact with the residues of U5, a greater number of NCs can bind the 5′ leader RNA. Thus, AUG has a critical role in genome dimerization and genome packaging. In addition, it is suggested that the residues ranging from the end of Ψ to the start of AUG form base-pairing with the residues of the 3′ terminus of PBS. That is also proposed by earlier work. In fact, ) indicate these residues are indispensable both for genome dimerization and genome packaging in vivo.It is suggested that the HIV-1 genome’s dimerization and packaging are controlled by dynamic changes of the 5′ leader’s conformation as indicated in MoMLV (;; ). Ψ is indicated to be important for genome packaging, however, it is not clear how Ψ works in the model suggested.

This will be the next question to answer for a better understanding of the molecular mechanism of HIV-1 genome packaging. Human Immunodeficiency Virus Type 2Human immunodeficiency virus type 2 (HIV-2) is one of two human lentiviruses that can cause AIDS. HIV-1 and HIV-2 exhibit approximately 55% nucleotide sequence identity. However, they differ significantly in their 5′ UTR. For example, the 5′ UTR of HIV-2 (HIV-2 ROD) consists of 535 nucleotides, whereas that of HIV-1 (HIV-1 NL432) consists of 335 nucleotides.

The 5′ leader RNA of HIV-2 contains three unique RNA elements referred to as Ψ-1, Ψ-2, and Ψ-3 in addition to a series of RNA stem-loops, TAR, poly(A), PBS, DIS, SD, and AUG, those are commonly observed in the 5′ leader of HIV-1 (Figure; ). The structure of HIV-2 NC also exhibits some different features. A major difference in NC between HIV-2 and HIV-1 is the structure of the N-terminal flanking domain (;; ). The N-terminal domain of HIV-1 NC forms an alpha-helix, whereas that of HIV-2 NC is too short to do so. Another difference is the intra-molecular interaction of two zinc knuckles of HIV-2 NC, by which HIV-2 NC forms a more globular structure than HIV-1 NC.

These structural differences of NCs between HIV-1 and HIV-2 may affect RNA recognition. (A) Genomic organization of HIV-2. (B) Representative secondary structures predicted for the 5′ UTR of HIV-2.

Variations among recent predictions for AUG (green) are shown. The residues of AUG have been suggested to form a stem-loop structure and show long range interaction with the residues of U5. It has been suggested that HIV-2 genome’s dimerization is employed the same mechanism as HIV-1. However, it remains to be clear whether NC preferentially binds the 5′ leader RNA containing the U5–AUG interaction. It has been suggested that Ψ-3 can be bound to NC (; ). A study using RNase protection assays suggested that the NC–Ψ-3 ribonucleoprotein exhibited strong protection at the loop of Ψ-3. This was supported by NMR experiments.

The guanosine residue of the UUAGAC loop is inserted into the hydrophobic cleft of the C-terminal zinc knuckle. The N-terminal zinc knuckle does not bind the RNA fragment of Ψ-3. However, virological study has suggested that Ψ-3 is not essential for either genome dimerization or genome packaging. In agreement with that, a recent study showed that NC binds the RNA fragment of Ψ-3 with 100 times lower affinity than HIV-1 NC binds the RNA fragment of HIV-1 Ψ.The RNA fragment of DIS is bound to NC with high affinity ( K d = 100 nM), equivalent to the affinity of HIV-1 Ψ RNA and the cognate NC. HIV-2 NC recognizes the inner bulge of DIS, and does not bind to the loop.

HIV-1 DIS is weakly bound to the cognate NC (; ). Interestingly, HIV-2 TAR and poly(A) is bound to the NCs with relatively high affinity (TAR–NC Kd = 450 nM, poly(A) – NC Kd = 550 nM). All the tight NC-binding sites DIS, TAR, and poly(A) are located upstream of SD. The regions responsible for the genome packaging of retroviruses are generally located downstream of SD, by which the genomic RNA is selectively packaged from an excess amount of spliced viral RNA.

It is suggested that HIV-2 genome packaging is primarily mediated by cis packaging mechanism (;; ). The Gag packages genomic RNA from which it is translated.

Therefore, the HIV-2 Gag is not required to distinguish the genomic RNA from viral spliced RNAs. Recent studies, however, indicated that HIV-2 genome packaging is primarily mediated by trans packaging mechanism.

TAR contains a palindromic sequence and has been suggested to form a dimer (; ). The dimerization of TAR may also function for efficient genome packaging. Further study will be needed to determine how the RNA elements located upstream of SD are involved in genome packaging.Despite significant differences in the recognition of NC by the 5′ leader RNA, HIV-1, and HIV-2 may employ similar mechanisms for genome packaging.

Observed that the RNA fragment of the 5′ leader region forms two alternative conformations as observed for the HIV-1 RNA fragments of the 5′ leader region. The long range interaction of U5–AUG is observed for the BMH conformation though it is not clear whether the LDI/BMH riboswitch mechanism is utilized for genome packaging in vivo. Recent study also indicated the U5–AUG interaction by SHAPE analysis. Of note, it is suggested that the U5–AUG interaction regulates the HIV-2 genome dimerization as indicated in HIV-1. Suggest that the 5′ leader RNA containing the U5–AUG interaction of HIV-1 is preferentially bound to the cognate NC. A major question is whether the U5–AUG interaction of HIV-2 also promotes the NC-binding to the 5′ leader RNA (Figure B). Phylogenetic and computational analyses suggested that the U5–AUG long range interaction is widely conserved among retroviruses.

The long range interaction of U5–AUG may be important for retroviral genome dimerization and genome packaging. PerspectivesIt has been indicated that the MoMLV NC domain of Gag binds predominantly to dimeric genomes (; ). HIV-1 was indicated to have a similar system.

MoMLV showed proximal NC-binding motifs in Ψ CES, implying that Gag–Gag multimerization is initiated by genomic RNA. A major question is whether the proximity of NC-binding motifs is observed for other retroviruses such as HIV-1 and HIV-2.

In addition, is the proximity of NC-binding motifs essential for retroviral genome packaging? Further study for these questions will lead to a better understanding for the molecular mechanism underlying retroviral genome packaging. Conflict of Interest StatementThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

AcknowledgmentsThis work was supported in part by a Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science (ID no. Reviewed by:, Kumamoto University, Japan, Osaka University, JapanCopyright: © 2011 Miyazaki, Miyake, Nomaguchi and Adachi. This is an open-access article distributed under the terms of the, which permits non-commercial use, distribution, and reproduction in other forums, provided the original authors and source are credited.Correspondence: Yasuyuki Miyazaki, Department of Microbiology, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto, Tokushima 770-8503, Japan. E-mail: ymiyazaki@basic.med.tokushima-u.ac.jp.

The key difference between lentivirus and retrovirus is that lentivirus is a type of retrovirus while retrovirus is an that contains the enzyme reverse transcriptase to convert RNA genome into DNA prior to insertion into the host organism.Furthermore, retroviruses are a group of viruses that belong to family Retroviridae. In this family, there are seven genera: Alpharetrovirus, Betaretrovirus, Gammaretrovirus, Deltaretrovirus, Epsilonretrovirus, Lentivirus and Spumavirus. Hence, lentiviruses are a type of retroviruses. What is a Virus?Viruses are.

Though viruses have a genetic element, they lack several key features of living organisms such as cell structures and independent replication. Hence, they belong to the non-living category. Humans have concern about viruses due to their disease-producing potential. Some examples for disease-causing viruses include Hepadnavirus, Herpes simplex virus, HIV, Enterovirus, and Filoviruses and the diseases they cause include Hepatitis B (viral), Herpes, AIDS, Polio, and Ebola, respectively.All viruses contain a enclosed in a protein coat. These particles do not have a cytoplasm. The nucleic acid present in the viruses can be either DNA or RNA, and their genome is linear or circular; single-stranded or double-stranded. Moreover, depending on their symmetry, viruses can be bihelical, icosahedral, binal, and polymorphic.

Based on the type of nucleic acid, there are different categories of viruses. What is Lentivirus?Lentivirus is a genus that comes under the Family Retroviridae. It is a type of retroviruses that cause chronic and deadly diseases characterized by a long incubation period. Lentivirus is an enveloped virus which is pleomorphic.

What is Retrovirus?Retrovirus is an RNA virus. However, unlike other RNA viruses, retrovirus possesses the unique ability to reverse transcribe its RNA genome into a double-stranded DNA.

It is due to the enzyme reverse transcriptase present in retroviruses. After transforming into DNA, viral DNA integrates with the host DNA. Thus, this unique ability has been used to develop certain applications like of inherited diseases. Unlike most other viruses, retrovirus vectors are capable of infecting a wide variety of cells. Because of this ability, retrovirus has been widely applied in clinical trials and cancer vaccine strategies. Retroviruses infect actively dividing cells.